key: cord-355931-3mvmetuv authors: nie, chuanxiong; ma, lang; luo, hongrong; bao, jinku; cheng, chong title: spiky nanostructures for virus inhibition and infection prevention date: 2020-07-16 journal: nan doi: 10.1016/j.smaim.2020.07.004 sha: doc_id: 355931 cord_uid: 3mvmetuv abstract the outbreak of a novel highly infectious virus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has aroused people’s concern about public health. the lack of ready-to-use vaccines and therapeutics makes the fight with these pathogens extremely difficult. to this point, rationally designed virus entry inhibitors that block the viral interaction with its receptor can be novel strategies to prevent virus infection. for ideal inhibition of the virus, the virus-inhibitor interaction has to outperform the virus-host interaction. in our view, the morphology of the inhibitor should be carefully designed to benefit virus-inhibitor binding, especially that the surfaces of viruses are mostly rough due to the existence of surface proteins for receptor-binding. in this perspective article, we would like to discuss the recent progress of designing inhibitors with spiky topography to maximize the interactions between viruses and inhibitors. we also would like to share our idea for the future study of inhibitors to prevent virus infection. flu' in 1918, the 'h2n2' outbreak in 1957, and the recent 'h1n1' outbreak in 2009, each of them has caused massive damage to the human society. [3] flu vaccines have been developed to prevent the infection, but the high error rate of rna polymerase enables iav to mutate with unpredictable antigen shifts and drifts, which makes it challenging to prepare the correct vaccine for the coming flu outbreak. [4] therefore, inhibitors that prevent virus infection are urgently needed. the infection cycle of iav starts with binding to the sialic acid receptors on the host cell surface. [5] blocking the virus binding with a decoy has been realized as an effective strategy to inhibit the virus infection at the beginning. [6] [7] [8] [9] [10] [11] to design such an inhibitor, maximizing the inhibitor-virus interaction to compete virus-cell interaction is the key to achieve potent virus inhibition. to this point, functional group valency, inhibitor size, and scaffold stiffness are reported to be the main parameters to match the receptor binding sites of targeted virions. [12, 13] additionally, the morphological matching of two subjects also benefits the binding, e. g. most of the plant weeds have evolved a spiky surface to adhere to a host for long-distance spreading. [14] as the virus-inhibitor binding is the key for inhibition, this morphological matching principle can be considered for the design of inhibitors to robustly bind to the virions and compete with the virus-host binding as shown in scheme 1. the general concepts of using spiky nanostructures for inhibiting virus binding to cell membranes. in a recent report by nie et al., [15] the principle of morphology-matching for iav inhibition was introduced. they firstly obtained the morphological details from cryogenic transmission electron microscopy (cryo-tem) images as shown in figure 1 . despite the difference between spherical and filament morphologies, the virions of iav exhibit similar surface nanostructure. the hemagglutinin (ha) and neuraminidase (na) generate a rough surface as shown in figure 1a . from the reconstruction of the geometry models, they found a gap around 10 nm for the surface proteins of iav. in order to match the surface nanostructure of iav virion, geometry models of spiky nanoparticles are generated with similar size but different spiky length and space as shown in figure 1b ,c. compared with smooth nanoparticles, these spikes increase the interface area by inserting into the gap of the proteins as shown in figure 1b . the binding patterns for the spiky nanoparticles are also different for different spike structures as shown in figure 1c . if the spikes are too long, the virion interacts only with the tip, resulting in decreased area. when the spikes are too dense, they no longer fit the gap, which decreases the interface area. at a suitable spike length and spacing (10 nm length, 10 nm spacing), the nanoparticle can tightly bind to iav virion with a maximized contact area. interaction between iav virion and spiky nanoparticles with different geometry parameters. reprinted with permission from [15] . copyright (2020) american chemical society. as an experimental proof to this idea, spiky nanostructures (sns) with similar morphologies with bioinert sio 2 cores are synthesized as shown in figure 2a . nanoparticles with a ~120 nm core and spike length of 0-30 nm are synthesized. the virion-binding abilities are compared via a centrifuge-western blot assay as shown in figure 2b . it is clear that the spikes enhance the binding between the nanoparticle and iav virion. when the spikes are 5-10 nm, maximized virus binding is achieved, which is in agreement with the geometry simulation results in figure 1c . to check the binding patterns between the spiky nanoparticles and iav virion, cryo-tem images are also acquired (figure 2c ). for the smooth nanoparticle (sns-0), the virion interacts with the nanoparticle with a very limited area. as expected, for sns with 10 nm spikes (sns-10), the spikes insert into the gap of the protein, forming a conjunction to benefit the binding. however, for sns with 30 nm spikes (sns-30), tip-to-tip interactions are noticed, for which the interaction area is smaller than sns-10. however, it is noticed that even the spiky nanoparticles show viral binding abilities, they are not able to inhibit the infection of iav. this is probably because the virus-inhibitor binding is not strong enough to compete with the virus-host binding. to achieve potent virus inhibition, the surface of the nanoparticles should be functionalized with binding motifs towards iav. erythrocyte has a natural display of sialic acid as the binding target for several subgroups of iav (figure 3a) . as a proof of concept, the authors coat the spiky nanostructures with erythrocyte membrane (em) as a targeting shell towards iav virion. during the coating process, all the membrane components are transferred to the surface of spiky nanoparticles. with em coating, the viral binding is further enhanced, and an inhibitor that can effectively reduce the viral binding to the host cells is obtained. in the cellular infection assay, all the inhibitors show a reduced number of infected cells as shown in figure 3b , c. sns-10 is the best inhibitor, by which >90% of cellular infection is inhibited. they also check if the inhibitors can prevent virus replication by plaque assay (figure 3d, e) . 99.9% inhibition is achieved with 1 mg/ml dose of the inhibitor. be co-used with na inhibitor, more potent (>99.99%) inhibition of virus replication is achieved. ha and na regulate iav binding in mucus and to host cells. briefly speaking, ha interacts with sialic acid to trigger virus entry, and na cleaves the ha-sialic acidbinding for virus release after the replication. [16] to achieve a better inhibition with sialic acid-based compounds, the negative effects of na on the binding should be minimized. in another report by this group, a heteromultivalent iav inhibitor based on the spiky nanoparticles is developed. the surface of the spiky nanoparticle is functionalized covalently with sialyllactose (sal) and zanamivir (zanamivir), via a linear polyglycerol linker as shown in figure 4a , b. [17] with zanamivir on the surface, the activity of na is inhibited (figure 4e) , the virus-inhibitor binding is further enhanced (figure 4c, d) , which results in nearly 100% blocking of virion interaction with the host cells. in a cellular infection assay, it is noticed that in the presence of the spiky inhibitors, there are no infected cells. even being used after the first cycle of infection, the inhibitor, vlnp-sal/zan, still shows a >99.9999% inhibition of virus titre (figure 4f ). they also report that the inhibitor is active against three human iav strains, which are a/x31 (h3n2), a/pr/8/34 (h1n1) and a/panama/2007/1999 (h3n2), as shown in figure 4g . [17] . copyright (2020) wiley. as most of the virus starts the replication with the binding to the receptors and most of the virus exhibit similar spiky morphology as iav, it is envisioned that such a strategy can also be used for other viral strains, e. g. coronaviruses. for a potent virus inhibition, the concept of geometry matching can be incorporated with different antiviral strategies. the nanostructures in these studies are hollow mesoporous structures, which are capable of loading antiviral cargos as delivery systems to further increase the virus inhibitor performance. on the other hand, enzyme-mimicking nanostructures have also been developed as a powerful tool to combat pathogen infections. these nanostructures exhibit the ability to produce highly 'toxic' reactive oxygen species, including oh , o 2 -, and etc., which show the ability to prevent wound infections. [18] [19] [20] these enzyme-mimic nanostructures have been fabricated into a self-disinfection mouth mask for the combating of the respiratory pathogen infections. [21] it is envisioned that combining functional materials cores with the spiky surfaces, a more potent virus inhibitor with multiple modes of action can be produced. however, the drawback of such spiky inhibitors is also clear: the non-specific interaction with the biological molecules will facilitate the uptake and clearance by immune systems. [22] therefore, the surface of the inhibitor should be also be functionalized with highly antifouling or bio-stealth groups to avoid the rapid clearance after the intaking. cellular membrane coating can be a good solution, which enables the nanoparticle to bypass the immune system for long-term circulation. in such a system, the source of host cells needs to be carefully selected to avoid the side effects of cellular membrane antigens. [23] [24] [25] another solution can be smart nanostructures that are able to change the morphology upon stimuli. [26] spiky nanostructures can also be obtained via other approaches, e. g. coating of a small particle onto an existing core material, which offers more possibilities to control the surface morphology and functionalization. [27] nevertheless, the geometry matching can be a universal approach to benefit the binding between the two subjects. not only for virus inhibition, this idea can also be used as a general approach for the combat with bacteria and tumors. [28] [29] [30] [31] the authors declare that they have no conflict of interest. virological assessment of hospitalized patients with covid-2019 h1n1 2009 influenza virus infection during pregnancy in the usa biomimetic nanoparticles as universal influenza vaccine the pathology of influenza virus infections pathogen inhibition by multivalent ligand architectures phage capsid nanoparticles with defined ligand arrangement block influenza virus entry nanostructured glycan architecture is important in the inhibition of influenza a virus infection linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo antiviral agents from multivalent presentation of sialyl oligosaccharides on brush polymers sialyllactose-modified three-way junction dna as binding inhibitor of influenza virus hemagglutinin highly efficient multivalent 2d nanosystems for inhibition of orthopoxvirus particles multivalent flexible nanogels exhibit broad-spectrum antiviral activity by blocking virus entry 56. xanthium strumarium l spiky nanostructures with geometry-matching topography for virus inhibition influenza a virus hemagglutinin-neuraminidase-receptor balance: preserving virus motility reverse design of an influenza neutralizing spiky nano-inhibitor with a dual mode of action core-shell-structured mof-derived 2d hierarchical nanocatalysts with enhanced fenton-like activities augmenting intrinsic fenton-like activities of mof-derived catalysts via n-molecule-assisted self-catalyzed carbonization metal-organic-framework-derived 2d carbon nanosheets for localized multiple bacterial eradication and augmented anti-infective therapy metal-organic frameworks with photocatalytic bactericidal activity for integrated air cleaning facile synthesis of uniform viruslike mesoporous silica nanoparticles for enhanced cellular internalization cell membrane-derived nanomaterials for biomedical applications generating giant membrane vesicles from live cells with preserved cellular properties nonchemotherapic and robust dual-responsive nanoagents with on-demand bacterial trapping, ablation, and release for efficient wound disinfection a versatile surface bioengineering strategy based on mussel-inspired and bioclickable peptide mimic silica nanopollens enhance adhesion for long-term bacterial inhibition physical activation of innate immunity by spiky particles inorganic nanozyme with combined self-oxygenation/degradable capabilities for sensitized cancer immunochemotherapy ultrasound-targeted microbubble destruction augmented synergistic therapy of rheumatoid arthritis via targeted liposomes spiky nanoparticles that match the surface topography of virions are believed to be ideal scaffolds for virus inhibitors key: cord-310780-0k8owwf8 authors: iwai, atsushi; shiozaki, takuya; miyazaki, tadaaki title: relevance of signaling molecules for apoptosis induction on influenza a virus replication date: 2013-11-22 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2013.10.100 sha: doc_id: 310780 cord_uid: 0k8owwf8 abstract apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. the severity of influenza a virus (iav) infection is also closely related to dysfunction of apoptosis regulation. in the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by iav proteins to enable effective virus replication. the modulation of the intracellular signaling pathway inducing apoptosis by the iav infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of iav infections. this review focuses on apoptosis related molecules involved in iav replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed. apoptosis is classified into type-i programmed cell-death, where morphological features of the cells ongoing apoptosis are subject to cytoplasmic shrinkage, plasma membrane blebbing, dna fragmentation, and chromatin condensation. finally, the cells form cell fragments, termed apoptotic bodies, and are removed by phagocytic cells. apoptosis is able to remove cells without a trace, and is an important physiological mechanism to maintain homeostasis in mammals. because of its physiological significance, the execution of apoptosis must be strictly regulated, and a large number of molecules are involved in controlling apoptosis. apoptosis is closely related to a number of aspects of influenza a virus (iav) infection. for instance, apoptosis plays a pivotal role in iav elimination through the removal of the virus infected cells, and tissue damage during the course of iav infection including multiple organ dysfunction is caused by apoptosis [1, 2] . further, abnormal induction of lymphocyte apoptosis is related in the disease symptoms of influenza, and apoptosis is also important to terminate inflammation through the induction of activation-induced cell death (aicd). in this review, focusing on the relationship between iav proteins and apoptosis related host cellular molecules, strategies of iav for effective viral propagation by the regulation of apoptosis are discussed. normally, apoptosis induction is accompanied by activation of caspases (cysteine-aspartic acid proteases) [3] . caspases are classified into initiator caspases and effector caspases. initiator caspases are responsible for initiating apoptosis through the activation of downstream effector caspases by proteolytic cleavage, and effector caspases are necessary for activation or inactivation of molecules, such as parp (poly (adp-ribose) polymerase) [4] and cad (caspase-activated dnase) [5] to execute apoptosis. there are two major pathways to activate caspase cascades for apoptosis induction, one is the death receptor pathway, and the other is the mitochondrial pathway (fig. 1) . death receptors, such as tnfar1 (tumor necrosis factor a receptor 1), fas, dr4 (death receptor 4, also called trailr1) and abbreviations: akt, v-akt murine thymoma viral oncogene homolog 1; apaf-1, apoptotic protease activating factor 1; bax, bcl-2-associated x protein; bcl-2, b-cell lymphoma 2; bh, bcl-2 homology; caspases, cysteine-aspartic acid protease; dap3, death-associated protein 3; dr4, death receptor 4; dr5, death receptor 5; fadd, fas-associated death domain; iav, influenza a virus; ifn, interferon; ips-1, inf-b promoter stimulator protein 1; irf3, ifn regulatory factor 3; jnk, c-jun aminoterminal kinase; m, matrix protein; nf-jb, nuclear factor-jb; na, neuraminidase; np, nucleoprotein; ns1, non-structural protein 1; pi3k, phosphatidylinositol-3 kinase; rig-i, retinoic acid-inducible gene-i; tnf-a, tumor necrosis factor a; tnfar1, tnf-a receptor 1; tradd, tumor necrosis factor receptor type 1-associated death domain protein; trail, tnf-related apoptosis inducing ligand; xiap, x-linked inhibitor of apoptosis protein; zaps, zinc finger antiviral protein, short form. dr5 (death receptor 5, also called trailr2), are defined by the death domain of its cytoplasmic region. the death domain is responsible for activating caspase-8. after the stimulation with the ligands of death receptors, fadd (fas-associated death domain) is directly or indirectly bound to the death receptors, and then caspase-8 is activated by fadd through the activation of a self proteolytic cleavage. therefore, fadd plays a pivotal role in death receptor-mediated apoptosis induction. it has been reported that fas is able to directly bind to fadd [6] , tnfar1 requires tradd (tumor necrosis factor receptor type 1-associated death domain protein) [7] , and dr4 and dr5 require dap3 (death-associated protein 3) for recruitment of fadd [8] . mitochondria are an important organelle in determining cell destiny, and generally the mitochondrial membrane potential is disrupted in the course of apoptosis induction. after disruption of the mitochondrial membrane potential, the cytochrome c in the mitochondrial inner membrane is released into the cytoplasm, and caspase-9 is activated by apaf-1 (apoptotic protease activating factor 1) which is known to be a cytoplasmic sensor molecule for cytochrome c [9] . the mitochondria membrane potential is mainly regulated by bcl-2 (b-cell lymphoma 2) family proteins [10] , a family of proteins characterized by bh (bcl-2 homology) domains, and bcl-2 are classified into three types. one is a pro-apoptotic multidomain sub-family of proteins including bax (bcl-2-associated x protein) and bak (bcl-2 homologous antagonist killer). these proteins have bh1, bh2, and bh3 domains, and activate apoptosis induction through the increment of permeability of the mitochondrial outer membrane. another is the anti-apoptotic sub-family proteins which have bh1, bh2, bh3, and bh4 domains, and include bcl-2 and bcl-xl (b-cell lymphoma-extra large). these proteins exhibit anti-apoptotic functioning through the inhibition of the pro-apoptotic multidomain bcl-2 sub-family function. the third is pro-apoptotic bh3 only proteins, such as bid (bh3 interactingdomain death agonist) and bad (bcl-2-associated death promoter). these proteins are antagonists to the anti-apoptotic bcl-2 subfamily protein function, and promote apoptosis. two major pathways for induction of apoptosis and involvement of caspase-3 activation for iav replication. death receptor pathway and mitochondria pathway are crucial for the activation of caspase-8 and caspase-9 respectively. disruption of mitochondrial membrane potential causes cytochrome c release, and activates caspase-9 by the complex formation with cytochrome c recognition protein, apaf-1. after the activation of these pathways, both activated caspase-8 and caspase-9 activate caspase-3 by proteolytic cleavage. both activated caspase-8 and caspase-9 activate common downstream caspases, mainly caspase-3, by proteolytic cleavage, and then the apoptosis takes place. importantly, apoptosis through these two major pathways is crucial for the elimination of iav through the removal of the virus infected cells from the body, as well as it is involved in the effective replication of the iav. previous study has demonstrated that activation of caspase-3 is important for the effective replication of the iav through the activation of exportation of the viral rnp (ribonucleoprotein) complex from the nucleus to the cytoplasm [11] . this may indicate that the iav utilizes apoptosis signaling molecules, crucial for host immune response to eliminate the virus, conversely for the effective replication of the iav. the death receptor-mediated signaling pathway is thought to be closely related to the pathology of iav infection, and tnfa, fasl (fas ligand), and trail (tnf-related apoptosis inducing ligand) are known to be death ligands, and recognized by tnfar1, fas, dr4, and dr5, respectively. abnormally elevated expression of these death ligands is frequently found in lethal iav infections, and aberrant induction of apoptosis caused by these death ligands is thought to be related in the onset of the multi organ disorders occurring in severe iav infections. death receptors play pivotal roles in the activation of caspase-8, and also work to activate nf-jb (nuclear factor-jb) and jnk (c-jun amino-terminal kinase) like other non-death receptors belonging to the tnf-superfamily receptors (fig. 2) . overall, death receptor-mediated signaling pathway contributes to apoptosis induced by iav infection through the activation of caspases mediated by fadd and jnk, and death receptor-mediated activation of nf-jb is crucial for enhancement of death receptor-mediated signaling in both autocrine and paracrine manner. generally, nf-jb activation increases cell viability, and competes with apoptosis induction. it has been reported that the ns1 (non-structural protein 1) protein encoded in the iav genome, is involved in the activation of nf-jb through the activation of the pi3k (phosphatidylinositol-3 kinase)/akt (v-akt murine thymoma viral oncogene homolog 1) pathway [12, 13] . in addition, iav neuraminidase (na) protein is also involved in activation of akt through binding with ceacam6 (carcinoembryonic antigen-related cell adhesion molecule 6) protein which is known to be related in activation of src (sarcoma viral oncogene homolog)/akt signaling pathway [14] . at the same time nf-jb is also an important transcriptional factor for activation of the immune response through fig. 2 . modulation of iav replication by death receptor-mediated signaling pathway. in addition to caspase 8 activation, death receptors activate nf-jb and jnk signaling pathways. iav ns1 and na are involved in activation of nf-jb through the activation of akt, and ns1 also has a function to inhibit the jnk activation. the induction of a number of inflammatory cytokines including tnf-a, fasl, and trail. previously, the importance of nf-jbdependent expressions of trail, fas, and fasl has been reported [15] . therefore, activation of nf-jb is thought to be important to prevent immediate execution of apoptosis and to increase cell viability for effective virus replication, as well as it is crucial for death ligand expression to enhance caspase activation. the jnk is known to be an important kinase which regulates expression of genes involved in stress responses. previous reports demonstrated that jnk activation is involved in production of type i interferons (ifns), and is that jnk activation is inhibited by the influenza virus ns1 protein [16, 17] . it has also been reported that treatment with jnk inhibitor exhibits antiviral activity against iav infections [18] . generally, the jnk signaling pathway is related to stress induced apoptosis through activation of the mitochondrial pathway. however, jnk activation is also implicated in cell proliferation and survival under some conditions. it is assumed that transient activation of jnk is involved in increasing cell viability, and that sustained activation of jnk is associated with apoptosis induction. the dual phased function of jnk might explain these controversial phenomena. in addition, since jnk activation emphasizes death receptor mediated apoptosis induction, the inhibition of jnk activation could be effective to protect from the apoptosis caused by abnormally elevated expressions of inflammatory cytokines including death ligands after iav infection [19] . the rig-i (retinoic acid-inducible gene-i) is known to be an intracellular pattern recognition receptor responsible for recogniz-ing the viral rna in iav-infected cells, and is crucial for activation of host anti-virus responses through the induction of type i ifns and inflammatory cytokines. the rig-i recognizes viral 5 0 -tri-phosphorylated rnas, and is activated through conformational changes and then binds to ips-1 (ifn-b promoter stimulator protein 1; also called mavs, cardif, or visa) [20] , an adapter protein localized in the mitochondria outer membrane. the ips-1 is responsible for activating downstream signaling molecules for the induction of type i ifns and inflammatory cytokines. further, ips-1 is also involved in apoptosis induced by viral infection [21, 22] . the irf3 (ifn regulatory factor 3), the downstream transcriptional factor activated by ips-1, is involved in ips-1-mediated apoptosis induction. the ips-1-mediated activation of irf3 leads to the expression of noxa/puma. noxa/puma inhibits the function of mitochondrial anti-apoptotic bcl-2 family proteins by binding, and induces apoptosis through the destruction of the mitochondrial membrane potential. further, irf3 is able to induce apoptosis independently from the function as a transcriptional factor. the ips-1-mediated irf3 phosphorylation leads to the irf3 binding to bax, and activates the bax-mediated apoptosis induction. the ips-1-mediated signaling pathway for the production of type i ifns is critical to the activation of the early defensive mechanism of the host against viral infections. because of the physiological significance of ips-1-mediated signaling pathway, this pathway is inhibited by many viruses, such as hepatitis c virus, ebora virus, and severe acute respiratory syndrome coronavirus [22, 23] . inhibition of the ips-1-mediated signaling pathway is also assumed to be important for the effective replication of iav. the iav has several viral components to inhibit the ips-1mediated signaling pathway (fig. 3) . the ns1 protein of the iav binds to rig-i and inhibits rig-i functioning through its rna binding activity [24] . in addition, the function of trim25 (tripartite motif-containing protein 25), the e3 ubiquitin ligase crucial for the rig-i mediated ifn production, is also inhibited by the ns1 protein [25] . the viral polymerase complex binds to ips-1 and inhibits the ips-1 function for type i ifn production [26] . further, pb1-f2, a viral protein encoded in the second open reading frame of iav segment 2 mrna, is also involved in the inhibition of the ips-1-mediated production of type i ifn by binding to ips-1 [27] . results of the comprehensive proteomic analysis indicate that the viral polymerase complex may bind to zaps (zinc finger antiviral protein, short form) [28] , the positive regulator of the ips-1-mediated signaling pathway by binding to rig-i [29] . this finding suggests that the viral polymerase complex may modulate its function. although the functions of these viral molecules on ips-1mediated induction of apoptosis have not been well confirmed, these molecules are thought to inhibit the function of ips-1 leading to induction of apoptosis and also the induction of type i ifns. a previous report has demonstrated that overexpression of bcl-2 effectively inhibits iav replication [30] . bcl-2 is an anti-apoptotic protein, functional against the pro-apoptotic proteins such as bax, noxa, and puma. these results suggest that the ips-1mediated activation of the mitochondrial pathway for apoptosis induction may be also functional for the effective replication of the iav. a previous report demonstrated that the siva-1, a pro-apoptotic protein, is crucial for effective replication of the iav [31] . since the function of siva-1 in the virus replication completely disappears after treatment with a pan-caspase inhibitor, z-vad fmk, the siva-1 appears to modulate iav replication through the controlling activation of caspases (fig. 4) . several apoptosis-related molecules involved in siva-1 mediated apoptosis induction were reported, and one of these is xiap (x-linked inhibitor of apoptosis protein) [32] . the xiap is known to be an anti-apoptotic protein, and it directly inhibits proteolytic activity of caspases including caspase-3 by binding [33] . siva-1 is considered to be involved in the inhibition of xiap function, and a previous report demonstrated that overexpression of xiap inhibits iav replication [11] . this may suggest the significance of the siva-1-mediated signaling pathway on the virus propagation. some molecules involved in the death receptor mediated signaling pathway are also important for the ips-1-mediated signaling pathway. for instance, fadd, an adaptor molecule responsible for activating caspase-8 in the death receptor signaling pathway, is important for nf-jb activation in the ips-1-mediated signaling pathway [34] . the tradd, an adapter molecule crucial for the recruitment of fadd to tnfar1, is also involved in the activation of the ips-1-mediated signaling pathway for type i ifn fig. 4 . other apoptosis induction factors involved in iav replication. siva-1 is involved in the effective iav replication through the activation of caspases. dap3 is essential for death receptor-mediated apoptosis induction and ips-1-induced anoikis, suggesting that dap3 is an important molecule to regulate apoptosis induced by iav infection. production [35] . in addition, dap3 is known to be crucial for apoptosis induced by trail stimulation through the recruitment of fadd to dr4 and dr5, and is also related to other ips-1 functions. a previous report has demonstrated that ips-1 is also involved in induction of anoikis [36] , which is known to be a form of apoptosis induced by anchorage-dependent cells detaching from the surrounding extracellular matrix. in anoikis induction, dap3 binds to ips-1 and recruits fadd for activation of caspase-8, and then apoptosis is executed. the dap3 function in anoikis induction is inhibited by akt-dependent phosphorylation [37] , and as described in the previous section, akt is activated by the ns1 protein of iav. therefore, the dap3 function inducing anoikis is thought to be inhibited in iav infected cells. the role of the dap3 and ips-1-mediated signaling pathway for apoptosis induction on iav infection is not fully understood. however, dap3 is also crucial for apoptosis induced by tnf-a, fasl, and trail stimulation [8, 38] . therefore, dap3 and dap3 related molecules, such as lkb1 (liver kinase b1) [39] , and dele (death ligand signal enhancer) [40] , are thought to be implicated in the virus replication and regulation of apoptosis caused by the iav infection. it has been reported that matrix protein (m) 1 and 2 of influenza a virus are involved in regulation of apoptosis induction [41, 42] . iav m1 protein is involved in cell susceptibility to apoptosis through binding to an anti-apoptotic protein, hsp70, and m2 protein is indirectly involved in apoptosis induction through inhibition of type ii programmed cell death, macroautophagy. further, involvement of iav pb1-f2 and np (nucleoprotein) proteins for apoptosis induction was reported [43, 44] . pb1-f2 protein induces apoptosis through direct disruption of mitochondrial membrane potential, and np protein is indirectly involved in apoptosis induction through binding to a host-cellular anti-apoptotic protein, clusterin. these virus proteins and host-cellular molecules are thought to be also important for controlling apoptosis induced by iav infection. this review has mainly discussed intracellular mechanisms for the regulation of apoptosis by iav. induction of apoptosis is thought to be important for both inhibition and activation of iav propagation, and the issues involved in a full understanding of the viral strategy for effective propagation by controlling apoptosis appear to be complicated. further investigation of the molecular mechanism regulating apoptosis by iav is required, and may provide insights to enable the development of novel anti-virus drugs. apoptosis: a mechanism of cell killing by influenza a and b viruses outbreak of avian influenza a(h5n1) virus infection in hong kong in 1997 human ice/ced-3 protease nomenclature identification and inhibition of the ice/ced-3 protease necessary for mammalian apoptosis a caspase-activated dnase that degrades dna during apoptosis, and its inhibitor icad fadd, a novel death domain-containing protein, interacts with the death domain of fas and initiates apoptosis the tnf receptor 1-associated protein tradd signals cell death and nf-kappa b activation a gtp-binding adapter protein couples trail receptors to apoptosis-inducing proteins apaf-1, a human protein homologous to c. elegans ced-4, participates in cytochrome c-dependent activation of caspase-3 the bcl2 family: regulators of the cellular life-or-death switch caspase 3 activation is essential for efficient influenza virus propagation influenza a virus ns1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling characterization of the interaction of influenza virus ns1 with akt influenza a virus neuraminidase protein enhances cell survival through interaction with carcinoembryonic antigen-related cell adhesion molecule 6 (ceacam6) protein nf-kappab-dependent induction of tumor necrosis factorrelated apoptosis-inducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation influenza virus-induced ap-1-dependent gene expression requires activation of the jnk signaling pathway the influenza a virus ns1 protein inhibits activation of jun n-terminal kinase and ap-1 transcription factors small molecule inhibitors of the c-jun nterminal kinase (jnk) possess antiviral activity against highly pathogenic avian and human pandemic influenza a viruses inhibition of c-jun n-terminal kinase rescues influenza epitope-specific human cytolytic t lymphocytes from activation-induced cell death ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction proapoptotic signaling induced by rig-i and mda-5 results in type i interferon-independent apoptosis in human melanoma cells viral apoptosis is induced by irf-3-mediated activation of bax mavs-mediated apoptosis and its inhibition by viral proteins ebola virus vp35 protein binds doublestranded rna and inhibits alpha/beta interferon production induced by rig-i signaling rig-i-mediated antiviral responses to single-stranded rna bearing 5 0 -phosphates influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i influenza a virus polymerase inhibits type i interferon induction by binding to interferon beta promoter stimulator 1 influenza virus protein pb1-f2 inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential comprehensive proteomic analysis of influenza virus polymerase complex reveals a novel association with mitochondrial proteins and rna polymerase accessory factors zaps is a potent stimulator of signaling mediated by the rna helicase rig-i during antiviral responses influenza a virus replication is dependent on an antioxidant pathway that involves gsh and bcl-2 requirement for siva-1 for replication of influenza a virus through apoptosis induction siva1 is a xiap-interacting protein that balances nfkappab and jnk signalling to promote apoptosis x-linked iap is a direct inhibitor of cell-death proteases identification of mavs splicing variants that interfere with rigi/mavs pathway signaling tradd protein is an essential component of the rig-like helicase antiviral pathway ips-1 is crucial for dap3-mediated anoikis induction by caspase-8 activation functional role of death-associated protein 3 (dap3) in anoikis structure-function analysis of an evolutionary conserved protein, dap3, which mediates tnf-alpha-and fasinduced cell death lkb1 is crucial for trail-mediated apoptosis induction in osteosarcoma identification of dele, a novel dap3-binding protein which is crucial for death receptor-mediated apoptosis induction cell death regulation during influenza a virus infection by matrix (m1) protein: a model of viral control over the cellular survival pathway matrix protein 2 of influenza a virus blocks autophagosome fusion with lysosomes pb1-f2, an influenza a virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes influenza a virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein clusterin key: cord-324696-htx0ul4o authors: chothe, shubhada k.; bhushan, gitanjali; nissly, ruth h.; yeh, yin-ting; brown, justin; turner, gregory; fisher, jenny; sewall, brent j.; reeder, deeann m.; terrones, mauricio; jayarao, bhushan m.; kuchipudi, suresh v. title: avian and human influenza virus compatible sialic acid receptors in little brown bats date: 2017-04-06 journal: sci rep doi: 10.1038/s41598-017-00793-6 sha: doc_id: 324696 cord_uid: htx0ul4o influenza a viruses (iavs) continue to threaten animal and human health globally. bats are asymptomatic reservoirs for many zoonotic viruses. recent reports of two novel iavs in fruit bats and serological evidence of avian influenza virus (aiv) h9 infection in frugivorous bats raise questions about the role of bats in iav epidemiology. iavs bind to sialic acid (sa) receptors on host cells, and it is widely believed that hosts expressing both sa α2,3-gal and sa α2,6-gal receptors could facilitate genetic reassortment of avian and human iavs. we found abundant co-expression of both avian (sa α2,3-gal) and human (sa α2,6-gal) type sa receptors in little brown bats (lbbs) that were compatible with avian and human iav binding. this first ever study of iav receptors in a bat species suggest that lbbs, a widely-distributed bat species in north america, could potentially be co-infected with avian and human iavs, facilitating the emergence of zoonotic strains. with a wide host range, ability to undergo genetic recombination and cross species barrier, influenza a viruses (iavs) continue to spread globally, causing huge economic losses to the poultry industry and threatening public health. unlike the low pathogenic avian influenza viruses (lpaivs), highly pathogenic avian influenza viruses (hpaivs) cause very severe disease in gallinaceous poultry often leading to 100% mortality within 2-3 days 1 . hpaivs of h5n1 subtype are of particular concern as certain contemporary eurasian lineage h5n1 viruses can carry an alarming case fatality rate of up to 50% in humans 2 . negative strand segmented rna genomes contribute to the genetic variability of iavs. in addition, iavs can infect a wide range of avian and mammalian host species resulting in the emergence of novel subtypes with altered species tropism and/or virulence. it is widely known that aquatic birds such as ducks, gulls, and shorebirds serve as a natural reservoir of most known iavs 3 . iavs are known to infect a wide range of avian and mammalian hosts, and it is highly likely that their host range could be broader than currently known, with more reservoirs to be revealed. for example, northwest atlantic gray seals have recently been suggested to be an endemically infected wild reservoir population for diverse iavs 4 . from these natural reservoirs, iavs can evolve into novel variants which can potentially lead to human pandemics. influenza pandemics occur time to time with the most recent pandemic being in 2009. it is undisputed that the next influenza pandemic will happen, but the only question is when it will happen. despite several reports investigating the basis of genetic variability of iavs, the precise mechanism of pandemic iav generation still remains an unresolved mystery. it is possible that additional iav reservoirs are yet to be identified which will help to see the full picture of iav ecology and evolution. bats (order: chiroptera) are one of the ancient mammals, and their speciation occurred long before the development of most modern mammals. bats are globally distributed, relatively long lived 5 and represent approximately 24% of all known mammalian species. further, bats are one of the most diverse families of mammals found in nearly every habitat/continent around the world except antarctica. more importantly, certain old world bat species are known to be natural reservoirs of zoonotic viruses that cause some of the deadliest diseases in humans including filoviruses (such as ebola and marburg viruses), lyssaviruses, severe acute respiratory syndrome (sars)-related coronaviruses and henipaviruses (e.g. hendra and nipah viruses) 6, 7 . in addition, bats also act as a major natural reservoir for hepaciviruses and pegiviruses (hepatitis c virus and gb virus b) 8 . notably, all the zoonotic viruses of bat-origin identified to date are rna viruses 5 . however, it is believed that the actual diversity of viruses in bats could be much more than what is currently known, as most of the investigations have focused on searching for specific viruses of interest and many additional viruses must have been overlooked 9 . the prospects for bats contributing to iav epidemiology came to light after the identification of two novel influenza-like viruses in fruit bats by next generation sequencing 10 . these two viruses are genetically distinct from all previously known iavs and hence are designated as novel subtypes, namely h17n10 and h18n11. these novel iavs have been recently recovered in cell culture using synthetic dna 11 . however, these ha and na subtypes have not been identified in birds serologically or virologically. consequently, the reservoir(s) of these novel iav subtypes is still undefined. phylogenetic studies raised a possibility that bats have the capacity to harbor more influenza virus genetic diversity than all the other mammalian and avian species combined 10 . in addition, it was demonstrated that little yellow-shouldered bats in central america could constitute a potential sylvatic mammalian reservoir of influenza 12 . susceptibility of bats to iavs has been confirmed by recent serological evidence of aiv h9 subtype in about 30% of frugivorous bats from africa 13 . it is worth noting that detection of antibodies against one aiv subtype in 30% of the bats tested is very significant. influenza viruses bind to sialic acid (sa) residues that are bound to glycans through α2,3 or α2,6 linkage on the host cells 14 . the expression of the appropriate host cell receptor to which viral haemagglutinin (ha) can bind is the key determinant of the ability of iavs to infect a host 15 . avian influenza viruses (aivs) preferentially bind to sa receptors that are linked to galactose by an α 2,3 linkage (sa α2,3-gal), while human and classical swine influenza viruses show preference to α2,6 linked sas (sa α2,6-gal). a key source of iav genetic diversity could be from the replication of iavs in a non-native host species that initiate evolution of new virus variants 16 . hosts that co-express both sa α2,3-gal and sa α2,6-gal receptors such as chickens, ducks and pigs have been hypothesized to potentially support re-assortment of iavs and hence play a major role in the evolution of iavs 14, 17 . while the role of bats in iav evolution is not yet known, recent evidence raises a primary question, "can bats support co-infection of avian and human iavs?". consequently a logical first unknown which needs to be addressed is whether bats express appropriate sa receptors compatible with avian and human iav binding. to resolve this enigma, we investigated for the first time the distribution of sa receptors in little brown bats (lbbs) (myotis lucifugus), a widely-distributed bat species in north america and their compatibility to support avian and human iav binding. tissues sections from a total of 10 juvenile and 10 adult lbbs were subjected to lectin histochemistry for the detection of influenza virus receptors. no differences in sa receptor distribution between the juvenile and adult lbbs were found. co-expression of both sa α2,3-gal and sa α2,6-gal receptors in lbb respiratory tract. abundant co-expression of avian (sa α2,3-gal) and human (sa α2,6-gal) type influenza receptors was found throughout the lbb respiratory tract (fig. 1) . the relative abundance of avian and human type influenza receptors were distinctly different in the upper and lower airway of lbb. while sa α2,3-gal receptors were predominant on the mucosal lining of the trachea (fig. 1a) , predominance of sa α2,6-gal receptors was found in the alveolar epithelium, alveolar duct and visceral pleura of the lung (fig. 1b) . further, abundant expression of sa α2,6-gal receptors was found in the lamina propria and submucosa of the tracheal tissue (fig. 1a ). expression of sa α2,3-gal receptors was found in the stomach and intestines of lbb (fig. 2) . cells lining the mucosa of stomach exhibited co-expression of both avian and human receptors ( fig. 2a) . the luminal epithelium of intestines primarily expressed sa α2,3-gal receptors whereas considerable expression of sa α2,6-gal receptors were found on the goblet cells, lamina propria, muscularis and serosa of the intestine (fig. 2b ). ing assays were performed on lbb trachea and lung sections using an lpaiv h5n2 (a/h5n2/chicken/ pennsylvania/7659/1985) virus. extensive binding of avian h5n2 virus to lbb trachea, lung and intestines was observed (fig. 3) . h5n2 virus binding pattern was in accordance with the relative abundance of sa α2,3-gal receptors in tissues such that greater virus binding to the tracheal ( in addition to the virus binding assay using antibody-based detection, we also visualized virus binding using scanning electron microscopy (sem) which also confirmed abundant binding of avian h5n2 virus (fig. 5a ) and human h1n1 virus (fig. 5b ) to lbb trachea (fig. 5) . it has been shown that lectins from different suppliers may show different binding specificities; in particular the source of maa has been shown to significantly affect specificity. lectins (sna and maaii) used in this study were purchased from vector laboratories which were shown to bind to the appropriate sialic acid linkages with a high degree of specificity by glycan microarray screening (http://www.functionalglycomics.org). we treated sections with sialidase a (n-acetylneuraminate glycohydrolase; prozyme, san leandro, ca), for 2 h at 37°c, which cleaves all non-reducing terminal sialic acid residues in the order α(2,6) > α(2,3) > α(2,8) > α (2, 9) . sialidase a treatment completely abrogated lectin binding (supplementary figure s1 ) and h5n2 influenza virus binding (supplementary figure s2) which further confirmed the specificity of the lectins used in this study to appropriate sialic acid linkages. in summary, we found that the sa receptors on lbb tissues supported binding of avian h5n2 and human h1n1 influenza viruses. further, the virus binding pattern was in accordance with the relative distribution of sa α2,3-gal and sa α2,6-gal receptors in tissues. with the continued recognition of the role of various wild animals in influenza virus evolution, the interest in understanding the tissue sa distribution in wild animals has intensified 18 . among the various factors, the ability to bind to sa receptors on host cells is considered a key feature of iav pathogenicity. there is serological evidence of avian influenza virus infection in certain frugivorous bats 13 and pteropus alecto bat cells were found to be susceptible to iav infection and reassortment 19 . in addition bats have been shown to harbor novel influenza-like viruses 10 . however, to date, the sa receptor distribution in any bat species is completely unknown. for the first in contrast, sa α2,6-gal receptors gradually increases towards the lower respiratory tract. tissue sections were stained with biotinylated maaii (red -specific for avian type receptor, sa α2,3-gal) and fitc labelled sna (green -specific for human type receptor, sa α2,6-gal) lectins, and dapi nuclear stain (blue). a": haematoxylin and eosin (h and e) stained tracheal tissue section. 1. respiratory epithelium, 2. lamina propria, 3. submucosa, 4. hyaline cartilage b": h and e stained lung tissue section. 5. alveolar duct, 6. visceral pleura, 7. pulmonary blood capillary. time, this study demonstrated that little brown bats (lbbs), a widely-distributed bat species in north america, co-express both avian and human type influenza receptors in their respiratory and gastrointestinal systems. co-expression of avian and human type influenza receptors was found in lbb trachea and lung. however avian type (sa α2,3-gal) receptors were predominant in tracheal mucosa similar to ducks 14 and some species of passeriformes and charadriiformes 20 . in contrast sa α2,6-gal receptors are predominant in the tracheal mucosal lining in chickens, pigs and humans 14, 17 . we found co-expression of both avian and human type receptors in the mucosal lining of stomach and to a lesser degree in intestines. sa α2,3-gal receptors were predominant throughout the digestive tract much like chickens and ducks 14, 21, 22 . in contrast, other mammalian species such as humans and pigs predominantly express sa α2,6-gal receptors in the gi tract 17 . unlike chickens and ducks, low levels of sa α2,6-gal receptor expression was also observed in lbb digestive tract which decreased progressively from stomach to intestines. we did not find any difference in the receptor distribution among various locations in intestines as it is difficult to distinguish large and small intestine in most bat species 23 . similarly, no major differences in the receptor distribution pattern between large and small intestine were found in most other species including chickens, ducks and pigs 14, 17 . a fair amount of sa α2,6-gal receptor expression was found in the intestinal crypts, lamina propria, muscularis and serosa of lbb intestine. it is worth noting that a similar co-expression pattern of influenza receptors is observed in various wild birds of the families columbiformes, anseriformes and gruiformes 20 , which constitute the natural influenza virus reservoir, and also in pigs 17 . virus binding assays with an avian h5n2 and a human h1n1 virus confirmed that the sa receptors found in lbb tissues are compatible with avian and human iav binding. bats act as natural reservoirs for a variety of zoonotic viruses and they coexist with viruses through several mechanisms including elevated metabolism and body temperature 24 . it is believed that this unique feature of bats leads to the selection of viruses that adapt better at higher body temperature, and hence are more virulent to humans 24 . bats carry a number of rna and dna viruses asymptomatically, and the detection rate of new viruses or virus sequences seems to be much higher in bats than any other mammals 25 . the perfect equilibrium between various zoonotic viruses and bats have been studied extensively in the past two decades 7, 26 . iavs have been isolated from more than 105 different bird species belonging to 26 different families 27 . although anseriforms (ducks, geese, and swans) and charadriiforms (gulls, terns, and shorebirds) are considered to be the major influenza reservoirs, iavs have also been isolated from gaviiformes (loons), podicepediformes (grebes), procellariiformes (shearwaters and petrels), pelecaniformes (pelicans and cormorants), ciconiiformes (ibis and herons), and gruiformes (moorhen and coots) 28 . many of these species may indeed be reservoirs of iavs, but no systematic surveys have been conducted, leaving a gap in our current understanding of the natural reservoirs of iavs. we showed for the first time that lbbs co-express both the avian (sa α2,3-gal) and human (sa α2,6-gal) influenza receptors in their respiratory and gastrointestinal system that are compatible with avian h5n2 and human h1n1 virus binding. in addition, there is strong evidence that cell lines from a range of bat species support productive iav replication 16 . the sum of this evidence suggests that bats could play an important role in iav epidemiology and zoonotic iav emergence. as the novel bat influenza viruses are different from other iavs, it was proposed that these viruses would therefore require significant changes before they can infect and spread among humans 11 . however, a recent study rescued these viruses using reverse genetics in cell culture and found that the novel bat influenza viruses can infect a range of mammalian cells including canine cells 29 . all the existing data suggests that bats could be susceptible to many different iav subtypes and even support co-infection of avian and human iavs. it is believed that the novel bat influenza viruses found in fruit bats are probably the ancient influenza viruses from which the modern world iavs have been derived over time 30 . evidence of high seroprevalence of avian influenza in frugivorous bats together with the evidence of abundant sa receptors in lbbs found in this study, raises a strong possibility that bats could be a major influenza virus reservoir. despite many rigorous scientific pursuits, we have been unable to understand the mechanism by which new pandemic influenza viruses emerge. consequently, we do not yet have sufficient scientific understanding needed to accurately predict which iav strains may cause the next pandemic. the extensive diversity of bat species globally and the limited understanding of the role of bats in iav biology raises an urgent need for comprehensive epidemiological surveillance of iavs across different bat species. experimental animals. little brown bat respiratory and gastrointestinal tissues were collected and provided by pa game commission from an ongoing collaborative study with bucknell university, on little brown bats from the state of wisconsin and new york. the study (dmr-17) was approved by the bucknell university institutional animal care and use committee and all methods were carried out in accordance with relevant guidelines and regulations. a total of 10 adult and 10 juvenile male little brown bat tissues were included in the study. lectin histochemistry. lectin histochemistry on paraffin embedded tissues was performed as previously described 14 . briefly, 5 µm thick tissue sections were deparaffinized in xylene and rehydrated in ethanol. following 10 min presoaking of the rehydrated sections in tris-buffered saline (tbs), sections were incubated with biotinylated maackia amurensis (maaii) and fitc labelled sambucus nigra (sna) lectins specific to sa α2,3-gal and sa α2,6-gal receptor respectively each at a concentration of 10 μg/ml, overnight at 4°c. both lectins were purchased from vector laboratories (burlingame, ca, usa). following three washes with tbs, sections were incubated with streptavidin alexafluor 594 conjugate for 2 h at 4°c. the sections were washed three times with tbs and mounted in prolonggold antifade reagent with nuclear stain 4′,6-diamino-2-phenylindole, dihydro-chloride (dapi). negative controls were performed omitting the primary reagents. following 24 h of curing at room temperature (rt), sections were imaged using olympus fluoview ™ fv1000 confocal microscope. settings of the confocal microscope were determined using negative controls to avoid any background fluorescence and the same settings were used to scan all the other sections for consistency. to rule out nonspecific binding of the lectins and iavs, control tissue sections were treated, prior to lectin staining or virus binding, with sialidase a (n-acetylneuraminate glycohydrolase; prozyme, san leandro, ca), for 2 h at 37°c, which cleaves all non-reducing terminal sialic acid residues in the order α(2,6) > α(2,3) > α(2,8) > α (2, 9) . sialidase treated and control sections were further subjected to lectin staining or virus binding. virus binding assay. virus binding assays with a low pathogenic aiv (lpaiv) h5n2 (a/h5n2/chicken/ pennsylvania/7659/1985) and human pandemic h1n1 virus (a/h1n1/virginia/2009) were performed as previously described 14 . virus binding assays were performed following strict biosafety level-2 (bsl-2) practices. briefly deparaffinised tissue sections were incubated with a 250 μl of lpai h5n2 virus (10 6 tcid 50 /ml) or human h1n1 virus (10 6 tcid 50 /ml) for 2 h at rt. tissues incubated with pbs served as mock treated controls. sections were washed with tbs before blocking with inactivated goat serum for 30 min and immunostained with primary mouse monoclonal antibody to influenza hemagglutinin h5 (ab82455, abcam) or influenza nucleoprotein (ab20343, abcam). following 40 min of incubation with the primary antibody, sections were washed and incubated with a secondary goat anti mouse igg-cy5 antibody (ab6563, abcam). after 40 min of incubation with secondary antibody at rt, sections were washed with tbs and mounted in prolonggold antifade reagent with dapi. following 24 h of curing, the sections were imaged using olympus fluoview ™ fv1000 confocal microscope. scanning electron microscopy (sem). virus binding was also visualized using scanning electron microscopy. deparaffinized sections were incubated with lpai h5n2 virus or human h1n1 virus for 2 h as described above. subsequently the sections were washed with distilled water and fixed using increasing ethanol concentrations (50%, 70%, 90% and 100%). the sections were then dried by a critical point dryer (leica em cpd300). gold was coated over the dried sections by applying sputter coating for 10 seconds (cressington, 108 auto sputter coater) and sem images were taken by a field emission sem (fe-sem, merlin zeiss) under 5 ev. image processing and pseudo coloring of virus particles bound to tissues was performed using adobe photoshop cc. experimental infection of chickens, ducks and quails with the highly pathogenic h5n1 avian influenza virus mammalian innate resistance to highly pathogenic avian influenza h5n1 virus infection is mediated through reduced proinflammation and infectious virus release highly pathogenic avian influenza 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viruses? current opinion in virology 1 bat flight and zoonotic viruses global patterns of influenza a virus in wild birds susceptibility of north american ducks and gulls to h5n1 highly pathogenic avian influenza viruses synthetically derived bat influenza a-like viruses reveal a cell type-but not species-specific tropism supplementary information accompanies this paper at doi:10.1038/s41598-017-00793-6competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-335871-zieuc7vk authors: brazee, patricia l.; sznajder, jacob i. title: targeting the linear ubiquitin assembly complex to modulate the host response and improve influenza a virus induced lung injury date: 2020-05-13 journal: arch bronconeumol doi: 10.1016/j.arbres.2020.04.019 sha: doc_id: 335871 cord_uid: zieuc7vk abstract influenza virus infection is characterized by symptoms ranging from mild congestion and body aches to severe pulmonary edema and respiratory failure. while the majority of those exposed have minor symptoms and recover with little morbidity, an estimated 500,000 people succumb to iav-related complications each year worldwide. in these severe cases, an exaggerated inflammatory response, known as “cytokine storm”, occurs which results in damage to the respiratory epithelial barrier and development of acute respiratory distress syndrome (ards). data from retrospective human studies as well as experimental animal models of influenza virus infection highlight the fine line between an excessive and an inadequate immune response, where the host response must balance viral clearance with exuberant inflammation. current pharmacological modulators of inflammation, including corticosteroids and statins, have not been successful in improving outcomes during influenza virus infection. we have reported that the amplitude of the inflammatory response is regulated by linear ubiquitin assembly complex (lubac) activity and that dampening of lubac activity is protective during severe influenza virus infection. therapeutic modulation of lubac activity may be crucial to improve outcomes during severe influenza virus infection, as it functions as a molecular rheostat of the host response. here we review the evidence for modulating inflammation to ameliorate influenza virus infection-induced lung injury, data on current anti-inflammatory strategies, and potential new avenues to target viral inflammation and improve outcomes. tratamiento dirigido al complejo de ensamblaje de cadenas lineales de ubiquitina para modular la respuesta del huésped y mejorar el daño pulmonar inducido por el virus de la gripe a la infección por el virus de la gripe se caracteriza por síntomas que van desde la congestión leve y los dolores corporales hasta el edema pulmonar grave y la insuficiencia respiratoria. aunque que la mayoría de las personas expuestas presentan síntomas leves y se recuperan con poca morbilidad, se estima que cada año 500 000 personas en todo el mundo fallecen por las complicaciones relacionadas con esta infección. en estos casos graves, se produce una respuesta inflamatoria exagerada, conocida como "tormenta de citoquinas", que causa daños en la barrera epitelial respiratoria y el desarrollo del síndrome de distrés respiratorio agudo (sdra). los datos de estudios retrospectivos en humanos, así como de modelos animales experimentales de infección por el virus de la gripe, resaltan la delgada línea que existe entre una respuesta inmune excesiva y una inadecuada, cuando la respuesta del huésped debe mantener el equilibrio entre el aclaramiento viral y la introduction seasonal influenza a viral infection affects a significant proportion of the population worldwide, with an estimated 500,000 people succumbing to iav-related complications each year. while most patients infected with influenza a virus (iav) recover without major sequelae, severe viral pneumonia is one of the most common causes of acute respiratory distress syndrome (ards) [1] [2] [3] [4] . clinically, ards presents with bilateral pulmonary infiltrates, hypoxemia, pulmonary edema and widespread lung inflammation that lead to high mortality rates due respiratory and to multiple organ failure [1, [4] [5] [6] . ards patients can be sub-grouped based on the severity of the inflammatory response, where patients with hyper-inflammation have worse clinical outcomes, spending more days on mechanical ventilation, experiencing increased incidence of organ failure and a higher mortality rate compared to hypo-inflammatory ards patients [7] . impairment of gas exchange in iav-induced ards, in large part, is due to damage to the respiratory epithelial barrier and edema accumulation [1, [4] [5] [6] [7] [8] . during iav infection, an exaggerated inflammatory response, known as "cytokine storm", can occur leading to the development of hyper-inflammatory ards, increasing iav-induced morbidity and mortality ( figure 1 ) [4] . post-mortem studies of lungs from iav-infected patients show extensive diffuse alveolar damage characterized by edema, cellular infiltration, thickening of alveolar walls, and necrosis [9] . interestingly, a study of critically ill patients showed no differences in pulmonary viral load between those who died and those who recovered, while mortality directly correlated with exuberant inflammation, further supporting a maladaptive host response as the major driver of iav-induced lung injury [10] [11] [12] [13] [14] . similar observations are being reported in patients with severe coronavirus disease (covid19) , where severe lung damage is associated with increased pro-inflammatory cytokines and respiratory failure from ards is the leading cause of mortality [15, 16] . with no virus-specific treatment options currently validated, therapies which target the inflammatory response are currently being considered for patients with severe covid-19 [17, 18] . however, as it has been reported for severe iav infections, current antiinflammatory drugs have pleiotropic effects and may lack the specificity needed to carefully calibrate the host response. respiratory epithelial cells, as primary targets for iav infection and replication, initiate inflammatory signaling [19] [20] [21] [22] . in response to respiratory epithelial derived cytokines, innate immune cells, such as neutrophils, monocyte-derived inflammatory macrophages, and natural killer (nk) cells are recruited to the airspace [23] . together with tissue resident alveolar macrophages, recruited innate immune cells are critical for control of viral replication through both lysis and clearance of virus-infected cells [23] [24] [25] [26] . however, in addition to controlling viral spread, innate immune cells contribute to the overproduction of pro-inflammatory cytokines that enhance iav-induced lung injury ( figure 2 ). the pulmonary immune response must be carefully balanced, simultaneously promoting viral clearance and limiting excessive inflammation to maintain proper lung function. findings from animal models of iav infection have shown that modulation of the host immune response is associated with reduced lung injury and improved survival [11, 14, 27, 28] . blockade of specific immune cell subsets has been shown to improve outcomes in mouse models of severe iav infection. for example, genetic deletion of the chemokine receptor ccr2 inhibited the recruitment of monocyte-derived inflammatory macrophages during iav infection and resulted in reduced lung injury with improved survival. however, loss of this myeloid cell population resulted in a delay in viral clearance [29, 30] . moreover, adoptive transfer of nk cells from iav-infected lungs, as compared to nk cell from naïve lungs, resulted in increased mortality of influenzainfected mice [31] , suggesting that inflammation-dependent activation, rather than recruitment, drives the observed pathology. taken together data from these animal models suggest that the determinant of influenza severity be orchestrated by respiratory epithelial cells. the linear ubiquitin assembly complex (lubac) is a multi-protein e3 ubiquitin ligase complex composed of two stabilizing proteins, the heme-oxidized iron responsive element binding protein 2 ubiquitin ligase-1l (hoil-1l) and shank-associated rh domain-interacting protein (sharpin), and a catalytic component, hoil-1-interacting protein (hoip) [32] [33] [34] (figure 3 ). the proteins within the heteromeric complex contain multiple domains for interactions within the complex, ubiquitin binding, as well as catalytic activity [32] [33] [34] [35] [36] . lubac is an essential regulator of nf-κb activation and has been shown to act as a molecular rheostat, regulating the amplitude of the epithelial-driven inflammatory response dung iav infection [35, 36] . the respiratory epithelium actively participates in the first line of defense against pathogens by orchestrating host innate immunity [23, [37] [38] [39] . as iav replicates within the respiratory epithelium cells, the cytosolic pattern recognition receptor (prr), rig-i, is activated and initiates formation of a signaling platform to which lubac is recruited. lubac covalently attaches met-1 linked linear ubiquitin chains to the nf-κb essential modulator (nemo), a component of the inhibitor of nf-κb (iκb) kinase (ikk) complex along with ikkα and ikkβ [40, 41] . due to the high affinity of nemo's ubiquitin binding domain for linear chains, linear ubiquitination of nemo facilitates the recruitment of additional ikk complexes, which results in the efficient trans-autophosphorylation and activation of proximal ikkβ followed by the phosphorylation and degradation of iκbα and robust nf-kb activation ( figure 3 ) [34, 35, 40, 42, 43] . recent reports show that destabilization of respiratory epithelial lubac, via loss of the non-catalytic component hoil-1l, dampens the host response during severe influenza and promotes survival with reduced lung injury as well as reduced viral titers. however, when lubac activity is abolished through deletion of hoip, the alveolar epithelia driven inflammatory response is inhibited and mortality is increased. these findings highlight the fine line between an excessive and an inadequate immune response and suggest that therapeutic modulation of lubac activity may be crucial, as it functions as a rheostat regulating the amplitude of the host response to iav infection. lubac covalently attaches linear ubiquitin chains to nemo, which facilitates the recruitment of additional ikk complexes. stably docked ikk complexes result in the efficient transautophosphorylation and activation of proximal ikkα/β, followed by the phosphorylation and degradation of iκbα. nf-κb translocates to the nucleus to stimulate transcription of inflammatory genes. current anti-influenza strategies are limited to yearly vaccination or administration of antiviral drugs, however, short therapeutic windows, viral mutation, and resistance to current therapies limit their effectiveness. despite available vaccination and anti-viral drugs, the most recent pandemic in 2009 resulted in an estimated 151,700 -575,400 deaths in its first year of circulation worldwide [44] . the pandemic strain contained a novel assortment of viral genes not previously identified in animal or human populations. from its first detection in april 2009, it was only 3 months until resistance to anti-viral drugs was reported, and it took an additional 3 months before the first vaccine offering protection from the pandemic strain was administered [45] . in addition to emerging pandemic strains, between 291,000 and 646,000 people worldwide die from seasonal influenza-related respiratory illnesses each year [46] . novel mutations and reassortments of the virus will inevitably lead to the next iav pandemic; therefore, the use and development of therapeutics that target conserved host pathways, rather than the virus itself, hold promise to curtail the impact of viral infection. moreover, a heterogeneous response to iav with the same virulence exists within the population, suggesting that host factors play a crucial role regulating the host response and determining the severity of lung injury [2, 3, 47] . additionally, experimental evidence from human studies and animal models of severe iav show that viral titers do not always correlate with severity of disease, but rather ards induced "cytokine storm" is the major driver of morbidity and mortality [4, 28, 38, 48] . severe iav infection is associated with inflammatory cytokines in humans and mice. due to their pleiotropic and redundant effects, targeting of individual cytokines may not be a suitable approach to reduce pathology during iav infection. instead, dampening of the immune response may be more effective, as was the case with lubac destabilization noted above [36] . fda-approved anti-inflammatory drugs, including corticosteroids and statins, have been proposed for the treatment of "cytokine storm" associated with severe iav infection [49] . moreover, current data regarding their efficacy is limited to mouse models and retrospective patient observations [49, 50] . corticosteroids have been shown to be effective in limiting the inflammation in in some lung pathologies [49] [50] [51] . however, observational studies of the impact of corticosteroid treatment of iav-infected patients suggest against their use; with administration associated with higher incidence of hospital-acquired pneumonia, longer duration of mechanical ventilation, and increased mortality [50] . similarly, clinical evidence does not support corticosteroid treatment for covid-19 lung injury [52] . statins are another class of drugs recognized for their ability to dampen inflammation [49] . while experimental evidence from mouse models using statins during iav infection have been inconclusive regarding their benefit [49, 53, 54] , retrospective analysis of patient data suggests an association between statin treatment and lower iav mortality rates [55] . these examples highlight the need for new avenues of drug discovery and validations, as no currently available immune modulators have convincingly demonstrated their ability to improve outcomes during influenza infection. lubac represents a potential new target for limiting the pathological inflammation that occurs during iav infection. several chemical inhibitors as well as peptides that bind hoip have been used to inhibit lubac activity in cell culture [56] [57] [58] [59] and in vitro assays [60, 61] and support the specific targetablility of lubac (figure 4) . currently, lubac inhibitors fall into two categories: those that target the catalytic activity of hoip (ie. bay117082, gliotoxin, hoipin) or those that disrupt the interaction between lubac components to destabilize the complex (ie. stapled peptides). bay117082, a small molecule commonly used as an inhibitor of nf-κb activation. it has been observed that treatment of raw 264.7 macrophages with bay117082 prevented il-1 stimulated formation of linear ubiquitin chains. further investigation revealed that bay117082 irreversibly inhibits lubac through a chemical reaction with cysteine residues in the active site of hoip, the catalytic unit of lubac [58] . while bay117082 represents a potent inhibitor of lubac activity, it targets multiple components of the ubiquitin system, including inhibition of e2 ubiquitin conjugating enzymes, and possible proteasome inhibition [58] . as such, use of bay117082 is not suitable for the study of lubac-dependent physiological functions or therapeutic targeting of lubac activity in disease. gliotoxin, a fungal metabolite, was identified in a highthroughput screening for lubac inhibitors using a time-resolved fret-based screening system. while gliotoxin is known to have multiple cellular targets, it is able to inhibit lubac activity and downstream activation of nf-κb at 10x lower concentrations [61] . gliotoxin's strong, irreversible binding to the catalytic site of hoip makes it a selective inhibitor of lubac activity [61] . interestingly, the potency of gliotoxin has been shown to vary between cell types, with myeloid and lymphoid cells being more sensitive to gliotoxin-mediated nf-κb inhibition than epithelial cells [61, 62] . however, this irreversible inhibition may quench the inflammatory response and increase susceptibility to secondary infections. hoipins are synthetic small molecules that reversibly inhibit lubac though targeting of hoip activity, displaying both lubac specificity as well as low cytotoxicity. several derivatives have been made with varying degrees of efficacy (hoipin-1-8), with hoipin-8 showing significantly enhanced ability to prevent lubac-mediated nf-κb activation in response to tnf-α without cytotoxicity compared to the other derivatives in vitro [57] . conversely, stapled α-helical peptides developed based on specific lubac structures disrupt interactions necessary for stable complex formation [56, 63] . stapled peptides are a class of synthetic macrocycles where the secondary α-helix structure is stabilized by the introduction of a hydrophobic bridge or "staple" that rigidifies specific areas to inhibit protein:protein interactions [56] . stapled peptides based on the hoip ubiquitin binding domain have been shown to successfully inhibit lubac activity in vitro by disrupting its interaction with hoil-1l and destabilizing the overall complex [56, 63] . while several inhibitors of lubac have been developed and shown promise in vitro, no data is available detailing their efficacy in vivo. further investigation in to these compounds which target lubac stability to modulate the degree of lubac activity is warranted as they may be therapeutically beneficial for the treatment of hyper-inflammatory response during viral infection, where a graded host response is necessary. within the past 150 years, iav has been the causative agent of at least five pandemics (1889, 1918, 1957, 1968, 2009) [47, 64] . in addition to iav, novel viral threats, such as the coronavirus outbreaks of 2003, 2015 and 2019, quickly spread worldwide before virus-specific vaccines or pharmacological options could be developed. thus, therapies that target conserved host pathways may provide a novel universal treatment strategies, regardless of viral sequence. findings from animal models of iav infection have shown that inhibition of the exuberant host immune response is associated with reduced lung injury and improved survival [11, 14, 27, 28] . however, current fda-approved anti-inflammatory drugs, such as corticosteroids and statins, have failed to show benefit during severe iav infection [49, 50] . beyond viral infections, the amplitude of the inflammatory response has been shown to be a critical determinant in outcome during bacterial sepsis in community acquired pneumonia, a common complication post iav infection [51, 65] . analysis of a cohort of patients showed that reductions in the inflammatory response during bacterial pnumonia, both due to host inability to mount a response or the administration of anti-inflammatory steroids, lead to increased mortality [51, 65] . these clinical observations highlight the need to balance the inflammatory response during viral infection, not only to improve lung injury during the primary viral infection, but also to prevent poor outcomes to secondary infections. in addition to the seasonal threat of influenza, we must also be cautious in the regulation of inflammation in treatment of the ongoing covid-19 pandemic. while a subgroup of patients with severe covid-19 develop 'cytokine storm' [15, 16, 18] , it must be remembered that current anti-inflammatory drugs have pleiotropic effects and lack the specificity needed to carefully calibrate the host response. as such, newly developed pharmacologics such as those that target lubac, a molecular rheostat of inflammatory signaling, have the potential to fine tune inflammation and moderate the host response. further investigation of compounds which modulate lubac activity is warranted, as they may be therapeutically beneficial for the treatment of hyperinflammatory response during viral infections, where a milder host response should improve outcomes. nonventilatory strategies for patients with life-threatening 2009 h1n1 influenza and severe respiratory failure complications of seasonal and pandemic influenza pathogenesis of influenza-induced acute respiratory distress syndrome an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals mortality trends of acute respiratory distress syndrome in the united states from 1999 to 2013 subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials gas exchange disturbances regulate alveolar fluid clearance during acute lung injury. front immunol lung pathology in fatal novel human influenza a (h1n1) infection suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus ccr2+ monocyte-derived dendritic cells and exudate macrophages produce influenza-induced pulmonary immune pathology and mortality role of host immune response and viral load in the differential outcome of pandemic h1n1 (2009) influenza virus infection in indian patients fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china baricitinib as potential treatment for 2019-ncov acute respiratory disease covid-19: consider cytokine storm syndromes and immunosuppression retinoic acid inducible gene-i and mda-5 are involved in influenza a virus-induced expression of antiviral cytokines actin and rig-i/mavs signaling components translocate to mitochondria upon influenza a virus infection of human primary macrophages cell type-specific involvement of rig-i in antiviral response pathogenic potential of interferon alphabeta in acute influenza infection respiratory epithelial cells orchestrate pulmonary innate immunity innate immune sensing and response to influenza evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice activation mechanisms of natural killer cells during influenza virus infection dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection fxyd5 is an essential mediator of the inflammatory response during lung injury. front immunol lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed tnfrelated apoptosis-inducing ligand contrasting effects of ccr5 and ccr2 deficiency in the pulmonary inflammatory response to influenza a virus nk cells exacerbate the pathology of influenza virus infection in mice ring-between-rings--keeping the safety on loaded guns sharpin forms a linear ubiquitin ligase complex regulating nf-kappab activity and apoptosis lubac, a novel ubiquitin ligase for linear ubiquitination, is crucial for inflammation and immune responses. microbes infect role of linear ubiquitination in health and disease linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection the airway epithelium: soldier in the fight against respiratory viruses alveolar epithelial cells: master regulators of lung homeostasis alveolar edema must be cleared for the acute respiratory distress syndrome patient to survive recruitment of the linear ubiquitin chain assembly complex stabilizes the tnf-r1 signaling complex and is required for tnf-mediated gene induction analysis of nuclear factor-kappab (nf-kappab) essential modulator (nemo) binding to linear and lysine-linked ubiquitin chains and its role in the activation of nf-kappab involvement of linear polyubiquitylation of nemo in nf-kappab activation the e3 ligase hoip specifies linear ubiquitin chain assembly through its ring-ibr-ring domain and the unique ldd extension estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study h1n1 pandemic timeline estimates of global seasonal influenza-associated respiratory mortality: a modelling study hospitalized patients with 2009 h1n1 influenza in the united states influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions immunomodulatory therapy for severe influenza corticosteroids for severe influenza pneumonia: a critical appraisal transcriptomic signatures in sepsis and a differential response to steroids. from the vanish randomized trial clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury effect of statin treatments on highly pathogenic avian influenza h5n1, seasonal and h1n1pdm09 virus infections in balb/c mice the effect of rosuvastatin in a murine model of influenza a infection association between use of statins and mortality among patients hospitalized with laboratory-confirmed influenza virus infections: a multistate study biophysical and biological evaluation of optimized stapled peptide inhibitors of the linear ubiquitin chain assembly complex (lubac) small-molecule inhibitors of linear ubiquitin chain assembly complex (lubac), hoipins, suppress nf-kappab signaling the anti-inflammatory drug bay 11-7082 suppresses the myd88-dependent signalling network by targeting the ubiquitin system essential role of the linear ubiquitin chain assembly complex in lymphoma revealed by rare germline polymorphisms fragment-based covalent ligand screening enables rapid discovery of inhibitors for the rbr e3 ubiquitin ligase hoip gliotoxin suppresses nf-kappab activation by selectively inhibiting linear ubiquitin chain assembly complex (lubac) in vitro and in vivo effects of gliotoxin, a fungal metabolite: efficacy against dextran sodium sulfate-induced colitis in rats cooperative domain formation by homologous motifs in hoil-1l and sharpin plays a crucial role in lubac stabilization the pathology of influenza virus infections genomic landscape of the individual host response and outcomes in sepsis: a prospective cohort study key: cord-102471-dtukacm7 authors: xu, y.; lewandowski, k.; downs, l.; kavanagh, j.; hender, t.; lumley, s.; jeffery, k.; foster, d.; sanderson, n.; vaughan, a.; morgan, m.; vipond, r.; carroll, m.; peto, t.; crook, d.; walker, s.; matthews, p.; pullan, s. title: nanopore metagenomic sequencing of influenza virus directly from respiratory samples: diagnosis, drug resistance and nosocomial transmission date: 2020-04-22 journal: nan doi: 10.1101/2020.04.21.20073072 sha: doc_id: 102471 cord_uid: dtukacm7 background: influenza virus presents a significant challenge to public health by causing seasonal epidemics and occasional pandemics. nanopore metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid diagnosis of infection, rationalising antimicrobial therapy, and supporting interventions for infection control. this study aimed to evaluate the applicability of this sequencing approach as a routine laboratory test for influenza in clinical settings. methods: we conducted nanopore metagenomic sequencing for 180 respiratory samples from a uk hospital during the 2018/19 influenza season, and compared results to routine molecular diagnostic testing. we investigated drug resistance, genetic diversity, and nosocomial transmission using influenza sequence data. results: metagenomic sequencing was 83% (75/90) sensitive and 93% (84/90) specific for detecting influenza a viruses compared with the diagnostic standard (cepheid xpress/biofire filmarray respiratory panel). we identified a h3n2 genome with the oseltamivir resistant s331r mutation in the na protein, potentially associated with the emergence of a distinct intra-subtype reassortant. whole genome phylogeny refuted suspicions of a transmission cluster in the infectious diseases ward, but identified two other clusters that likely reflected nosocomial transmission, associated with a predominant strain circulating in the community. we also detected a range of other potentially pathogenic viruses and bacteria from the metagenome. conclusion: nanopore metagenomic sequencing can detect the emergence of novel variants and drug resistance, providing timely insights into antimicrobial stewardship and vaccine design. generation of full genomes can contribute to the investigation and management of nosocomial outbreaks. 4 and vaccine design. generation of full genomes can contribute to the investigation and management of nosocomial outbreaks. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 21.20073072 doi: medrxiv preprint influenza a viruses (iav) are enveloped viruses of the orthomyxoviridae family, with a segmented, ~13kb rna genome [1, 2] . iav can cause both seasonal epidemics and occasional pandemics, presenting a significant challenge to public health [3] . seasonal epidemics are estimated to cause half a million deaths globally each year, primarily among young children and the elderly [4] . estimates suggest a future pandemic could infect 20% to 40% of the world population and cause over 30 million deaths within six months [5, 6] . tracking and characterization of circulating influenza viruses, in both human and animal populations, is critical to provide early warning of the emergence of novel variants with high virulence and to inform vaccine design. direct-from-sample metagenomic sequencing can potentially identify all viral and bacterial pathogens within an individual clinical sample. the genomic information generated can comprehensively characterize the pathogens and enable investigation of epidemiology and transmission. oxford nanopore technology (ont) is a third generation sequencing technology that can generate long-read data in real-time, which has been successfully applied in the real-time surveillance of ebola, zika, and lassa outbreaks [7] [8] [9] . ont metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid and accurate diagnosis of infection [10] , informing antimicrobial therapy [11] [12] [13] , and supporting interventions for infection prevention and control [14] . we have recently demonstrated proof-of-principle for a direct-from-sample nanopore metagenomic sequencing protocol for influenza viruses with 83% sensitivity and 100% specificity compared to routine clinical diagnostic testing [15] . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 21.20073072 doi: medrxiv preprint here we describe nanopore metagenomic sequencing directly from clinical respiratory samples at a uk hospital during the 2018/19 influenza season, evaluating the applicability of this approach in a routine laboratory as a test for influenza, and investigating where further optimisation is still required before the assay can be deployed in clinical practice. we assessed the performance of this experimental protocol head-to-head with routine clinical laboratory tests, and used the influenza sequence data to investigate drug resistance, genetic diversity, and nosocomial transmission events, demonstrating the diverse benefits that can be gained from a metagenomic approach to diagnostics. residual material was collected from anonymised throat swabs, nasal swabs, and nasopharyngeal aspirates that had been submitted to the clinical diagnostic laboratory at the oxford university hospitals nhs foundation trust during the 2018/19 influenza season. prior to metagenomic sequencing, samples had been tested in the diagnostic laboratory based on a standard operating protocol using either xpert xpress flu/rsv assay (cepheid, sunnyvale, ca, usa, that detects influenza a/b and respiratory syncytial virus), or biofire® filmarray® respiratory panel 2 assay (biofire diagnostics, salt lake city, ut, usa, that detects a panel of viral and bacterial respiratory pathogens). xpert reports a quantitative diagnostic result (ct value) for the detected pathogen, while biofire® rp2 reports a binary result (pathogen detected or not all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the first laboratory diagnosis of influenza in our hospital laboratory in the 2018/19 season was made on 30th october 2018, and our sample collection ran until 5th (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. methods for sample processing (sequence independent single primer amplification as described in [15] ), nanopore sequencing, and genomic and phylogenetic analyses are described in supplementary material. for the nanopore metagenomic sequencing, the sample processing and library preparation time in our protocol was eight hours, the sequencing time was 48 hours, and thus total turnaround time for each sample was <72 hours. with a team of three members, we prepared the sequencing libraries for 180 samples within 9 days and completed the sequencing runs for the 90 influenza-positive samples within 12 days of commencing sequencing ( figure 1a ). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 21.20073072 doi: medrxiv preprint nanopore sequencing generated between 4.9x10 3 and 4.1x10 6 (mean 4.3x10 5 ) total reads per sample (table s1 ). we retrieved hazara virus reads (spiked as an internal control at 10 4 genome copies/ml) from 147/180 (82%) samples. the 33 samples in which hazara virus reads were not identified were all influenza negative and had comparatively low total cdna concentrations following amplification. therefore, we repeated sequencing of the 18/33 samples that had sufficient remaining material with the addition of linear polyacrylamide as a carrier, which produced hazara virus reads in 16/18 samples. taken together, we therefore retrieved hazard internal control in 163/180 (91%) samples (15 were not possible to re-test with carrier). the xpert xpress influenza-positive samples (sensitivity 83%), ranging from 1 to 171,733 reads ( figure 2a ). iav reads were present in all 58 samples with ct ≤31, and up to a maximum ct value of 36.3 (sample 48, 12 iav reads). there was a strong correlation between ct value and both iav read numbers (r 2 =0.43, p<0.0001; figure 2a ) and the ratio of iav:hazara virus reads (r 2 =0.54, p<0.0001; figure 2b ). the remaining 15 influenzapositive samples for which iav reads were not generated by nanopore sequencing had lower viral titres, reflected by higher ct values (range 31.7-39.0). iav reads were not present in 84/90 influenza-negative samples (specificity 93%). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. among the 75 samples for which we generated iav reads, we could determine the ha subtype of 59/75 (79%) samples; 40 were h1 and 19 were h3 (designated as blue vs red dots in figure 2 ). we could determine ha subtype for all samples with ct ≤27, and up to a maximum of ct 36.3 (sample 48) ( figure 2a ). we retrieved 28/75 (37%) consensus sequences with genome coverage ≥70%, among which 18 were h1 and 10 were h3 subtype ( figure 2c ). the genome coverage for samples with ct value between 20 and 25 showed substantial variation, which was not associated with any sample attributes that we were able to measure, including sample type, or percentage of human or bacterial reads (data not shown). from consensus sequences covering drug-resistant positions, we identified the s31n amino acid mutation in the m2 protein in 20/20 h1n1 and 11/11 h3n2 sequences, which is known to be widespread, conferring reduced inhibition by amantadine [16] . 1/13 h3n2 sequences (sample 5) carried the s331r amino acid mutation in the na protein, which has been reported to confer reduced inhibition by oseltamivir [17] . analysing mapping data for sample 5, 51/53 (96%) reads carried the s331r mutation. other drug resistance mutations, such as h275y in the na protein associated with oseltamivir resistance [18] , were not present in our dataset. the majority of our h3 sequences were clustered within clade 3c.2a1b, with one sequence in clade 3c.3a ( figure 3a ). comparison of the h3 and n2 phylogenies all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 21.20073072 doi: medrxiv preprint showed that ha and na segments of each individual sample were clustered within the same clade, except sample 5 had a distinct genotype with the h3 segment clustered within clade 3c.2a1b and the na segment within clade 3c.2a2 (denoted subsequently as 'r-genotype'), suggesting intra-subtype reassortment ( figure 3a and 3b) . interestingly, the s331r mutation occurred in the same sample (sample 5), motivating us to further investigate the prevalence of this mutation in seasonal iav using all published h3n2 sequences from the last two influenza seasons (2017/18 and 2018/19). in the 2017/18 dataset, 13/7129 (0.2%) sequences carried the s331r mutation, with ha and na segments from clade 3c.2a2 or 3c.2a3. in 2018/19, the proportion of sequences with the s331r mutation increased to 139/9274 (1.5%), and all belonged to the r-genotype. these results suggest a potential association between the increase in prevalence of the s331r mutation and the emergence of this distinct rgenotype. we included a putative clinical cluster of eight influenza-positive samples (group 3) collected from patients on the infectious diseases ward over a 30 day period, aiming to investigate potential nosocomial transmission events ( figure 4a ). we could determine the ha subtype of six samples, three being h3 and three h1. among these, two h3n2 (samples 53 and 55) and one h1n1 consensus sequences had >70% full genome coverage. a minimum spanning tree (mst) of our h3n2 sequences showed that samples 53 and 55 differed by 25 snps (figure 4b ), despite being collected on the same day from patients on the ward. these results refuted the suspicion that these all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. figure 4b ) also demonstrated: • three sequences were identical (cluster 1), from one patient on the infectious diseases ward (sample 53), one who had been recently on the infectious diseases ward and then under the care of emergency assessment unit (eau) (sample 62), and one who had been on the eau for a couple of days and then in the complex medicine unit until discharged (sample 58). • two identical sequences (cluster 2) differed from cluster 1 by 3 snps, and were from patients on the respiratory ward, taken two days apart. • one sequence (sample 24) differed from cluster 1 by 4 snps, and was from an acutely admitted patient in the eau three weeks later. • the remaining four sequences, including sample 55 from the refuted cluster and three from patients elsewhere in the hospital, were separated from cluster 1, cluster 2, and each other by ≥25 snps. these results suggested that cluster 1 patients on the infectious diseases ward and cluster 2 patients on the chest ward likely reflected nosocomial transmission. there was no clear link between cluster 1 patients, cluster 2 patients, and the acutely admitted eau patient (sample 24). one patient in cluster 1 (sample 58) and this eau patient were positive for influenza on the first day of their admission to the hospital, suggesting these samples may be associated with a predominant strain circulating in the community. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. phylogenetic analysis of the h1 segment showed that our sequences clustered within clade 6b.1 (figure s2a ). at the full genome level, we found no evidence of phylogenetic clustering of ph1n1 iavs recovered from our hospital, suggesting these represent independent introductions. rather, our ph1n1 genomes were closely related to other genomes recovered during the uk 2018/19 season ( figure s2b ). 12 of our ph1n1 genomes had their most closely related sequence within <15 snps, 11 of these most closely related sequences were from the uk. among the 90 influenza-negative respiratory samples we sequenced, 55 had tested positive for another virus in the clinical diagnostic laboratory. from this small dataset, our metagenomic sequencing dataset was >80% sensitive for hmpv, rsv, and piv, but only 30% sensitive for coronavirus and enterovirus; specificity was high at >94% for all five viruses ( table 1) . in five influenza-positive samples for which iav reads were generated by sequencing, we also retrieved reads for other viruses, including human coronavirus (table s1 ). for the 90 influenza-negative all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (table s1 ). while these organisms may represent agents of respiratory infection, they can also be commensal or colonising flora. in the absence of detailed clinical metadata, we were unable to explore their likely contribution to pathology. in this study, we conducted nanopore metagenomic sequencing of iav directly from clinical respiratory samples at a uk hospital during the 2018/19 influenza season, reporting a head-to-head comparison with routine clinical diagnostic tests. the total turnaround time for metagenomic sequencing of each sample was <72 hours. we processed 180 clinical samples and generated sequencing data for 90 clinical samples over a 12 day period. while the turnaround time is still slower than other laboratory diagnostic tests, and the approach requires an investment in labour and sequencing/analysis time, there is potential to reduce this further, through simplification of the wet laboratory protocol and implementation of real-time bioinformatic analysis of reads as they are generated. timeliness is crucial for the deployment of international vaccine strategies. each february, who determines influenza vaccines for use in the following northern all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (figure 1 ), due to the substantial increase of 3c.3a viruses in several regions since november 2018 associated with low vaccine effectiveness (5%) [19] . this one-month delay raised concerns about the timeliness of vaccine manufacturing and distribution for the upcoming influenza season. within our cohort, a clade 3c.3a h3n2 sample was collected on 27th january 2019, and if we had conducted rapid-turn-around sequencing as a routine assay then the complete genome sequence could be available in <72 hours, while the first strain from this clade in the country was not officially reported by public health england until 14th march. this timeline illustrates how routine laboratory sequencing would allow earlier genetic characterization, providing translational advantages in influenza surveillance, monitoring change in the proportion of genetically diverse strains, and contributing to timely insights into seansonal epidemiology vaccine design. our sequencing data showed 83% sensitivity for iav compared with existing laboratory diagnostic tests, consistent with our previous study with a smaller dataset [15] . further optimization is needed to improve the sensitivity of our protocol for clinical samples with lower viral titres (ct values >30). potential methods include depletion of host and bacterial rna to reduce the amount of non-target nucleic acid present, and enrichment of the target via probes or primer amplification. our data show that addition of a carrier can improve the detection of internal spiked control in samples with low total cdna, which is likely due to the improved purification and reduced degradation of lower all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 21.20073072 doi: medrxiv preprint concentration rna, thus we intend to incorporate this approach as a routine part of the protocol in future. the s331r na mutation in h3n2 iav has been associated with reduced susceptibility to oseltamivir since the 2013/14 influenza season [17, 20, 21] . among 1,039 h3n2 iavs tested globally during the 2018/19 season, one strain from south korea showed reduced susceptibility to oseltamivir due to this mutation [22] . our analysis demonstrates that iavs carrying this mutation from the 2018/19 season belong to a distinct genotype generated through intra-subtype reassortment between clades 3c.2a1b and 3c.2a2. a previous study reported a similar observation that the emergence and rapid global spread of adamantane resistant h3n2 iavs (conferred by a s31n mutation in the m2 protein) was associated with a single genotype generated through intra-subtype reassortment [23, 24] . s31n now occurs in almost all circulating iav globally, causing the cessation of use of adamantane to treat influenza [16] . the genesis, prevalence, distribution and clinical impact of the s331r mutation merits additional study to evaluate potential implications for the clinical usefulness of oseltamivir, which is widely used as a first-line agent when treatment is indicated [20] . whole genome sequencing can provide high resolution characterization of the spatiotemporal spread of viral outbreaks [7, 8] . previous studies have used targeted enrichment combined with next generation sequencing to investigate nosocomial transmission of influenza [14, 25] , and our study demonstrates the application of nanopore metagenomic sequencing for this purpose. our sequencing data allow us to all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.21.20073072 doi: medrxiv preprint refute the suspicion of a single transmission cluster on the infectious diseases ward, although the small number of whole genomes generated limits the extent to which we could draw conclusions about transmission among this specific patient group. furthermore, our dataset reveals two clinical clusters that likely represent nosocomial transmission on the infectious diseases ward and the chest ward, showing proof of concept that nanopore metagenomic sequencing can identify nosocomial transmission with the potential to inform infection prevention and control practices. based on a small exploratory dataset, our protocol shows >80% sensitivity for the detection of human metapneumovirus, parainfluenza, and respiratory syncytial virus compared to routine clinical diagnostic testing. the lower sensitivity for enterovirus and coronavirus could be due to low viral titres in these samples, although we are not able to confirm this as the biofire® rp2 assay is a non-quantitative test. another possibility is that the sispa method is less sensitive for certain viruses [26] . moreover, no influenza b virus reads are present in our 90 influenza-positive samples, congruent with the global low level of influenza b virus during the 2018/19 season. further work is needed to determine the limits of detection and optimize the laboratory and bioinformatic protocol to improve the sensitivity for a wider range of potential pathogenic organisms. this study included a limited cohort, with samples stratified by clinical diagnostic results, collection time, and the observation of a putative clinical cluster. we were not able to systematically sequence all influenza-positive samples from the clinical all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.21.20073072 doi: medrxiv preprint diagnostic laboratory due to limited manpower and laboratory resources. generalisability is limited by this sampling approach, as well as by other confounding influences which we were unable to control, including both laboratory and clinical influences (e.g. diverse sample types, sample exposure to freeze/thawing, underlying immunocompromise, symptom duration prior to sample collection). while metagenomic data holds the promise for simultaneous detection of all pathogens from an individual clinical sample, it poses general challenges to analyze and distinguish between pathogens, commensal flora and potential contaminants. accurate interpretation is based upon the clinical context of the patient, type and quality of the sample, the absolute and relative abundance of the organism in the metagenome, genome coverage and mapping depth, and the occurrence of the organism in samples on the same run (if multiplexed) and historical runs in the same laboratory. expert caseby-case appraisal is currently required if the data are to be used for clinical decisionmaking. in summary, we demonstrate the feasibility of applying nanopore sequencing in clinical settings to simultaneously detect influenza and other respiratory viruses, identify drug resistance mutations, characterize genetic diversity, and investigate potential nosocomial transmission events. while work is still needed to refine and streamline the sequencing protocol and bioinformatic analysis, nanopore metagenomic sequencing has the potential to become an applicable point-of-care testing for infectious diseases in clinical settings. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the study of anonymised discarded clinical samples was approved by the london -queen square research ethics committee (17/lo/1420). following removal of human reads, our sequencing data have been uploaded to the european bioinformatics institute https://www.ebi.ac.uk/, project reference prjeb….. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. hazara virus was spiked as an internal control at 10 4 genome copies/ml. c) coverage of the iav consensus sequence against ct value. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. distance between each pair of sequence is denoted by number adjacent to the branch. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.21.20073072 doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04. 21.20073072 doi: medrxiv preprint evolution and ecology of influenza a viruses the biology of influenza viruses are we ready for pandemic influenza? science estimates of global seasonal influenza-associated respiratory mortality: a modelling study the origin of the 1918 pandemic influenza virus: a continuing enigma the lancet infectious diseases. how to be ready for the next influenza pandemic real-time, portable genome sequencing for ebola surveillance metagenomic sequencing at the epicenter of the nigeria 2018 lassa fever outbreak multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis diagnostic tests for influenza infection. current opinion in pediatrics review of rapid diagnostic tests for influenza rapid molecular tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review of diagnostic accuracy and clinical impact studies use of whole-genome sequencing in the investigation of a nosocomial influenza virus outbreak nanopore sequencing of influenza virus direct from clinical respiratory samples structure and inhibition of the drugresistant s31n mutant of the m2 ion channel of influenza a virus global update on the susceptibility of human influenza viruses to neuraminidase inhibitors community transmission of oseltamivir-resistant a(h1n1)pdm09 influenza spread of antigenically drifted influenza a(h3n2) viruses and vaccine effectiveness in the united states during the 2018-2019 season. the journal of infectious diseases global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals global update on the susceptibility of human influenza viruses to neuraminidase inhibitors recommended composition of influenza virus vaccines for use in the 2019-2020 northern hemisphere influenza season the genesis and spread of reassortment human influenza a/h3n2 viruses conferring adamantane resistance the origin and global emergence of adamantane resistant a/h3n2 influenza viruses whole-genome sequencing provides data for stratifying infection prevention and control management of nosocomial influenza a single primer isothermal amplification (spia) combined with next generation sequencing provides complete bovine coronavirus genome coverage and higher sequence depth compared to sequence-independent single primer amplification (sispa) key: cord-338070-y8zi8iz9 authors: liu, wei; ren, xiaojuan; wang, qian; zhang, yan; du, junfeng title: pharmacological inhibition of poly (adp-ribose) polymerase by olaparib ameliorates influenza-virus-induced pneumonia in mice date: 2020-08-31 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-020-04020-5 sha: doc_id: 338070 cord_uid: y8zi8iz9 treatments against influenza a viruses (iav) have to be updated regularly due to antigenic drift and drug resistance. poly (adp-ribose) polymerases (parps) are considered effective therapeutic targets of acute lung inflammatory injury. this study aimed to explore the effects of parp-1 inhibitor olaparib on iav-induced lung injury and the underlying mechanisms. male wild-type c57bl/6 mice were intranasally infected with iav strain h1n1 to mimic pneumonia experimentally. olaparib at different doses was intraperitoneally injected 2 days before and 5 consecutive days after virus stimulation. on day 6 post-infection, lung tissues as well as bronchoalveolar lavage fluid (balf) were sampled for histological and biochemical analyses. olaparib increased the survival rate of iav mice dose-dependently. olaparib remarkably reduced iav mrna expression, myeloperoxidase (mpo) level, and inflammatory cell infiltration in iav lungs. moreover, olaparib significantly reduced the level of interleukin (il)-1β, tumor necrosis factor (tnf)-α, interferon (ifn)-γ, il-6, and il-4 and increased il-10 in iav lungs. also, olaparib efficiently reduced il-6, monocyte chemotactic protein (mcp)-1, granulocyte colony-stimulating factor (g-csf), tnf-α, chemokine (c–x–c motif) ligand (cxcl)1, cxcl10, chemokine (c–c motif) ligand (ccl)3, and regulated on activation, normal t cell expressed and secreted (rantes) release in iav balf. olaparib decreased parylated protein content and p65, iκbα phosphorylation in iav lung tissues. this study successfully constructed the pneumonia murine model using iav. olaparib decreased iav-induced mortality in mice, lung injury, and cytokine production possibly via modulation of parp-1/nf-κb axis. electronic supplementary material: the online version of this article (10.1007/s10096-020-04020-5) contains supplementary material, which is available to authorized users. influenza is an acute infectious disease affecting respiratory tracts accompanied with different clinical manifestations ranging from wild to lethal. influenza causes seasonal, unpredictable epidemics and it is now one of the major public health concerns worldwide [1, 2] . according to the report of the world health organization (who) posted online in 2018, only influenza a virus (iav) has caused pandemics up to now, and most human influenza cases are due to the infection of two iav strains, h1n1 and h3n2. iav have laid heavy burdens on global population and economy these years [3] . although vaccine inoculation and antiviral drug administration have been proved to be effective ways to control influenza, epidemics occur sometimes as a result of antigenic drift, which urges the development of novel anti-influenza drugs [4] . poly (adp-ribose) polymerases (parps) family is composed of 18 members which are involved in the bioprocesses, including dna repair, cell cycle regulation, transcription, and so on [5, 6] . abnormal expression of parps is correlated with necrotic cell death, cancer, and some inflammatory disorders [7] . parp-1 activation has been regarded as one of the critic mechanisms underlying lung inflammation in the context of lipopolysaccharide (lps) and elastase stimulations experimentally [8, 9] . evidences indicated the abnormal increased expression of parp-1 in non-pulmonary cells, alveolar epithelial cells, and lung tissue after iav infection [10, 11] , suggesting its potential as the target for the treatment of iav infection. one of the parp inhibitors, olaparib, is reported to ameliorate acute lung injury induced by elastase and lps [9, 12] . yet the role of olaparib on iav-induced lung injury has rarely been reported. this study aimed to explore the effects of parp inhibition by olaparib on iav infection. animals specific-pathogen-free 8-to 9-week-old male wild-type (wt) c57bl/6 mice, weighing 25 to 30 g, were purchased from the nanjing model animal center and kept at 25°c in a 12-h light/ dark cycle, with free access to food and water. animals were allowed to acclimate to the housing environment for 1 week before experimental procedures. all the animal-involved experiments were performed in accordance with the animal care and use committee of cangzhou central hospital. the a/font monmouth/47(h1n1, fm1) mouse-adapted influenza virus (chinese center for disease control and prevention) was first plaque purified in the madin-darby canine kidney mdck cells, followed by replication in 9-day-old chicken embryos. the virus pool was pretitrated in mice before further studies in order to determine a suitable challenge dose. the murine model of viral pneumonia was constructed by intranasal infection with h1n1 according to previously described [13] . briefly, ketamine (50 mg/kg weight) and pentobarbital (30 mg/kg weight) were intraperitoneally injected to induce the mild anesthesia in mice. next, a 50-μl influenza virus in ice-cooled pbs, containing fifteen 50% mouse lethal challenge doses (mld50), was infected intranasally. mice were kept on a 37°c thermal insulation blanket for 20 min to recover. the day of virus infection was defined as experimental day 1 and 0-days post-infection. two grouping methods were applied in this study. for the part of survival rate analysis, mice were randomly divided into 5 groups, that is, the influenza a virus (iav) group (h1n1 virus + normal saline), the ola groups (h1n1 virus + 2.5, 5, 10 mg/kg olaparib), and the positive control group (h1n1 virus + 10 mg/kg oseltamivir). for the part of biochemical detection, mice were divided into 3 groups, the control group (normal saline), the iav group (h1n1 virus + normal saline), and the iav + ola group (h1n1 virus + 10 mg/kg olaparib). olaparib (selleck chemicals, houston, tx, usa) at different doses was intraperitoneally injected 2 days before and 5 consecutive days after h1n1 virus challenge. the day of virus challenge was defined as experimental day 1. oseltamivir (hoffmann-la roche, basel, switzerland) was administered in a similar manner to olaparib. after anesthetization, mice were infected intranasally with 50-μl influenza virus in ice-cooled pbs, containing five mld50, at experimental day 1 (0 days post-infection). then mice in different groups were followed for 15 consecutive days post-infection with the number of deaths being monitored daily. the severity of pulmonary edema induced by h1n1 virus challenge was quantified by lung indexing. five days after virus infection, the body weight of mice was measured using an electronic analytical balance. then lungs were dissected, washed in pre-cooled pbs, and weighed. the lung index was calculated by the following formula: lung weight/body weight × 100%. at day 6 post-infection, lungs were dissected and fixed in 4% paraformaldehyde for further histopathological analysis. paraffin sections in 5-μm thickness were stained with hematoxylin and eosin (h&e) and examined using a light microscope. the severity of the lung injury was assessed by scoring the h1n1-induced lung histopathological changes using the scoring system previously described [14] . in brief, the grading was conducted in a blinded manner with the grader unaware of the concrete group or mice being reviewed. the scores of 0 to 4 were defined to represent normal, mild, severe, and very severe lung injury, respectively. in concrete, 0 was valued for normal lung, 1 for lower than 25%, 2 for 25-50%, 3 for 50-75%, and 4 for higher than 75% lung involvement, respectively. at day 6 post-infection, lung homogenates and balf were sampled from different groups of mice. for lung homogenates, the lungs were dissected and homogenized in ice-cold ripa lysis (sigma) supplemented with protease inhibitor (sigma) on ice for 2 h. after 4°c centrifugations at 4000 g for 20 min, supernatants were collected and stored at − 80°c for further biochemical analysis, including myeloperoxidase (mpo), cytokines, and targeted protein levels. total protein content of lung homogenates was determined using a bca assay kit (beyotime, shanghai, china). for balf sampling, bronchoalveolar lavage was conducted according to previously described by yashiro m et al. [15] . briefly, after anesthesia, the left main bronchus was ligated and the right lung was quickly lavaged twice by 1 ml of cold sterile pbs. then the collected balf was centrifuged at 2000 rpm for 10 min at 4°c, and the supernatant was stored at − 80°c for measurement of cytokine levels. mpo activity was assessed by 3,3′,5,5'-tetramethylbenzidine (tmb, sigma-aldrich, st. louis, mo, usa) method according to previously described [16] . in brief, equal amount of sample (10 μl) was sequentially mixed with 0.75-mm hydrogen peroxide (80 μl), 2.9-mm tmb dissolved in 14.5% dimethyl sulfoxide (110 μl), and 150-mm sodium phosphate buffer (ph 5.4) in a 96-well plate for 5-min incubation at 37°c. then 2-m sulfuric acid (sigma) was added to stop the reaction and absorbance at 450 nm was measured to evaluate mpo activity in each sample. the mpo levels were expressed as pmol mpo per milligram of lung tissue (pmol/mg protein). cytokines and chemokines in lung homogenate and balf samples were measured using commercial assay kits according to the recommended protocols. interleukin-1 β (il-1β), interleukin-4 (il-4), interleukin-6 (il-6), interleukin-10 (il-10), interferon-γ (ifn-γ), and tumor necrosis factor-α (tnf-α) were measured by enzyme-linked immunosorbent assay (elisa) kits (r&d system, minneapolis, mn, usa). monocyte chemotactic protein-1 (mcp-1), granulocyte colony-stimulating factor (g-csf), chemokine (c-x-c motif) ligand 1 (cxcl1), chemokine (c-x-c motif) ligand 10 (cxcl10), chemokine (c-c motif) ligand 3 (ccl3), and regulated on activation, normal t cell expressed and secreted (rantes) were measured using a mouse multi-cytokine/chemokine magnetic bead panel (millipore, billerica, ma, usa) and analyzed on a luminex 100 system (luminex, austin, tx, usa). the level of target factors was expressed as pg/mg protein for lung homogenate detection and pg/ml for balf detection. real-time quantitative polymerase chain reaction (rt-qpcr) viral load was determined by rt-qpcr according to previously described by li y et al. [17] with slight modifications. the total rna of isolated lung tissues at day 6 post-infection was extracted using trizol method (life technologies, carlsbad, ca, usa) according to the manufacturer's instructions. then equal amount of rna for each sample was transcribed into complementary dna (cdna) using the first strand cdna synthesis kit (takara, dalian, china) following the instructions. quantitative pcr (qpcr) was performed using a sybr green suit (takara) to determine the mrna expression levels of target genes, iav m gene, and gapdh, in an abi real-time pcr system (applied biosystems, new york, ny, usa), with the following amplification procedures: 1-min preincubation at 98°c, 40 cycles of 98°c for 10 s, 56°c for 20 s, and 72°c for 30 s. each sample was performed in triplicate in one single technical repetition. the primer sequences used in this study were as follows: iav m, 5'-aatggtgcaggcgatagag-3′ (forward) and 5'-tacttgcggcaacaacgagag-3′ (reverse); gapdh, 5'-tgaggtcaatgaaggggtcg-3′ (forward) and 5'-tgaggtcaatgaaggggtcg-3′ (reverse). the relative quantitation of iav was calculated using the comparative 2 −δδct method. gapdh was used as the inner control. the parylated protein content in the lung tissues was analyzed by western blot. briefly, − 80°c stored lung homogenate samples were thawed on ice. for each sample, 25-μg protein was separated through 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (millipore), followed by 1-h block in 5% nonfat milk at room temperature. then the membranes were incubated at 4°c overnight with primary antibodies against par (1:1000, abcam, cambridge, uk), phospho-p65 (p-p65, 1:2000, abcam), p65 (1:2000, abcam), phospho-iκbα (p-iκbα, 1:1500, abcam), iκbα (1:1500, abcam), or β-actin (1:2000, santa cruz, ca, usa), respectively. after the 2-h incubation with peroxidase-conjugated secondary antibodies (abcam) at room temperature, the enhanced chemiluminescence kit (millipore) was used to detect the proteins of interest in the uvp biospectrum imaging system (biospectrum, ca, usa). βactin served as the inner control. each experiment was performed for at least three times. data were expressed as mean ± sd for each assay. statistical analysis was conducted by a one-way analysis of variance (anova) test using the spss software (chicago, il, usa). p < 0.05 was considered to be statistically significant. for the body weight change analysis, the two-way anova followed by tukey's multiple comparison test was used. this study aimed to investigate whether olaparib (chemical structure in fig. 1a ) possessed protective effects against influenza virus challenge. from day 3 post-infection, the iavinfected mice began to exhibit clinical symptoms, and on day 6 post-infection, the symptoms got worse, which included but not limited to the following, such as weight loss (fig. s1) , inactivity, rapid shallow breathing, and poor appetite, indicating the successful construction of viral pneumonia model experimentally. figure 1b showed that the iav-only mice began to die on day 4 post-infection until day 12 post-infection. the positive control, oseltamivir, improved the survival rate of infected mice significantly compared with those in the iavonly group. to the expectations, mice in the olaparib group showed higher survival rate compared with that in the iav group in a dose-dependent manner, indicating that olaparib could powerfully protect against influenza virus challenge in by h&e staining and the quantitative analysis of histological changes in the lung tissues (e) (n = 8 for each group). data were presented as mean ± sd. ## p < 0.01 and ### p < 0.001 compared with the control. * p < 0.05 and ** p < 0.01 compared with iav mice. additionally, olaparib-treated iav mice exhibited lower weight loss compared with untreated ones, suggesting it might possess lighter side effects (fig. s1) . also, the dose of 10 mg/kg olaparib was chosen for further investigation as a result of the highest survival rate among all olaparib groups. as olaparib treatment evidently reduced the virus-induced mortality, pathological changes were further explored to assess the influences of olaparib on the lung injury severity. in comparison with the control group, the lung index of mice in the iav group was significantly higher than that in the control group, and olaparib treatment remarkably reduced the lung index, suggesting that olaparib could alleviate the pulmonary edema induced by iva infection (fig. 2a) . as shown in fig. 2b , higher mrna expression of iav was detected in iav lungs, while no virus was detected in the control lung tissues, indicating the direct relationship between iav infection and pathological manifestations of mice model. unsurprisingly, less iav was detected in the lungs of olaparib-treated iav group, indicating the antiviral effect of this drug functions in the iav model mice. virus infection always correlated with leukocyte filtration to the target organs. we next determined the mpo levels and the marker of leucocytes, in lung tissues. figure 2c showed that iav infection elevated the mpo levels compared with that in the control group, whereas olaparib evidently reduced mpo levels, illustrating that olaparib reduced leucocyte infiltration to the lungs of iav mice. morphologic analysis was performed using h&e staining of lung tissues. as shown in fig. 2d , extensive inflammatory cell infiltration, especially around bronchioles, was presented in iav lungs, signifying lung edema might lead to the breathing difficulty in infected mice. and olaparib treatment attenuated the pathological abnormalities in iav lungs. lung histopathological grading numerically pointed that olaparib rectified the lung injury caused by iav infection (fig. 2e) . virus-infected pneumonia always comes along with abnormal release of inflammatory cytokines. next the level of inflammatory cytokines in lung homogenate was detected to investigate whether olaparib could influence the release of inflammatory cytokines in lung tissue. as shown in fig. 3a , b, c, d, e, compared with those in the control group, pro-inflammatory cytokines, il-1β, tnf-α, ifn-γ, il-6, and il-4, were significantly elevated, and anti-inflammatory cytokine, il-10, was remarkably descended in the iav group, while olaparib treatment obviously rectified the abnormal release of the above inflammatory cytokines, meaning that olaparib might possess anti-inflammatory effect in murine lung tissue under the iav context. the detection of cytokine/chemokine in balf samples at day 6 post-infection showed that il-6, mcp-1, g-csf, tnf-α, cxcl1, cxcl10, ccl3, and rantes were remarkably increased in the iav group compared with those in the control group, while olaparib treatment significantly reduced the abnormal increased levels of the above cytokine/chemokines, which was similar with the results obtained from lung tissue (fig. 4a-h) . to explore the mechanisms underlying the protective effect of olaparib against iav-induced injury to murine lungs, western blot was performed to detect the parps, the marker of apoptosis. iav infection increased the parylated protein content in lung homogenate samples compared with the control group, while parp inhibitor olaparib significantly reduced the parylated protein content in iav-infected samples (fig. 5a and c) . nf-κb genes are reported to be the targets of olaparib in the context of lps stimulation to the lungs [12] . as shown in fig. 5b , d, and e, iav remarkably elevated the p-p65 and p-iκbα protein expression in lung homogenates compared with those of the control group, while olaparib obviously decreased the abnormal increase of the two proteins, suggesting that olaparib inhibits the activation of nf-κb signaling pathway in the condition of iav infection. and there were no significant differences of the total expressions of p65 and ikba among different groups (fig. 5b) . the highly transmissible human pathogen iav mainly attacks human respiratory epithelium with its hemagglutinin binding with sialic acid to initiate endocytosis [18] . severe symptoms after iav infection are commonly found in the infant, elderly, pregnant, as well as the immunocompromised populations [19] [20] [21] [22] . iav can cause serious pandemics in a short time, with shocking and heart-wrenching facts in history, e.g., the 1918 great influenza killed almost 1/30 of the global population only in 1.5 years. also iav is capable of generating new strains adapted to human beings. that is the very reason that influenza vaccines have to be updated regularly. also antiviral drugs have to be replaced as a result of the increasing drug resistance developed by viable iav subtypes. thus, it is urgent to explore novel anti-iav drugs to enrich the available antiviral drug bank. this study investigated for the first time the possible role of olaparib on iav infection in a murine model and found that parp-1 inhibitor olaparib remarkably relieved iav-induced lung injury and lung inflammation possibly via inhibition of parp, as well as p65, and iκbα phosphorylation. parp has been indicated to be the target of treatment strategy against cancers and inflammatory disorders [23, 24] . the pro-inflammatory effect of parp-1 has been highlighted in a variety of non-pulmonary and pulmonary inflammatory diseases, including arthritis, allergic encephalomyelitis, asthma, acute lung injury (ali), and so on [25] [26] [27] . parp-1 is activated and involved in the lungs of allergen-induced asthma animal models via modulating immune cell recruitment, airway modeling, as well as cytokine production, mainly th2 cytokines [27] [28] [29] . parp-1 is reported to play a critical role in lps-induced and mechanical ventilation-induced ali in mice [30, 31] . hence parp-1 might be a convincing target for the prevention and treatment of lung inflammatory injury. genetic as well as pharmacological measures modulating parp-1 activity were proved to be effective to alleviate the lung inflammation and injury experimentally from the aspects of reducing neutrophil infiltration and macrophage accumulation [32] , blocking the activation of nf-κb and ap-1 [33] . additionally, parp-1 inhibition can relieve the secondary kidney injury induced by ali [34] . as a potent parp inhibitor, olaparib has already been approved to be used in cancer patients for clinical trials with acceptable toxicity. olaparib downregulates nf-κb-related genes including tnf-α and il-1β, decreases neutrophil, and alleviates edema of lpsstimulated murine lungs [12] . our study found that olaparib possesses protective effects against iav-induced lung inflammation, suggesting that this drug might exert non-specific anti-inflammatory roles. as the outbreak of severe coronavirus infection since december 2019, it is urgent to explore the mechanisms of virus-induced damage to lung tissues or other organs, as well as develop more effective antiviral drugs or therapies to prevent related damages to patients. our study western blot analysis of parylated protein content (a) and p-p65, p65, p-iκbα, and iκbα (b) in lung samples from each experimental group. the relative expressions were normalized to control (c, d, e). data are presented as mean ± sd. ## p < 0.01 and ### p < 0.001 compared with the control. ** p < 0.01 and *** p < 0.001 compared with iav explored the protective effects of olaparib in experimental virus-induced pneumonia, suggesting its possible application in viral pneumonia treatment in the future. there are some shortcomings in our study. we did not perform toxicity analysis about the influence of olaparib on normal physiology of mice. the relationship between parp-1/nf-κb and iav-induced lung inflammation as well as the concrete mechanisms was not well illustrated. related experiments would be performed in the future study. the present study successfully constructed the pneumonia murine model using iav. olaparib decreased iav-induced mortality in mice, lung injury, and cytokine production, possibly via modulation of parp-1/nf-κb axis. this study indicates the non-specific anti-inflammatory effect of olaparib in lung disorders and would shine light on the development for treatment against pneumonia infected by viable iav subtypes. conflict of interest the authors declare that they have no competing interests. ethical approval all the animal-involved experiments were performed in accordance with the animal care and use committee of cangzhou central hospital. informed consent not applicable. the genomic and epidemiological dynamics of human influenza a virus global circulation patterns of seasonal influenza viruses vary with antigenic drift rationale and opportunities in estimating the economic burden of seasonal influenza across countries using a standardized who tool and manual variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies poly (adp-ribose) polymerases in double-strand break repair: focus on parp1, parp2 and parp3 role of poly (adp-ribose) polymerase in cell-cycle checkpoint mechanisms following gamma-irradiation the functional role of poly (adpribose) polymerase 1 as novel coactivator of nf-kappab in inflammatory disorders activation of poly(adp-ribose) polymerase-1 is a central mechanism of lipopolysaccharide-induced acute lung inflammation parp-1 inhibition ameliorates elastase induced lung inflammation and emphysema in mice infection of human retinal pigment epithelial cells with influenza a viruses erucic acid from isatis indigotica fort. suppresses influenza a virus replication and inflammation in vitro and in vivo through modulation of nf-kappab and p38 mapk pathway parp inhibitor, olaparib ameliorates acute lung and kidney injury upon intratracheal administration of lps in mice combined effect of anti-high-mobility group box-1 monoclonal antibody and peramivir against influenza a virus-induced pneumonia in mice critical role for cxcr2 and cxcr2 ligands during the pathogenesis of ventilator-induced lung injury redox-active protein thioredoxin-1 administration ameliorates influenza a virus (h1n1)-induced acute lung injury in mice measuring myeloperoxidase activity in biological samples oral administration of patchouli alcohol isolated from pogostemonis herba augments protection against influenza viral infection in mice influenza virus amino acid changes in ha and determinants of pathogenicity associated with influenza virus a h1n1pdm09 during the winter seasons impact of pregnancy on intra-host genetic diversity of influenza a viruses in hospitalised women: a retrospective cohort study more than just a common cold: endemic coronaviruses oc43, hku1, nl63, and 229e associated with severe acute respiratory infection and fatality cases among healthy adults influenza virus: small molecule therapeutics and mechanisms of antiviral resistance targeted therapy: ariel3 -broad benefit of parp inhibitors in ovarian cancer targeted therapy for cancer using parp inhibitors signaling mechanism of poly (adpribose) polymerase-1 (parp-1) in inflammatory diseases beyond dna repair, the immunological role of parp-1 and its siblings poly (adp-ribose) polymerase-1 in lung inflammatory disorders: a review gene knockout or pharmacological inhibition of poly (adp-ribose) polymerase-1 prevents lung inflammation in a murine model of asthma inhibition of poly (adp-ribose) polymerase prevents allergen-induced asthma-like reaction in sensitized guinea pigs lipopolysaccharide activates erk-parp-1-rela pathway and promotes nuclear factor-kappab transcription in murine macrophages inhibition of poly (adenosine diphosphate-ribose) polymerase attenuates ventilator-induced lung injury effects of parp-1 deficiency on airway inflammatory cell recruitment in response to lps or tnf: differential effects on cxcr2 ligands and duffy antigen receptor for chemokines parp-1 inhibitor, dpq, attenuates lps-induced acute lung injury through inhibiting nf-kappab-mediated inflammatory response inhibition of poly (adenosine diphosphate-ribose) polymerase attenuates lung-kidney crosstalk induced by intratracheal lipopolysaccharide instillation in rats publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-003598-m2fsrwvw authors: elbahesh, husni; gerlach, thomas; saletti, giulietta; rimmelzwaan, guus f. title: response modifiers: tweaking the immune response against influenza a virus date: 2019-04-12 journal: front immunol doi: 10.3389/fimmu.2019.00809 sha: doc_id: 3598 cord_uid: m2fsrwvw despite causing pandemics and yearly epidemics that result in significant morbidity and mortality, our arsenal of options to treat influenza a virus (iav) infections remains limited and is challenged by the virus itself. while vaccination is the preferred intervention strategy against influenza, its efficacy is reduced in the elderly and infants who are most susceptible to severe and/or fatal infections. in addition, antigenic variation of iav complicates the production of efficacious vaccines. similarly, effectiveness of currently used antiviral drugs is jeopardized by the development of resistance to these drugs. like many viruses, iav is reliant on host factors and signaling-pathways for its replication, which could potentially offer alternative options to treat infections. while host-factors have long been recognized as attractive therapeutic candidates against other viruses, only recently they have been targeted for development as iav antivirals. future strategies to combat iav infections will most likely include approaches that alter host-virus interactions on the one hand or dampen harmful host immune responses on the other, with the use of biological response modifiers (brms). in principle, brms are biologically active agents including antibodies, small peptides, and/or other (small) molecules that can influence the immune response. brms are already being used in the clinic to treat malignancies and autoimmune diseases. repurposing such agents would allow for accelerated use against severe and potentially fatal iav infections. in this review, we will address the potential therapeutic use of different brm classes to modulate the immune response induced after iav infections. influenza viruses (ivs) are responsible for significant morbidity and mortality in the human population with ∼500,000 annual deaths worldwide. ivs can cause severe acute respiratory disease especially in high-risk populations like children, the elderly and the immunocompromised. while both influenza a and b viruses (iav and ibv, respectively) cause annual epidemics, the majority of severe human infections are caused by iav. ivs have segmented negative-sense single-stranded rna genomes. the lack of proof-reading activity of the viral rna-dependent rna polymerase (rdrp) and successive replication can lead to the accumulation of nucleotide mutations which drive antigenic drift. in addition, the segmented nature of their genome allows genetic reassortment between iv's to take place, which can produce novel strains that have acquired alternative antigenically distinct hemagglutinin, also known as antigenic shift. both antigenic drift and antigenic shift contribute to the iv's ability to evade pre-existing host immunity induced by previous infections. early recognition and responses to iv infection are largely mediated by innate immune sensors expressed by its primary target, the alveolar epithelial cells (1, 2). recognition of ivs is mediated by pattern recognition receptors (prrs) that include toll like receptors (tlrs), retinoinc acid inducible gene-i (rig-i), and nucleotide oligomerization domain (nod)-like receptor family pyrin domain containing 3 (nlrp3); all of which can recognize viral rnas during various stages of the infection cycle (3-5). activation of these sensors triggers signaling cascades that lead to the production of interferons as well as pro-inflammatory cytokines and chemokines ultimately resulting in an antiviral state within the surrounding cells/tissue (6). accordingly, ivs have multiple mechanisms to evade these responses mediated by the viral nonstructural 1 protein (ns1), polymerase basic 1 protein (pb1), polymerase basic 2 protein (pb2), polymerase acidic (pa) and nucleoprotein (np) [ in otherwise healthy individuals, iav infections are mild and the ensuing pro-and anti-inflammatory responses are balanced. in contrast, a "cytokine storm" is typically associated with severe infections including those caused by highly pathogenic iv strains. during a cytokine storm, chemokine and cytokine responses are dysregulated in both intensity and kinetics resulting in excessive damage to the host due to infiltration of inflammatory immune cells. acute lung injury (ali) caused by this inflammatory response is typically characterized by significant damage or destruction of the respiratory epithelium leading to acute respiratory distress syndrome (ards) (7, 8). clinical treatment options for severe influenza virus infections remain limited and relying heavily on the administration of antiviral neuraminidase inhibitors (nais) and supportive critical care (9). however, nais have not been effective in patients with severe h7n9 or h5n1 infections and there is evidence that fatal outcomes are associated with development of antiviral resistance in patients (10-12). while virus-targeted therapies remain the standard approach, iv's mutability and adaptation to current antivirals has highlighted the need for new therapeutic options that target host factors that regulate iv infections and resulting immune responses. in either approach, the focus is to prevent or limit damage to the lung epithelium due to exaggerated or dysregulated immune cell responses. biological response modifiers (brms) can alter the immune response thereby offering an additional therapeutic approach to treating severe infections. in this review, we highlight several studies that have shown the viability of brms as potential treatment options. for clarity, brms are categorized based on the type of biological agent (table 1) . therapeutic antibodies iav infections and some vaccines elicit broadly-neutralizing antibodies (abs) that target the viral ha-stem. however, their abundance and immune-subdominance is overshadowed by abs targeting the ha-head domain. the effectiveness of these hastem abs against a broad range of iav subtypes, makes them an attractive target not only for vaccine development but also as antivirals. indeed, several ha-stem specific human monoclonal abs are now being evaluated in clinical trials [reviewed in davidson (34) ]. mhaa4549a, medi8852, and vis410 are human monoclonal abs that have been shown to control viral replication and improve symptoms of human patients in phase 2 clinical trials (13-15). while virus-specific abs aim to reduce antigenic load, abs to host targets aim at limiting the secondary wave of cytokines and reduce prolonged damaging cellular infiltration during severe infections. host-target directed antibodies have been utilized to target key regulators of this inflammatory wave and could potentially be used to dampen these overt responses. angiopoietin-like 4 (angptl4) is a soluble angiogenicregulating protein. following proteolytic cleavage, the cterminal portion (cangptl4) is involved in integrin-dependent wound repair and can regulate vascular permeability (35, 36) . angptl4 was significantly elevated in lung biopsies from iav-induced pneumonia patients (16). in mouse studies, neutralizing anti-angptl4 abs reduced pulmonary tissue leakiness significantly accelerating lung recovery and improved lung tissue integrity (16). neutrophil infiltration into the alveolar space occurs within 1 day following iav infections (37) . neutrophil extracellular traps (nets) released during iav-induced pneumonia into the alveolar space caused alveolar damage (38) . the complement protein c5a was shown to induce nets release and administration of anti-c5a abs (ifx-1) reduced h7n9-induced ali due to reduced infiltration of lung macrophages and neutrophils as well as reduction of viral load in african green monkeys (17, 39). tumor necrosis factor alpha (tnfα) is a key cytokine for controlling severe iav infections. it regulates two main antiviral functions: the induction of (i) the nfkb pathway, which ultimately controls expression of several inflammatory cytokines and (ii) apoptosis through multiple signaling cascades (40, 41) . tnf upregulation during iav infections correlates with infection severity, especially following highly pathogenic iav-infections (42) (43) (44) . mice treated with anti-tnf abs showed reduced disease burden; however, the authors of that study reported no effect on viral replication (20). tnf-related apoptosis inducing ligand (trail) can trigger apoptosis in iav-infected cells. iav-infected human epithelial cells are sensitized to trail-mediated apoptosis while peripheral blood mononuclear cells upregulate trail expression. moreover, administration of monoclonal abs against trail increases survival rate following iav infections in mouse studies (18, 19). antimicrobial peptides (amps) are host proteins that have direct antibacterial and antiviral activities and can modulate immune responses to infections. while the literature is largely focused on the antibacterial aspects of amps, several studies have highlighted the antiviral potential of amps against several viruses including ivs [reviewed in hsieh and hartshorn (45) anti-angptl4 -reduced pulmonary tissue leakiness, significantly accelerated lung recovery and improved lung tissue integrity in mice. -mouse-adapted laboratory iav (h1n1) c5a ifx-1 antibody -reduced viral load and virus-induced ali due to reduced infiltration of lung macrophages and neutrophils in iav-infected african green monkeys. -highly-pathogenic avian iav (h7n9) trail anti-trail -increased survival rate following iav infections in mouse studies. -improved symptoms and increased survival of iav infected mice. ed survival after h1n1 or h5n1 mouse infections. -1957 pandemic iav (h2n2); mouse-adapted laboratory iav (h1n1); 2009 pandemic iav (h1n1) (31) (32) (33) and albericio and kruger (46)]. ll-37 is a human cathelicidin derived amp that is found predominantly in neutrophils and its expression can also be induced in epithelial cells and macrophages (47) . aerosol administration of either human ll-37 or its mouse counterpart mcramp led to reduced morbidity and mortality to similar levels as the neuraminidase inhibitor zanamivir that is used for the treatment of human influenza patients (21). both cellular and viral fadd-like il-1β-converting enzymeinhibitory protein (cflip and vflip, respectively) protect cells from death receptor mediated apoptosis. kα2 is a vflip-derived peptide that consists of 10 amino acids from the α2 helix of the kaposi's sarcoma herpes virus (kshv) death effector domain 1 protein. a synthetic version of this peptide, tat-kα2, was generated by fusing kα2 to a portion of the hiv tat protein (22, 48) . in mouse challenge studies, intranasal administration of tat-kα2 at the time of infection with highly pathogenic avian h5n1 virus resulted in protection of the treated mice. no replicating virus was detected in the lungs at either 3 or 5 days after infection suggesting complete protection from infection (22). it should be noted that this effect is largely due to direct destabilization of the virions by the tat-kα2 peptide and it is likely that infection in treated mice was not established; the efficacy of this amp has not been determined during an established infection and warrants further investigation. host kinases regulate not only iav entry and replication but also initiate antiviral signaling cascades that regulate expression of pro-inflammatory chemokines and cytokines during infections and present viable targets for intervention (24, (49) (50) (51) (52) (53) (54) (55) (56) (57) (58) . iav infection has been shown to upregulate c-jun n-terminal kinases 1 and 2 (jnk1/jnk2). these kinases directly regulate the induction of pro-inflammatory responses. iav-induced jnk1/jnk2 activation mediates production of chemokines and cytokines including tnf-α, interferon β (ifn-β), and interleukin 6 (il-6) (24) . in vivo inhibition of jnk1/jnk2 resulted in reduced levels of pro-inflammatory cytokines and reduced viral titers (23, 24). the mitogen activated protein kinase (mapk), p38, regulates viral entry and replication (55, 59) . furthermore, p38 regulates ifn stimulated gene (isg) gene expression and ultimately cytokine production via stat1 phosphorylation (25) . using either of two specific p38 inhibitors (sb 202190 or sb 203580), mice were protected from lethal h5n1 infection exhibiting reduced mortality and pro-inflammatory responses (25) . activation of another mapk, mek, is required for efficient iav replication and its inhibition results in viral ribonucleoprotein (vrnp) retention and reduced titers of progeny virus (26, 60, 61) . importantly, treatment of mice with the clinically approved mek inhibitor (ci-1040) showed reduced lung viral load and mortality of mice following infection with a lethal dose of pandemic h1n1 iav; interestingly, this inhibitor significantly out-performed the clinically recommended oseltamivir in these studies (26) . another central regulator of immune responses at the epithelium as well as immune cells is the nf-κb signaling pathway. accordingly, iav has evolved several mechanisms to modulate this pathway to counteract antiviral responses including directly targeting the ikb kinase (ikk) (62, 63) . sc75741 is a potent nfkb inhibitor that functions by reducing the ability of the p65 subunit of the nfkb complex to bind dna; thereby limiting its transcription-regulating functions (64, 65) . in vivo administration of sc75741 at 4 days after lethal infection with either h5n1 or h7n7 avian viruses resulted in significant protection with most mice surviving and showing little to no clinical symptoms; similar results were obtained by prophylactic administration (27) . g-protein coupled receptor kinase 2 (grk2) is best known for its phosphorylation of gpcrs in cardiac tissue resulting in recruitment of β-arrestin to facilitate rapid receptor internalization and lysozomal degradation (66) . recent phosphoproteomic studies identified grk2 as a potentially proviral host protein for iav that plays a major role in virion uncoating (28) . although in vivo inhibition of grk2 using paroxetine led to a significant reduction in upper respiratory tract viral load and to a modest reduction in lower respiratory tract titers at 4 days post infection, this inhibition was not protective from lethal infections (28) . however, it is possible that the route of administration (intraperitoneal vs. intranasal) and dosing regimen influenced the results. sphingosin kinases (sphk) are lipid kinases that mediate conversion of sphingosine to bioactive lipid sphingosine 1phosphate (s1p) (67), a known modulator of central apoptotic pathways (68) . iav infections leads to increased expression and activation of sphk1 and sphk2 (29) and in vitro inhibition of sphk1 was shown to decrease iav rna synthesis via suppression of nfkb activation (69) . treatment of mice with specific inhibitors to either sphk1 or sphk2 or a pan-sphk inhibitor led to prolonged survival of mice following lethal iav infection (29) . peroxisome proliferator-activated receptors (pparα, pparβ, and pparγ) regulate metabolic homeostasis and are important mediators of the inflammatory response. several ppar agonists have been investigated for efficacy during iav infections with varying results. gemfibrozil (pparα agonist) not only improved symptoms when administered 4 days after infections with an h2n2 virus, but also increased survival of iav infected mice (31) . prophylactic treatment of h1n1-infected mice with pioglitazone (pparγ agonist) resulted in increased survival (32) . combined activation of pparγ and its downstream target ampk improved survival of mice infected with pandemic iav strains (33) . protease activated receptor (pars) link protease activity to inflammatory cellular responses (70) . par1 expression is upregulated in the mouse airways following iav infections (71) . intranasal administration of a par1 antagonist (sch79797) at the time of infection with various iav strains including highly pathogenic avian h5n1 and pandemic h1n1 viruses led to increased survival and a decrease in inflammatory responses. moreover, this effect was also observed when sch79797 was administered 48-72 h after infection (30) . the use of statins, angiotensin ii receptor blockers (arbs) and angiotensin converting enzyme inhibitors (acei) has been proposed to regulate the iav-induced cytokine storm in severe infections (72, 73) . retrospective studies conducted separately in mexico, netherlands, uk and usa reported an association of reduced iav-related pneumonia and lower case fatality due to lower respiratory tract iav infections with statin treatment (74) (75) (76) (77) . however, this association was contested in two additional studies that found no benefit of statin treatment on iavinduced disease burden (78, 79) . this uncertainty regarding the iav therapeutic potential of these widely used compounds warrants further investigations at the basic science level and in clinical trials. the continuous accumulation of adaptive mutations and the introduction of novel viruses in the human population continue to pose a threat to public health, especially to individuals at high risk to influenza. the emergence of strains resistant to existing classes of antiviral drugs and reduced vaccine effectiveness highlights the need for the development of alternative intervention strategies. therefore, therapeutic approaches that can diminish the potential for drug-resistance while being effective against multiple iav subtypes/strains are highly desirable. targeting host cell factors meets these criteria and is more likely to avoid overtly robust immune responses thereby reducing disease severity and improve patient outcome (figure 1) . a large effort has been made in recent years to identify host proteins to serve as intervention targets against iv infections. several genetic and proteomic screens have identified several promising hits with potential roles in the iv replication cycle (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) . in addition to these genome-wide screens, viral and host protein interactions can be mapped into networks that can also be used to identify host factors critical for iv replication (91, 92) . interestingly, meta-analysis of some these studies shows limited overlap in the genes/proteins identified as required host factors (87, (93) (94) (95) . this is likely due to study-specific variations in iv types/strains and cell-lines used, inclusion/exclusion criteria, limited hit-validations and methods used to "knock-down/out" these genes. local microenvironment within a given tissue can dictate the quality and intensity of an immune response. inhibition or activation of critical signaling pathways expressed in both respiratory tract epithelial and immune cells by brms can have opposite and unintended consequences. as discussed above, trail regulates immune cell-mediated apoptosis of infected cells and several studies have shown that blocking trail signaling by genomic deletion or depletion by monoclonal antibody administration can improve infection outcome in iavinfected mice. indeed inhibition of trail signaling in alveolar macrophages and other monocytes limits their ability to induce apoptosis in alveolar cells, prevents lung tissue damage and promotes survival (19, 96, 97) . however, cd8+ t cells from trail−/− mice are less able to protect mice from severe infections, consistent with impaired trail-mediated effector functions of cd8+ t cells (18). similarly, opposing beneficial and detrimental outcomes have also been observed in studies using bcl-2 inhibitors to treat iav infections (98, 99) . brm delivery should be guided by immune system "compartmentalization" to ensure they elicit balanced immune responses. ideally, mucosal delivery deposits brms that reduce viral titers at the site of iav replication; however, systemic delivery of certain brms might be required to dampen dysregulated responses. this not only depends on the brms used but also on the timing of their administration. moreover, the duration of treatment with brms must be considered because sustained inhibition of certain inflammatory responses can result in an immune status that increases susceptibility to secondary opportunistic infections. repurposing of clinically approved drugs could potentially be used as brms for the treatment of severe iav infectious and should be explored (86, 89, 90) . considering that susceptibility to severe iav infections is influenced by host genetics and hostspecific immune responses, selection of therapeutic brms should be carried out using in vivo model systems that are representative of the immune status spectrum and underlying conditions of high-risk influenza patients (young, immunocompromised, nonnaive, obese, pregnant, or aged). using these model systems will increase the likelihood of identifying brms with clinically relevant antiviral and immunomodulatory potentials. he, tg, gs, and gr conceptualized and composed the manuscript. gr and he oversaw all aspects of the manuscript preparation. this work was funded by the alexander von humboldt foundation in the framework of the alexander von humboldt professorship endowed by the german federal ministry of education and research. we apologize to any 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lethal influenza virus infection peroxisome proliferator-activated receptor and amp-activated protein kinase agonists protect against lethal influenza virus challenge in mice treating influenza infection, from now and into the future angiopoietinlike 4 interacts with matrix proteins to modulate wound healing role of angptl4 in vascular permeability and inflammation h5n1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis viable neutrophils release mitochondrial dna to form neutrophil extracellular traps molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression relevance of signaling molecules for apoptosis induction on influenza a virus replication role of host cytokine responses in the pathogenesis of avian h5n1 influenza viruses in 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abt-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice antiviral properties of chemical inhibitors of cellular anti-apoptotic bcl-2 proteins key: cord-267531-tqqj4cy0 authors: he, ying; lin, guang-yu; wang, qiong; cai, xiao-ying; zhang, yin-hui; lin, chuang-xing; lu, chang-dong; lu, xue-dong title: a 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen, china date: 2014-05-14 journal: influenza other respir viruses doi: 10.1111/irv.12257 sha: doc_id: 267531 cord_uid: tqqj4cy0 background: the epidemiology of local viral etiologies is essential for the management of viral respiratory tract infections. limited data are available in china to describe the epidemiology of viral respiratory infections, especially in small–medium cities and rural areas. objectives: to determine the viral etiology and seasonality of acute respiratory infections in hospitalized children, a 3-year study was conducted in shenzhen, china. methods: nasopharyngeal aspirates from eligible children were collected. influenza and other respiratory viruses were tested by molecular assays simultaneously. data were analyzed to describe the frequency and seasonality. results: of the 2025 children enrolled in the study, 971 (48·0%) were positive for at least one viral pathogen, in which 890 (91·7%) were <4 years of age. the three most prevalent viruses were influenza a (iav; 35·8%), respiratory syncytial virus (rsv; 30·5%) and human rhinovirus (hrv; 21·5%). co-infections were found in 302 cases (31·1%), and dual viral infection was dominant. rsv, hrv and iav were the most frequent viral agents involved in co-infection. on the whole, the obvious seasonal peaks mainly from march to may were observed with peak strength varying from 1 year to another. conclusions: this study provides a basic profile of the epidemiology of acute respiratory viral infection in hospitalized children in shenzhen. the spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern china and neighboring hong kong. acute respiratory tract infections (artis) are a persistent and pervasive public health problem in both developed and developing countries. they cause a great burden of disease worldwide. especially in developing countries including china, artis, mainly pneumonia, are the leading cause of death among children under the age of 5 years. 1,2 a great variety of pathogens can cause artis, and viruses have been considered as the predominant pathogens in this children population. 3, 4 the most frequently reported viruses include respiratory syncytial virus (rsv), influenza viruses a and b (iav, ibv), parainfluenza viruses (pivs), human rhinovirus (hrv) and adenovirus (adv), which are responsible for most episodes of artis in children. 1 in the past decade, several new viruses associated with artis such as human metapneumovirus (hmpv), novel strains of coronaviruses (sars-cov, hcov-nl63 and hkui), human bocavirus (bov), wu polyomavirus (wupoyv) and ki polyomavirus (kipoyv) have been discovered in human respiratory tract specimens. among them, some have been identified to be causative pathogens of artis. 1, 4, 5 currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 a better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of artis. studies showed that many viral respiratory infections exhibited predictable seasonal variations. however, the epidemiological profiles of viral respiratory infections from different climate zones or different countries in the same climate zone may be varied. [6] [7] [8] [9] [10] [11] [12] china is a large country crossing three climate zones, and great differences in climate are found from region to region. a better understanding of the epidemiology of artis in different regions could be helpful to develop effective surveillance, prevention and treatment strategies. although some studies on the epidemiology of artis have recently been reported in big cities such as beijing, shanghai and hong kong, [13] [14] [15] [16] the epidemic characteristics of viruses in artis are still not well established all around china, especially in other cities and rural areas. shenzhen is the largest migratory city of china with high population density and population mobility. it is located in southern china at 22°27 0 -22°52 0 n and 113°46 0 -114°37 0 e, immediately north of hong kong, with a typical subtropical monsoon climate. the annual average temperature and relative humidity of shenzhen are about 23°c (12-33°c) and 77%, respectively. the purpose of this study is to investigate the prevalence, seasonality and clinical characteristics of acute viral respiratory infections in hospitalized children in shenzhen and to provide insights into etiologies of artis in local infants and children. a consecutive 3-year prospective study from july 2007 to june 2010 was conducted in shenzhen, a coastal city neighboring hong kong. four hospitals including a children's hospital were chosen for the study. selected patients with artis admitted to the pediatric wards were enrolled. the inclusion criteria were as follows: less than 14 years old, acute fever (t ≥ 38°c), with any one of respiratory symptoms (such as sore throat, cough, wheezing and dyspnoea/ tachypnoea), normal or low leukocyte count, the onset of illness within 3 days before hospitalization. the diagnosis of pneumonia was based on the guideline of the management of childhood community acquired pneumonia (cap) issued by the chinese medical association in 2006. 17 in the guideline, the clinical symptoms and signs for the diagnosis of childhood cap include fever, cough, tachypnoea (defined according to different age), difficulty breathing and/or lower chest wall indrawing. x-ray evaluation has been carried out when necessary. the study protocol was approved by the medical ethical committees of the hospitals. written informed consent was obtained from the parents or legal guardians of the children. nasopharyngeal aspirates (npa) were obtained by trained personnel following standard operating procedures within 24 hour after admission. the specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at à80°c until further processing. total viral nucleic acids (dna and rna) were extracted from 200 ll of npa specimen using the axyprep body fluid dna/rna miniprep kit (axygen, union city, ca, usa), according to the manufacturer's instructions. purified dna and rna were stored at à80°c in aliquots for further pcr analysis. for each specimen, assays for ten common and newly identified viruses were performed. briefly, wupoyv and bov were tested using monoplex pcrs described previously. 18, 19 other viruses were tested using the luminex platform and multiplex xtag tm respiratory viral panel assay (rvp assay) according to the manufacturer's instructions. 20 all multiple infection samples were retested. if there was discordance between two tests, the sample was confirmed by monoplex pcr. statistical package for the social sciences (spss) for windows version 11.0 (spss inc. chicago, il, usa) was used. for comparison of categorical data, chi-square or fisher's exact test was used. all tests were two-tailed, and a p value below 0á05 was considered statistically significant. a total of 2025 specimens were obtained from 2025 eligible patients ranging from 15 days to 14 years old with a median age of 12 months, in which 89á6% of patients were < 4 years old. there were 964 (47á6%) females and 1061 (52á4%) males included. of all hospitalized children enrolled in this study, 84á0% involved lower respiratory infection and 16á0% had upper respiratory tract infection (table 1) . among 971 positive cases, 572 (58á9%) were diagnosed as pneumonia. about 971 of the 2025 cases (48á0%) were positive for at least one viral pathogen. among them, iav, rsv, hrv and pivs were detected in 348 (35á8%), 296 (30á5%), 209 (21á5%) and 169 (17á4%) cases, respectively. single infection was observed in 669 (68á9%) cases, and multiple infection was found in 302 (31á1%). our results also showed that rsv, iav and hrv were the main pathogens in single viral infection cases ( table 1) . the monthly positive rates varied from 32á5% to 75á0% with a mean of 45á1% ( figure 1 ). in the year 2009, when influenza a (h1n1) was pandemic worldwide, the positive rate started to increase in march and the highest positive rate 75á0% was observed in may. among the 971 positive cases, a total of 1335 viral pathogens were detected. the most frequently detected pathogen was iav (26á1%, 348/1335), followed by rsv (22á2%, 296/1335), hrv (15á7%, 209/1335), piv1 and piv3 (12á7%, 169/1335) ( about 302 co-infection cases were identified, accounting for 14á9% of all 2025 hospitalized children. during the h1n1 outbreak from march to august 2009, co-infection cases and co-infection rate increased significantly ( figure 2 ). 143 of 302 (47á4%) co-infection cases were detected during that time. among them, 121 cases were involved in iav infection, including 90 dual infection cases. of all co-infection cases, 247 (81á8%), 49 (16á2%) and 5 (1á7%) were infected with two, three and four potential viral pathogens, respectively. one multiple infection with five viruses was detected in a rsv iav hrv bov piv3 adv hmpv wupoyv piv1 ibv total a total cases 163 172 85 55 51 49 37 26 24 7 669 bronchitis 12 19 7 7 5 2 2 3 5 0 62 bronchiolitis 45 19 20 7 13 9 8 2 5 0 128 pneumonia 93 97 55 28 24 28 25 18 14 5 387 urti 13 37 3 13 9 10 2 3 0 2 *case number and percentage in all enrolled children. **incidence rate in this age group significantly higher than the other age groups. ***no significant difference between these three age groups. †no significant differences between these two age groups, but significantly lower than the other age groups. 6-month-old infant. iav, rsv and hrv were the three most frequently found viruses in co-infection and detected in 176, 133 and 124 cases with co-infection rates of 50á6%, 44á9% and 59á3%, respectively ( table 2) . various multiple infection patterns were observed in the study. a total of 152 (50á3%) co-infection cases involved at least two viruses of rsv, hrv and iav. co-infection rate of each individual virus detected varied significantly. the lowest and highest co-infection rates were observed in wupoyv (33á3%) and ibv (66á7%), respectively. 91á4% (276/302) of co-infection cases were tested in the age group of 4 years old or younger ( table 2) , but among all age groups, no statistical difference in co-infection rate was found (v 2 = 1á83, p = 0á8721). gender-specific difference in co-infection rate was not observed (v 2 = 2á17, p = 0á1404). there was no significant difference in co-infection rates between picu and non-picu cases. similarly, no significant difference in clinical symptoms was observed between co-infection and single cases (data not shown). in general, respiratory viruses were detected more often in the period of march to may than in other months (55á4% and 40á6%, respectively, v 2 = 28á06, p = 0á0000), and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. a weaker seasonal peak could also be distinguished in some winter months in different years ( figure 1) . the seasonality profile of each individual virus detected was diversified. a seasonal distribution of iav can be observed from late spring to summer (mainly march to may) and sometimes in fall (october, november or december). a wide seasonal peak of iav infection was detected from march to august 2009 ( figure 3a ). although rsv was tested almost a whole year, two yearly peaks were identified. one was found in november and/or december and the other stronger one was found in march to may of the year. the peak duration in 2009 was longer than those in other years. the seasonal trends of hrv and pivs were similar to that of rsv, but the peaks of these three viruses fluctuated and shifted mildly ( figure 3b ). although ibv and adv had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter. peaks in spring and summer were also observed in some years ( figure 3c ). our investigation did not find regular seasonality in bov infections. a sudden increase in bov infection was recorded in april and may 2010. although the positive rate of hmpv infection was only 4á8%, regular seasonality was observed from march to may of each year. of 39 patients with wupoyv infection, 36 were detected after july 2008. our data implied that peak months of wupoyv infection were from march to may ( figure 3d ). the positive rates of viral infections in male and female were 52á5% and 47á5%, respectively. no significant gender difference was revealed (v 2 = 0á012, p = 0á9118). the distribution of viral agents and infection patterns in different age groups are shown in table 2 . of all 971 positive children, 890 (91á7%) were 4 years old or younger. the positive rate in this age group was significantly higher than that in children more than 4 years old (v 2 = 8á26, p = 0á0041). children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14á8% (table 2) . very few long-term prospective studies were performed for viral etiologies of artis among hospitalized children. in this present study, the infection frequency, seasonality, co-infection pattern and clinical features of viral respiratory infections were investigated based on prospective analysis of three consecutive year's data from hospitalized children with artis. our results provided a distinctive epidemiological profile of viral respiratory infections in hospitalized children with artis in the study areas, which was different from those in the big cities in northern china such as beijing and shanghai and also different from that in adjacent hong kong. overall, 48á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 a similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as rome 23 and milan, 24 but it was different from other studies. [10] [11] [12] in china, the overall positive rate reported varied from 27á3 to 74á8% depending on different areas and detection methods. 15, 16, [25] [26] [27] [28] [29] [30] [31] the rate of respiratory viral infections varied worldwide, and many factors such as geographic distribu-tion, study design and detection protocols could lead to these variations. 1, 7, 8, 32 in our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. viruses not considered in the study, for example coronaviruses, would underestimate the positive rate. most studies showed that rsv or hrv was the most prevalent viruses in children with viral respiratory tract infection. 1 in this study, iav was the most frequently detected respiratory virus, followed by rsv and hrv. iav (h1n1) outbreak in 2009 could explain this shift. data showed that about 60% of iav infections were detected during the outbreak period. studies showed that the h1n1 outbreak could change viral distribution patterns. 24, 29, 33 regardless of the iav (h1n1) outbreak, rsv and hrv were the two most common viral pathogens in artis, which was consistent with most previous studies. 1, 10, 15, 16, 22, [25] [26] [27] [28] [29] our study further confirmed the importance of rsv and hrv in children with artis, especially in children < 4 years of age. 10, 14, 23 our results also showed that 12á7% of viral pathogens detected were piv1 and piv3, which implied that pivs played an important role in children with artis. similar findings were obtained in the studies conducted in shanghai, 14, 34 changsha, 26 harbin, 30 hong kong 13 and rome. 23 the prevalence of piv3 was twofold higher than that of piv1, particularly in infants, which was similar with other reports, 25, 26, 30, 35 implying that infants could be more vulnerable to infection with piv3 than piv1. hmpv has been proven to be one of the main viral pathogens responsible for artis in children. 5 the positive rate found in the study was consistent with previously published results. 10, 36, 37 in china, the infection rate of hmpv varied from 3á2 to 10á6%. 22, 26, 28, 29, 31 the seasonality of hmpv in this study was mainly from march to may, similar to that in hong kong, 36 but different from other places. 5, 37 in our study, 4á9% of cases were positive for bov, which coincided with 5á0% in hong kong 38 and higher than guangzhou 39 and eastern guangdong. 22 our result suggested that bov might be present throughout the year with no seasonal distribution. however, seasonal distribution was noted from september to february in hong kong 38 and may and june in guangzhou. 39 the use of multiple pcr made it possible to simultaneously detect a broad spectrum of viruses with excellent sensitivity, at the same time, with increased viral detection rate and co-infection rate for artis. 12,40 among our positive cases, co-infection rate was 31á1%, which was similar to 27á9% reported by do et al. 10 co-infection rate reported elsewhere varied widely from 25á4 to 47á9%. 40 the relatively lower co-infection rates ranging from 0á24 to 26á9% were reported in the studies conducted in various cities of china. 22, [25] [26] [27] [28] [29] [30] [31] in most of these studies, immunofluorescence kits were used to test a lower number of respiratory viruses. it was worth to note that in the study by peng et al. 32 in wuhan, china, 69á5% of co-infection rate was reported with immunofluorescence kit. these variations might be attributed to geographic differences, diagnostic methods for viral agents and study design. 12, 32, 34, 41, 42 pathogens in those negative patients need to be further investigated as only ten common and newly identified viruses were included in our study, which might underestimate positive rate or coinfection rate. it was notable that the correlation between co-infection rate and positive rate was not observed. of multiple infections, dual infection was predominant in this study whether or not considering the iav (h1n1) outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 similar with the studies conducted in the cities of guangzhou and wuhan, china, 28, 29 our study showed that iav, rsv and hrv were the main viruses involved in multiple infections. high co-infection rate between these three viruses could be explained from the overlap of their seasonal distributions. a variety of predominant multiple infection patterns between respiratory viruses were observed in different studies. 12, 32, 42, 43 for example, it was shown in martin et al.'s study 43 that adv and coronaviruses were the most common co-infection pattern. our study showed that rsv and hrv were the two most viruses involved in multiple infection, followed by iav and pivs, regardless of iav infection in the h1n1 outbreak period. it was difficult to explain the variations of coinfection patterns based only on seasonal distribution. a recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 as molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first. viral load detection could provide some clues for solving this issue. 43, 44 although high co-infection rates have been reported in various studies, the associations among multiple infections, hospitalization rate and severity of artis were still not clear with inconsistent results in different studies. 42, 43, 45 our data suggested that multiple infection had less association with the severity of disease, consistent with peng et al.'s study. 32 the relationship between co-infection rates and age group was also investigated in our study, and little correlation was observed. several previous studies observed that co-infection rates were more frequent in a certain age group, but results were varied. 32, 43 in contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 in this study, due to the high detection rate and similar seasonality of rsv, hrv, iav, piv and hmpv, an overall spring-summer seasonality of viral respiratory infections in children was concluded. studies conducted in hong kong showed that a clear seasonal peak was from april to september, 36, 46 with a longer duration than our study. the overall seasonality in this study was also different from the studies conducted in northern or central cities of china, in which the seasonality of most viruses presented in autumn-winter and/or winter-spring. 15, [25] [26] [27] 30 the winter-spring seasonality was also observed in guangzhou, a city about 150 kilometers north of shenzhen. 28 different seasonal onset and duration were observed in various studies conducted in (sub-) tropical regions. in these studies, ambient temperature, humidity and rainfall were widely used to explain these differences in seasonality, but inconsistent results were observed. 9, 46, 47 although most studies demonstrated that the seasonality of viral respiratory infections was correlated with increased rainfall, effects of climate factors such as humidity and temperature on the seasonality were complex and interactive. 9, 46, 48 the study areas have four indistinct seasons, and the coldest month usually emerges in january (average 12°c). during the period from march to may, the weather featured warm ambient temperature (average 18-25°c), high relative humidity (average 85%-90%) and increasing rainfall. these meteorological conditions were perhaps conducive to viral survival. 9, 48 in addition, intensive temperature fluctuations during seasonal alternation could increase the susceptibility to infections. 49 as reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for artis, compared with older children. this was particularly substantiated in rsv infection. our presumption was supported by other studies. 14,25-28 of course, this speculation needed to be validated by the population-based study. the findings reported elsewhere suggested that more males than females were affected by artis, which were not observed in our study. notably, our study occurred over a span of 3 years, which included the iav (h1n1) outbreak in 2009. the impact of the outbreak on the results should be considered. data showed that the detection rate of iav increased significantly and co-infection rate during outbreak months was much higher than average co-infection rate. unfortunately, we did not type these influenza strains based on the original study design. it was most likely that these strains contributed to the relatively high proportion of iav. relatively higher single and multiple infections of rsv, hrv and pivs were also observed during the outbreak of iav. increased susceptible population and awareness, intensive testing and altered patient and physician behavior could lead to these increases. these factors could partly explain the relatively high proportion of pneumonia cases in the study. furthermore, studies showed that the outbreak of iav (h1n1) could increase the risk of other viral infections such as rsv and hrv. 24, 33 other limitations also existed in this study. first, molecular methods allowed the detection of only viral nucleic acid even without virus replication, which complicates the interpretation of positive detection results. second, the subtype identification of some common respiratory viruses such as iav and hrv was not performed in our study, particularly during the iav (h1n1) outbreak in 2009. in summary, despite those aforementioned limitations, this three consecutive years' surveillance would provide a basic profile of the spectrum, seasonality, age and gender distribution, co-infection patterns as well as clinical association of viral respiratory infections in hospitalized children in the study sites. it could help the prediction, prevention and control of artis in children. respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology causes of deaths in children younger than 5 years in china in 2008 respiratory virus infection in infants and children emerging respiratory agents: new viruses for old diseases? ten years of human metapneumovirus research occurrence of respiratory virus: time, place and person epidemiology of respiratory syncytial virus infection in northern taiwan variation in timing of respiratory syncytial virus outbreaks: lessons 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chongqing during investigation on prevalence and mixed infection of childhood respiratory virus infection in guangzhou the epidemiology and etiology of influenza-like illness in chinese children from 2008 to 2010 respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin, china viral pathogens of acute respiratory infection in hospitalized children from suzhou multipathogen infections in hospitalized children with acute respiratory infections the impact of pandemic influenza a(h1n1) 2009 on the circulation of respiratory viruses high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in human parainfluenza virus type 4 infection in chinese children with lower respiratory tract infections: a comparison study children with respiratory disease associated with metapneumovirus in hong kong seasonal distribution and epidemiological characteristics of human metapneumovirus infections in pediatric inpatients in southeast china pediatric hospitalization of acute respiratory tract infections with human bocavirus in hong kong detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china coinfections with influenza and other respiratory viruses evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections mixed respiratory virus infections multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection evaluation of viral co-infections in hospitalized and nonhospitalized children with respiratory infections using microarrays epidemiology of respiratory syncytial virus infection among paediatric patients in hong kong: seasonality and disease impact incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus exposure to cold and respiratory tract infections we are most grateful to the clinicians and nurses for their assistance in sample collection. we also thank dr. yi-wei tang for reviewing this manuscript and dr. linda chui, dr leo su for their help in improving english in this paper. none of the authors has a conflict of interest. key: cord-000434-ff2zadol authors: zhao, rongmao; cui, shujuan; guo, li; wu, chao; gonzalez, richard; paranhos-baccalà, gláucia; vernet, guy; wang, jianwei; hung, tao title: identification of a highly conserved h1 subtype-specific epitope with diagnostic potential in the hemagglutinin protein of influenza a virus date: 2011-08-19 journal: plos one doi: 10.1371/journal.pone.0023374 sha: doc_id: 434 cord_uid: ff2zadol subtype specificity of influenza a virus (iav) is determined by its two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na). for ha, 16 distinct subtypes (h1–h16) exist, while nine exist for na. the epidemic strains of h1n1 iav change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. the recent introduction of pandemic a/h1n1 iav (h1n1pdm virus) into humans re-emphasizes the public health concern about h1n1 iav. several studies have identified conserved epitopes within specific ha subtypes that can be used for diagnostics. however, immune specific epitopes in h1n1 iav have not been completely assessed. in this study, linear epitopes on the h1n1pdm viral ha protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from h1n1pdm patients. one epitope, p5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza h1 ha [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. this previously unidentified linear epitope is located outside the five well-recognized antigenic sites in ha. a peptide elisa method based on this epitope was developed and showed high correlation (χ(2) = 51.81, p<0.01, pearson correlation coefficient r = 0.741) with a hemagglutination inhibition test. the highly conserved h1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against h1n1 iavs. influenza a viruses (iavs), members of the orthomyxoviridae family, are highly contagious to a variety of avian and mammalian species. iavs cause seasonal influenza epidemics annually and recurring pandemics with severe consequences for public health and global economy [1, 2] . at least three iav-pandemics emerged in the last century (1918 a/h1n1, 1957 a/h2n2, and 1968 a/h3n2). the 1918 spanish flu was the most serious influenza pandemic that killed over 50 million people worldwide [3] . the latter two pandemics, although mild compared to the 1918 incidence, resulted in significant mortality, with close to 2 million and 1 million deaths, respectively [4] . the latest pandemic influenza, and newest global health challenge, occurred in 2009 due to the emergence of an a/ h1n1 pandemic iav (h1n1pdm virus). the h1n1pdm virus has been detected in more than 214 countries and territories and has caused 18,389 deaths as of july 30, 2010 [5] . the viral genome of iav consists of eight single-stranded negative sense rna segments that encode at least 11 viral proteins, including two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) [6] . based on the antigenic properties of ha and na, iavs have been classified into 16 ha subtypes and 9 na subtypes [7] . all 16 ha subtypes have been identified in avian species, while only 6 ha subtypes (h1, h2, h3, h5, h7 and h9) are known to infect human beings [8, 9, 10] . h1, h2 and h3 subtypes have caused pandemics, while h1 and h3 also dominate seasonal epidemics together with influenza b virus. ha, encoded by segment 4 of the iav genome, is a glycoprotein of approximate 560 amino acid. the biologically active ha is a homologous trimeric molecule that is attached to the virion membrane through its carboxy terminus [11] . ha plays a critical role in the pathogenesis of iavs. ha mediates iavs' binding to the cellular receptor n-acetylneuraminic (sialic) acid as well as the subsequent membrane fusion process [12] . ha also stimulates host protective immunities, specifically the production of neutralizing antibodies. the generation of anti-ha neutralizing antibodies has been the major target for influenza vaccine development [11, 13] . due to its specificity in immune response, ha is also an important target for iav subtyping using immunoassays [7, 14] . active serological surveillance for viral antibodies is of great importance for influenza control and prevention. several iav subtype-specific serological tests have been developed. at present, subtyping of iav mainly relies on a hemagglutination inhibition (hi) test using ha and na subtype-specific reference sera [15] . however, there are a number of drawbacks to hi testing. this assay is 1) relatively laborious; 2) low in sensitivity; 3) requires preparation of antigen from viable viruses which are potentially hazardous and 4) contains low signal to noise ratio, e.g. the assay exhibits inter-variability and subtype cross-reactivity [16, 17] . moreover, the hi test can be confounded by steric hindrance from na antibodies, leading to nonspecific inhibition and misidentification [18] . microneutralizing test is an alternative method to type and subtype influenza viruses. however, due to the needs of cell culture process, this method is labor-intensive and requires biological safety containments (particularly for high pathogenic strains). as such, it is not suitable for large scale investigations [19, 20] . recently, subtyping of iav antibodies using different categories of elisa assays have also been reported [16, 17, 21] . however, present elisa assays mainly rely on an ha antigen, which can lead to nonspecific detection to some extent due to the possible cross-reaction of different subtypes [22, 23] . virus-derived epitopes are useful tools to accurately evaluate immune response and to differentiate which responses are specific or due to cross-reactivity [24, 25, 26] . several studies have reported the existence of ha subtype-specific as well as inter subtypeconserved epitopes [27, 28, 29] . elisa assays based on epitopes that are highly conserved and specific for one certain ha subtype will be useful for rapid and simple subtyping of iavs. such epitopes in iavs have not been fully addressed although many studies have been performed. in the present study, we report the successful identification of a new epitope, which is highly conserved among the majority of iav strains of h1 subtype. moreover, we developed an elisa assay for h1 antibody subtyping based on this epitope. results derived from this new assay correlate with results obtained through the use of hi test. to identify the immunodominant epitopes in the ha protein, a peptide scanning assay was performed. a set of 50 peptides with five residues overlapping with the adjacent peptides spanning the ectodomain sequences of the ha protein of the h1n1pdm virus strain a/california/04/2009 were synthesized. the binding between these peptides and the convalescent serum samples from 11 h1n1pdm patients were examined by elisa using these peptides as coating antigens. five of these peptides (p3, p5, p15, p16 and p31) were found to react well with the sera tested. these peptides corresponded to the sequences of amino acid (aa) residues 38-52, 58-72, 158-172, 168-182, and 318-332 in the ha molecule, respectively ( fig. 1a and table 1 ). among them, the p3 peptide reacted with 54.5% (6/11) of the sera, the p15 and p16 peptides reacted with 81.8% (9/11) of the sera, while the p5 and p31 peptides reacted with 100% (11/11) of the sera. these data indicated that these peptides may contain h1n1pdm virus b cell epitopes. to visualize the location of the peptides on the ha protein, we mapped the peptides on the crystal model of this protein (fig. 1b) . the various colors in figure 1b represent the different peptides. although p3 (residues 38-52, indicated by blue) and p31 (residues 318-332, indicated by red) are parts of ha1 in primary sequence, they are located in the middle of helix a and b in the trimeric structure and are partially surface exposed. p5 (residues 58-72, indicated by magenta) seems to be a dispatch that links the stem region and the globular region and is fully surface exposed (fig. 1b) . p15 and p16 (residues 158-172 and 168-182, indicated by orange) are located in the receptor binding domain [11] . to confirm the immunogenicity of these peptides in vivo, we analyzed sera derived from peptide-immunized mice. the five positive peptides and two control peptides (p6 and p30) were coupled with keyhole limpet hemocyanin (klh) and were used to immunize balb/c mice ( table 1 ). the antisera were collected five days after the third immunization and titrated by elisa using corresponding peptide as a coating antigen. our results showed that all of the peptide conjugates except p15 induced potent antibody titers. the endpoint titers of antisera in elisa from mice immunized with p3, p5, p6, p16, p30, and p31 peptides were 1:6,400, 1:51,200, 1:51,200, 1:12,800, 1:51,200, and 1:25,600, respectively ( fig. 2a) . these data indicate that most of the positive peptides elicite humoral immunity and are highly immunogenic in mice. to confirm that these antibodies can recognize the ha antigen, the reactivity of the anti-peptide sera were evaluated by western blot and elisa against the purified ha0 protein of h1n1pdm virus. our data demonstrate that sera against p3, p5, and p31 but not those against p6 and p30 (controls) react to the ha0 protein ( fig. 2b and 2c ). the anti-p16 sera did not react to the ha0 protein, although it exhibited a high elisa reactivity to the ha0 protein ( fig. 2b and 2c ). taken together, our results demonstrate that p3, p5, and p31 peptides contain dominant epitopes of h1n1pdm virus. we then characterized these three peptides in the following studies. to determine if the epitopes identified in this study can stimulate neutralizing antibodies, a ha-pseudotype neutralization test was performed against the anti-peptide sera using the h1n1pdm pseudotyped lentivirus. none of the sera against p3, p5, p16, and p31 could efficiently inhibit (90% inhibition [30] ) the entry of h1n1pdm ha pseudotypes ( figure 2d ), indicating that these epitopes do not contain neutralizing activity. western blot analysis was used to determine the specificity of the epitopes present in the peptides p3, p5, and p31. the h1-h16 recombinant ha proteins were obtained by transient expressions of corresponding genes by the pcaggs vector in 293t cells. the lysates of these cells were used to examine the specificity of antibodies elicited by peptide-conjugates. as shown in fig. 3a , the anti-p3 serum reacted with h1 (including 07h1 and 09h1 viruses), h2, h5, and h6 ha proteins, while anti-p5 and anti-p31 sera only reacted with the h1 ha proteins. these findings indicated that p5 and p31 may contain h1-subtype specific epitopes. to evaluate the subtype-specificity of epitopes in p5 and p31 further, additional ha proteins of three epidemic human strains from different years (1918, 1934 and 1977) as well as a swine strain were expressed by pcaggs vector in 293t cells. the reactivity of anti-p5 and p31 sera with the cell lysates was determined by western blot analysis. our results showed that the anti-p5 serum strongly reacted with all of the six h1 ha proteins in a manner similar to an antibody against the ha1 of h1n1pdm virus (fig. 3b ). we found that the anti-p31 sera reactivity was weak against ha proteins from the 1918 and 1977 virus strains (fig. 3b ). these data indicated that the epitopes in p5 and p31 peptides are relatively conserved among h1-subtype iavs though these viruses have circulated in the world for almost a century. to test if anti-p5 sera cross-react with influenza type b virus, the reactivity of anti-p5 sera with two representative influenza type b virus strains (b/hubeiwujiagang/158/2009,yamagata lineage and b/heilongjianghulan/116/2010, victoria lineage) and an influenza type a virus strain (a/h1n1/pr8/34) was examined by western blot analysis. the results showed that anti-p5 serum reacted well with a/h1n1/pr8/34 virus but not with influenza type b virus strains (fig. 3c) , further confirming the specificity of the epitope in p5. to determine the conservation of the identified epitopes among iavs, the aa sequences of p3, p5, and p31 were aligned with the corresponding aa sequences of all the 16 subtype has available in the genbank. fig. 4 is a representative of the alignment analysis, showing that p5 is identical to ha of h1 subtype strains. p3 is identical to ha of the h1 subtype, as well as highly identical to ha of the h2, h5, and h6 subtypes. these data are consistent with the specificity analysis by western blot (fig. 3a) . although anti-p31 antibody only recognizes the h1-subtype ha, it is similar to multiple subtypes. to assess the identity levels of p5 and p31 sequences among the known iav strains, in silico coverage analysis was performed. this analysis showed that the p5 peptide sequence could be identified in 91.6% (9860/10767) of the h1-subtype ha sequences available in the influenza research database (http://www.fludb.org) ( table 2) . notably, this sequence scarcely presented (3.3% 792/23895) among the has of h2-h16 subtype iavs. however, despite a high identity in the h1 ha proteins (93.1%), the peptide sequence of p31 also presented among the has of h2-h16 viruses (78.8%). taken together, these findings indicate that the p5 peptide is h1-subtype specific and is conserved among h1 virus strains. to define the epitope contained in p5 precisely, a peptideinhibition elisa was performed. this experiment is reliable and is a standard methodology to determine the fine specificity of antigen-antibody reactions [31, 32] . a panel of short peptides derived from p5 (n6-n14, with c-terminus truncation; and c6-c14, with n-terminus truncation) were used to block the binding of anti-p5 antibody to coated p5. as shown in fig. 5 , antibody induced by the p5-klh conjugate was inhibited by peptide n10-n14 and the parental peptide p5 to similar extents, whereas peptides n6-n9 only showed inefficient inhibition even at high molar concentrations. a similar pattern of inhibition was observed with the c-terminal conservative derivatives. peptides c12, c13, c14, and the parent peptide p5 demonstrated comparable and efficient inhibition, whereas only slight inhibition was observed in peptides c6-c11 (fig. 5b) . since the amino acid sequence lrgvapl overlapped both peptides n10 and c12 (fig. 5) , we speculate that this sequence met the minimum requirements of binding to the anti-p5 antibody. however, the synthetic peptide lrgvapl did not block the binding between p5 peptide and its antibody, nor did it directly bind to the p5 antibody (fig. 6 ). as p5 (1918, 1934, 1977, 2007, 2009 ) and a swine h1n1 strain. 293t cells transfected with an empty vector was used as a control (ctrl). b-actin was used as a loading control. for the backgrounds of various subtype iav strains, see inspired by the high specificity of p5 among the h1-subtype viruses, we developed an indirect elisa assay using the p5 peptide to evaluate its performance as a diagnostic tool for h1 antibodies. the hi test was used as a reference method. as shown in table 3 , the overall agreement of these two methods was 87%, showing that the two methods have good correlation (pearson correlation coefficient r = 0.741). the sensitivity and specificity of peptide-elisa versus hi test was 96.5% and 74.4%, respectively, indicating the potential of the peptide-elisa method in detecting antibody against h1-subtype iavs. in the present study, we identified immunodominant linear b cell epitopes on the h1n1pdm virus ha protein by a peptide scanning approach using h1n1pdm patients sera. we confirmed that an unidentified epitope was highly conserved among h1 subtypes viruses and showed a good correlation with results obtained using the hi test. these findings demonstrate the potential of epitope-based antibody detection in iav diagnosis and surveillance. iav escapes the human immune system by continuous antigenic drifts and occasional antigenic shifts [33] . attempts to develop universal vaccines and reliable diagnostic tools based on conserved epitopes of iav are big challenges. several epitopes that can elicit broad spectrum neutralizing antibodies have been identified recently. for example, sui et al. identified a universal neutralizing epitope for group 1 ha [34] . yoshida et al. reported a universal epitope in antigenic site b shared by h1, h2, h3, h5, h9, and h13 subtypes [35] . all these epitopes are conformationdependent. in this study, we identified two epitopes (p5 and p31) which have not been identified previously (fig. s1 ). the p5 (aa58-72) seems to be a dispatch that links the stem region and the globular region and is fully exposed on the surface, while p31 (aa 318-332d) is located in the middle of helix a and b on ha2. in contrast to previous studies, we found p5 to be a linear b cell epitope. our data demonstrate that this epitope is highly conserved among h1 viruses (9860/10767, 91.6%). because viral mutants that are resistant to conformational epitopes are more easily generated, the conserved linear epitope is more suitable for differentiating subtypes than conformational epitopes [33] . hence, the epitope in p5 provides a new target for reliable diagnostics of h1-subtype iavs. antigenic sites in iav ha proteins of h1, h2, and h3 subtypes had previously been characterized by sequence analysis on antigenic variants and amino acid substitutions. these previously identified antigenic sites were mainly located in the globular head in the three-dimensional structure of the ha1 subunit of the ha molecule [36, 37, 38, 39] . for instance, five antigenic sites have been identified in ha of influenza virus a/pr/8/34, a well-known reference strain of h1n1 iav [39] . recently, several epitopes were identified in the ha2 unit [34, 40, 41] . together with these reports, our results indicate that there are more epitopes than what we have imaged and the epitopes of iav need to be further characterized. the difference between our findings and previously identified epitopes can be explained by the difference of screening method used between our study and those of others. in previous studies, monoclonal antibodies from murine hybridoma cells were used to identify antigenic sites, while in this study we used a peptide scanning approach, which involves overlapping peptide library and human convalescent antisera-a strategy that is widely used for viral epitope identification [27, 42] . given the fact that viral antigen can be recycled and presented as short peptides with different conformation during humoral immune response and these short peptides can be selected by b cell clones [43] , and that convalescent sera from patients were much more complex than monoclonal antibodies from mice and can reflect the real immune responses during viral infection [44] , our approach adds to the available techniques currently being used to identify linear epitopes in serologic tests. because ha pseudotyped lentivirus has been widely applied in the study on neutralizing antibodies against iavs [30] , we used this method to evaluate if the epitopes identified in this study could stimulate neutralizing antibodies. our data showed that these epitopes could not elicit neutralizing antibodies in pseudovirion neutralizing assays due to their linear nature. previous studies have shown that most neutralizing epitopes are conformation dependent [34, 35] . the length of b cell epitopes can vary from 5 to 20 amino acids [45, 46] . to map the epitope contained in p5, we performed a peptide-inhibition elisa using a series of n-terminal and cterminal truncated peptides. however, we found that the fulllength p5 (15 aa in length) rather than truncated peptides showed strongest binding to the corresponding antibody ( fig. 5 and fig. 6 ). as peptides n10 and c12 are the shortest truncated p5 that can bind to anti-p5 antibody and share a core sequence of lrgvapl, we tested whether this sequence could be the epitope. however, the synthetic peptide lrgvapl did not block p5-antibody interactions nor bind p5 antibody (fig. 6) . thus, we speculate that adjacent amino acids to this sequence are also involved in the binding of antibody elicited by p5-klh conjugates. the concept of using linear epitopes in influenza virus diagnostics and control has not been extensively investigated. in a recent study, an epitope-blocking elisa, which can universally detect antibodies to human h5n1 virus, has been developed [17] . our results show that a peptide-elisa based on the highly conserved h1 subtype-specific epitope can also be used for the detection of h1 antibodies, displaying good correlativity with the hi test. our results indicate the potential of the p5 epitope in h1subtype iav diagnosis. however, the performance of this assay needs to be further evaluated in studies with large scale samples. in conclusion, our data provide evidence that the h1 subtype ha harbors more epitopes than what has been found previously. the conservation of an epitope (p5, aa 58-72) in the h1-subtype ha of iav and its near complete absence in other subtypes indicate that this epitope meets the critical requirements for diagnosis of h1 subtype influenza virus infections. the peptide-elisa developed in our study may be applicable for serodiagnosis and may serve as a universal diagnostic tool for h1subtype iav surveillance. to screen the h1-subtype specific epitopes, a set of 50 peptides spanning the amino acid sequences of the ha protein ectodomain of pandemic a/h1n1 2009 (h1n1pdm) influenza virus strain a/ california/04/2009 were synthesized. each peptide is 15 amino acids in length with five residues overlapping with the adjacent peptides [46] (fig. 1) . the peptides selected for immunization experiments are shown in table 1 . these peptides were conjugated with a carrier protein, the keyhole limpet hemocyanin (klh; sigma, st. louis, mo), to improve their immunogenicity [47] . as the water solubility of peptides p5, p15, p16 and p31 were too low to conjugate with klh directly, these peptides were first linked to 6-aminocaproic acid and then to the tripeptide kkc prior to being conjugated with klh [48] . in addition, a family of short peptide homologs to the p5 peptide was also synthesized to fine map the epitope contained in the p5 peptide (fig. 5) . all of the peptides and their conjugates used in this study were synthesized by sangon (shanghai) biotechnol co., ltd. (shanghai, china). each peptide was purified to homogeneity (.95% purity) by highperformance liquid chromatography and verified by mass spectrometry. the reference influenza virus strains a/pr8/34 (h1n1) (abbreviated pr8), b/hubeiwujiagang/158/2009 (yamagata lineage, abbreviated by) and b/heilongjianghulan/116/2010 (vic-toria lineage, abbreviated bv) were kindly provided by the beijing center for disease control and prevention. the viruses were grown in mdck cells as described elsewhere [5] . the titers of virus strains were determined by hemagglutination tests and expressed as hemagglutinating units (hau). for western blot analysis, the inactivated viruses were lyzed in a lysis buffer (50 mm tris-hcl, 150 mm nacl, 5 mm edta, 1% triton-x, ph 7.4) supplemented with a protease inhibitor cocktail (roche, indianapolis, in). serum samples were collected from 11 convalescent patients during the early 2009 h1n1 pandemic in beijing. the diagnostic criteria for h1n1 influenza virus infection of these patients fully followed the who's descriptions. sera from 10 healthy blood donors were used as negative controls. in addition, serum samples collected from 100 blood donors were recruited to evaluate the peptide-elisa assay developed in this study. all these samples were kindly provided by the beijing municipal center for disease control and prevention (beijing, china) and written informed consent was obtained from all participants. all samples were coded prior to analysis to ensure anonymity and the procedures were approved by the institutional medical ethic review board of the institute of pathogen biology, chinese academy of medical sciences (beijing, china). the full-length cdna fragments corresponding to h2-h16 ha subtypes of iav were inserted into the pcaggs vector (purchased from addgene) to express entire ha proteins (unpublished data). for h1 proteins, ha gene representing human iav strains from different years (1918, 1934, 1977, 2007 and 2009 ) and a swine influenza virus strain were expressed by inserting the corresponding cdna fragments into the pcaggs vector in a similar manner. for the details of these influenza virus strains, please refer to fig. 4 . recombinant plasmids were transfected into 293t cells (atcc number crl-11268) using lipofectamine 2000 (invitrogen, carlsbad, ca) according to the manufacturer's instructions. the cells were harvested and lyzed 72 hours after transfection. the expression of ha proteins was verified by western blot analysis using murine antibodies against corresponding ha1 proteins (unpublished data). the reactivities of the synthetic ha peptides or purified ha0 protein (eenzyme llc, montgomery village, md) with the convalescent-phase sera from h1n1pdm patients and the serum samples from mice immunized with peptide conjugates were determined by elisa. briefly, each peptide (1 mg/well) or protein (0.1 mg/well) was used to coat 96-well microtiter plates (corning costar, acton, ma) in 0.1 m carbonate buffer (ph 9.6) at 4uc overnight. after blocking with 1% bovine serum albumen (bsa), the plates were incubated with indicated diluted serum samples (human or mouse) at 37uc for 2 hr, then washed four times with pbs containing 0.1% tween 20 (pbs-t). bound igg antibodies were detected with horseradish peroxidase (hrp)-conjugated goat anti-human igg or anti-mouse igg (sigma) at 37uc for 1 hr. after four washes with pbs-t, the reaction was visualized by addition of the substrate 3,39,5,59-tetramethylbenzidine (tmb, sigma). color development was stopped by the addition of 50 ml/well of 2 m sulphuric acid after 15 min. the optical density at 450 nm (od 450 nm ) was measured by an elisa plate reader (molecular devices, sunnyvale, california). to evaluate the reactivity of the p5-derived short peptides with the anti-p5 antibody, peptide-inhibition elisa assays were performed by adding dilutions of the peptides to a constant amount of the antibody elicited by the p5-klh conjugates (1:5000 dilution). maximum binding to antigen-coated wells was observed in the absence of a peptide inhibitor. the antibody bound was expressed as a percentage of the maximum level of binding. female balb/c mice of 6-8 weeks old were immunized subcutaneously with various peptide-klh conjugates mixed with freund's complete adjuvant (sigma) at 100 mg per injection. boost injections were given at 2-week intervals with 50 mg antigen in freund's incomplete adjuvant (sigma) [49] . the antibodies were collected five days after the third boost. all the animal experiments were carried out in the facilities of the institute of laboratory animal sciences (ilas), chinese academy of medical sciences (cams). all the experimental procedures were approved (permit number slxkj2009-0007) and supervised by the animal protection and usage committee of ilas, cams. at 72 hr post-transfection, the cells transfected with haexpressing plasmids were harvested, pelleted, and lyzed in a lysis buffer (50 mm tris-hcl, 150 mm nacl, 5 mm edta, 1% triton-x, ph 7.4) supplemented with a protease inhibitor cocktail (roche, indianapolis, in). aliquots of cell lysates (50 mg) or virus lysates were blotted after 10% sds-page onto nitrocellulose membranes (pall, port washington, ny). the membranes were blocked with 5% non-fat milk and then incubated with the primary antibodies indicated in the figures at 4uc overnight. this was followed by incubation with the goat anti-mouse irdyeh fluor 800-labeled igg secondary antibody (1:10, 000) (li-cor, lincoln, ne). after washing, the membranes were scanned by the odysseyh infrared imaging system (li-cor) and analyzed with odyssey software. the molecular sizes of the developed proteins were determined by comparison with the pre-stained protein markers (fermentas, maryland, ca). hi test was carried out as described elsewhere [5] . rde treated serum samples were inactivated at 56uc for 30 min and two-fold serially diluted at an initial dilution of 1:10. twenty five ml of the diluted serum were incubated with 25 ml of the four hemagglutination units from reference influenza strains for 30 min at room temperature. the reference h1n1 iav strains for hi test were a/ tianjinjinnan/15/2009(h1n1) and a/california/04/2009 (h1n1), respectively. 50 ml of 1% chicken erythrocyte suspension was added to each well and incubated for 30 min at 4uc. positive reactions were recorded when the hi antibody titer was equal to or greater than 40. h1n1pdm virus pseudotyped lentiviruses were produced in 293t cells co-transfected with pnl4.3-r 2 e 2 , ha and na constructs using a polyethylenimine (pei)-based transfection protocol [50] . cell culture supernatants were collected 48 hr post-transfection, filtered through a 0.45 mm-pore size filter (millipore, billerica, ma ) and used in pseudotype neutralization test. serum samples, heat inactivated at 56uc for 30 min, were diluted 40-fold in culture medium and mixed with an equal volume of diluted h1n1pdm influenza pseudovirions. after incubation at 37uc for 1 hr, 100 ml of pseudovirions (containing 50 ng/ml of hiv p24 gag protein) and serum mixtures were added into 96-well plates that contained 293t cells grown for 24 hr at initial 1610 4 cells. infectivity was evaluated at 72 hr post-infection by luciferase assay. the percentage of infectivity of pseudovirions treated by tested serum to that of negative serum (as control) was calculated. 90% reduction in infectivity by serum addition is considered to be neutralizing activity [30] . tests were run at least as duplicates. to assess the identity of the ha epitopes in iavs, in silico analysis was performed by utilizing bioinformatics tools at the influenza research database (http://www.fludb.org) [51] . the two programs used in this study were search for protein sequences and identify short peptides in flu proteins. the former program was used to define the number of total sequences in ha proteins according to the subtype parameter (h1 or h2-h16). the latter program defined the number of hits (p5 or p31) in the h1 or h2-h16 total sequences. because there are no standards for evaluating short peptide sequence homology, we chose the fuzzy match analysis to represent the identical level of a peptide sequence to ha proteins. the analysis type was chosen as fuzzy match, which meant .50% of characters were identical to the searched aa string. for example, entering gilgfvftl may also find ailgfvfti but not aligfvfsi. the pearson correlation coefficient was calculated by pearson chi square test for crosstab tables using spss software. the sensitivity and specificity of the peptide-elisa versus hi test was determined by roc curve analysis using spss software. figure s1 localization comparison between the identified peptides and the classical five antigenic sites in stereo view. the ha monomer surface view of influenza virus a/pr/8/34 (pdb id:1ru7) is shown and colored to illustrate the five antigenic sites (sa, sb, ca1, ca2, and cb) and the identified peptides. from most membrane distal to proximal: p3 (blue), p31 (red), p5 (black), cb (green), ca1 (magenta), ca2 (rainbow), sa (yellow), and sb (cyan). (tif) influenza: lessons from past pandemics, warnings from current incidents influenza: old and new threats the origins of pandemic influenza-lessons from the 1918 virus the 2009 influenza a (h1n1) pandemic: what have we learned in the past 6 months fields virology: orthomyxoviridae. 5th edn characterization of a novel influenza a virus hemagglutinin subtype (h16) obtained from black-headed gulls continuing evolution of h9n2 influenza viruses in southeastern china avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome avian influenza a (h5n1) receptor binding and membrane fusion in virus entry: the influenza hemagglutinin the structure and function of the hemagglutinin membrane glycoprotein of influenza virus influenza vaccines for the future characterization of a novel influenza hemagglutinin, h15: criteria for determination of influenza a subtypes manual of diagnostic tests and vaccines for terrestrial animals development of blocking elisa for detection of antibodies against avian influenza virus of the h7 subtype development of epitope-blocking elisa for universal detection of antibodies to human h5n1 influenza viruses a laboratory manual for the isolation and identification of avian pathogens comparison of the hemagglutination-inhibiting and neutralizing antibody responses of volunteers given 400 chick cellagglutinating units of influenza a/new jersey/76 split-virus vaccine role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics development of rha1-elisa for specific and sensitive detection of h9 subtype influenza virus prokaryotic expression and purification of ha1 and ha2 polypeptides for serological analysis of the 2009 pandemic h1n1 influenza virus utility of pandemic h1n1 2009 influenza virus recombinant hemagglutinin protein-based enzymelinked immunosorbent assay for serosurveillance ab and t cell epitopes of influenza a virus, knowledge and opportunities epitope analysis for influenza vaccine design quantifying influenza vaccine efficacy and antigenic distance antigenic characterization of recombinant hemagglutinin proteins derived from different avian influenza virus subtypes epitope mapping of the hemagglutinin molecule of a highly pathogenic h5n1 influenza virus by using monoclonal antibodies evaluation of the subtype specificity of monoclonal antibodies raised against h1 and h3 subtypes of human influenza a virus hemagglutinins establishment of retroviral pseudotypes with influenza hemagglutinins from h1, h3, and h5 subtypes for sensitive and specific detection of neutralizing antibodies minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin one step closer to universal influenza epitopes structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses crossprotective potential of a novel monoclonal antibody directed against antigenic site b of the hemagglutinin of influenza a viruses antigenic structure of the haemagglutinin of human influenza a/h2n2 virus structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution structural identification of the antibodybinding sites of hong kong influenza haemagglutinin and their involvement in antigenic variation the antigenic structure of the influenza virus a/pr/8/34 hemagglutinin (h1 subtype) a simple slot blot for the detection of virtually all subtypes of the influenza a viral hemagglutinins using universal antibodies targeting the fusion peptide monoclonal antibodies against the fusion peptide of hemagglutinin protect mice from lethal influenza a virus h5n1 infection identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus the who, how and where of antigen presentation to b cells screening of random peptide library of hemagglutinin from pandemic 2009 a(h1n1) influenza virus reveals unexpected antigenically important regions predicting flexible length linear b-cell epitopes b cell epitope mapping using synthetic peptides approaches to augmenting the immunogenicity of melanoma gangliosides: from whole melanoma cells to ganglioside-klh conjugate vaccines universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza a viral hemagglutinins production of antipeptide antisera analysis of hemagglutinin-mediated entry tropism of h5n1 avian influenza biohealthbase: informatics support in the elucidation of influenza virus host pathogen interactions and virulence understanding sensitivity and specificity with the right side of the brain we thank dr. fang huang for her assistance in serum sample collection. key: cord-003870-hr99dwi7 authors: clohisey, sara; baillie, john kenneth title: host susceptibility to severe influenza a virus infection date: 2019-09-05 journal: crit care doi: 10.1186/s13054-019-2566-7 sha: doc_id: 3870 cord_uid: hr99dwi7 most people exposed to a new flu virus do not notice any symptoms. a small minority develops critical illness. some of this extremely broad variation in susceptibility is explained by the size of the initial inoculum or the influenza exposure history of the individual; some is explained by generic host factors, such as frailty, that decrease resilience following any systemic insult. some demographic factors (pregnancy, obesity, and advanced age) appear to confer a more specific susceptibility to severe illness following infection with influenza viruses. as with other infectious diseases, a substantial component of susceptibility is determined by host genetics. several genetic susceptibility variants have now been reported with varying levels of evidence. susceptible hosts may have impaired intracellular controls of viral replication (e.g. ifitm3, tmprs22 variants), defective interferon responses (e.g. gldc, irf7/9 variants), or defects in cell-mediated immunity with increased baseline levels of systemic inflammation (obesity, pregnancy, advanced age). these mechanisms may explain the prolonged viral replication reported in critically ill patients with influenza: patients with life-threatening disease are, by definition, abnormal hosts. understanding these molecular mechanisms of susceptibility may in the future enable the design of host-directed therapies to promote resilience. the normal response to infection with influenza a virus (iav) is to remain asymptomatic. during the 2009/2010 pandemic, serosurveillance studies revealed that a majority of volunteers who tested positive for antibodies to the new h1n1pdm09 virus did not report any symptoms [1] . the majority of people newly exposed to one of the most dangerous viruses to circulate in human populations in recent history, which in the same population created an overwhelming burden of critical illness [2] , did not notice any symptoms. wide variation in susceptibility is a general feature of human and animal populations exposed to any pathogen [3] . explaining the mechanisms of susceptibility may enable effective targeting of vaccine therapies, may reveal new therapeutic approaches [4, 5] , and, in theory, may contribute to future clinical risk prediction models. as with any infectious disease in a given host, the site of infection, the scale of the initial exposure, and the virulence, degree of pathogenicity, of the pathogen determine the nature of the disease in iav infection. although the alimentary tract is a common site of infection in other species (for example the natural hosts, water fowl [6] ), initial infection in humans is through the respiratory tract. the number of viable iav virions transmitted has a direct effect on the probability of symptoms, both in animal models [7] and human challenge studies [8] . this may explain a proportion of the variation in individual responses to the virus. the virulence of the virus itself varies greatly. perhaps fortunately, there is a general trend for the most virulent iav strains to be less transmissible; that is, those that cause the most severe disease are less likely to be passed on to others. while highly transmissible iav strains, such as h1n1pdm09, replicate well in the upper respiratory tract, viruses associated with higher rates of severe disease, such as h5n1 and h7n9 avian iav, exhibit tropism for the lower respiratory tract [9, 10] . within a given strain, not all iav viruses are the same. in fact, it is statistically unlikely that any two iav virus particles will have exactly the same genome sequence. small changes, such as a single amino acid change in the hemagglutinin protein, can significantly alter the tropism of the virus for example, increasing the likelihood of spread to the lower respiratory tract and establishing a more severe infection [11] . iav viruses change rapidly by two mechanisms: shift and drift. shift is the exchange of viral segments between strains, occasionally resulting in a new iav subtype to which a large proportion of the population does not have existing immunity. this shuffling of the viral genes contributes to the sudden and dramatic change in virulence that may occur from season to season, and to zoonoses, as iav jumps from its natural avian host to mammalian pig and human hosts. drift refers the accumulation of small mutations in the viral genome that occur on a continuum. because of the short genome (around 13,500 bases of rna are carried by a functional virion particle) and a very high error rate when this genome is replicated [12, 13] , viral quasispecies arise, leading to a heterogenous swarm of virions [14] . this variation enables iav to evolve extremely rapidly where a selective pressure exists. for example, it is likely that iav can evolve de novo resistance to antivirals during treatment of a single patient [15] [16] [17] . studies of viral whole genome sequence during outbreaks have failed to identify consistent viral factors associated with severe disease [18] . it is therefore likely that viral factors do not explain the vast spectrum of variation observed in the disease. variation attributable to the host previous exposure to iav due to the remarkable memory of the adaptive and innate immune systems, previous exposure to iav has a strong effect on future susceptibility. adaptive immune memory is highly strain-specific and provides targeted antibodymediated defence against iav [19] . the first iav strain to which a child is exposed has a profound effect on subsequent immunity-a concept known as original antigenic sin [20] . the host immune system is extensively programmed by this first iav exposure, such that the susceptibility of whole populations of adults can be predicted using the patterns of circulating iav in each patient's year of birth [21] . this has been proposed as one reason why the burden of mortality for the 2009/2010 outbreak was shifted towards patients younger than 65 years of age [22] -patients over 65 years old are more likely to have been exposed at a young age to an iav strain similar to the h1n1pdm09 strain, and were hence protected. interestingly, the lifelong immunity provided by this first iav exposure has broad protective effects against different iav strains [21] . cell-mediated immunity may play an important role in this protection. an iav challenge study in healthy volunteers found that preexisting cd4(+) t cell responses to iav nucleoprotein and matrix proteins were present prior to infection [23] . the magnitude of this cd4(+) t cell response when challenged correlated with reduced symptoms and reduced virus shedding. regardless of prior exposure, the most reliably quantified risk factors for life-threatening seasonal and pandemic iav are advanced age (> 65 years), obesity, immunosuppression, cardiovascular disease, and neuromuscular disease [24] . a number of well-recognised host factorsbest summarised by the broadly understood but poorly defined term, "physiological reserve"-increase the chance of organ failure and death following any severe injury or infection. these factors are extensively discussed elsewhere in the critical care literature; here, we focus on host factors that are thought to confer some element of specific susceptibility to iav (fig. 1) . studies dating back to the 1918-1919 pandemic have suggested that pregnancy, particularly in the third trimester, increases the risk of death from iav [25] . additionally, pregnant women have a higher rate of hospitalisation with seasonal iav [26] . however, in the largest systematic review of clinical risk factors for iav, pregnancy was not fig. 1 conceptual visualisation of variation in specificity of host susceptibility factors. factors predicted to confer more specific susceptibility to influenza are placed higher in the diagram independently associated with severe disease from either seasonal or pandemic iav [24] . the immunological changes that occur in pregnancy are theoretically compatible with increased severity of iav: in particular, an increase in innate immune activation and a decrease in the number and activity of cells associated with cytotoxic immunity-in which infected cells are killed to limit the dissemination of the virus [27] . these changes may lead to an increased propensity to develop ards [28] and a decreased ability to eliminate iav-infected cells, which is a core component of anti-iav immunity. some indices of severity used in epidemiological studies are themselves directly affected by pregnancy. the cardiovascular adaptations to pregnancy, combined with an increased metabolic rate, a decrease in functional residual capacity, and increased basal ventilation to perfusion mismatch, are expected to worsen hypoxaemic respiratory failure following any insult. in parallel, admission to hospital or critical care may be in part biased by elevated concern for a pregnant patient, and by the perception of high risk of severe iav [29] . obesity was identified as a risk factor for iav infection over a decade ago and confirmed during the swine flu pandemic [30, 31] when it was associated with an increased risk of death [32] . although comorbidities associated with obesity-specifically diabetes mellitus and cardiovascular disease-compromise pulmonary host defence and increase the chance of death following any severe systemic injury [33] , an independent association between obesity and severe iav is robust and replicated [24] . in parallel with the immune changes associated with pregnancy, obese patients are more likely to have impaired cell-mediated immunity and excessive chronic activation of the innate immune system [34] . this is reflected in a study which demonstrated that among vaccinated adults, those who are obese are more likely to suffer severe consequences of iav [35] . furthermore, it has been shown that obese adults have an impaired antibody response to iav vaccination [36] , and impaired cd4(+) and cd8(+) t cell responses iav in vitro [37] . obese patients have a prolonged period of viral replication and shedding, even in the absence of clinical disease [33] . extremes of age are well-recognised risk factors for severe disease. children under the age of 5 years, and particularly those under 2 years, have consistently been found to be at high risk for severe disease and serious complications following iav infection [38] [39] [40] . functional immaturity of the immune system, together with a failure to recognise iav-related antigens, may largely explain this effect. in industrialised countries, the group at highest risk of death from seasonal iav is those over 65 years of age [22, 41, 42] . senescence affects antiviral immunity in complex ways; it is difficult in clinical epidemiological studies to distinguish the effect of these immune changes from the effects of frailty and antigenic exposure. baseline markers of systemic inflammation are elevated [43] and circulating t cell counts are reduced. naive t cells, a key component of cell-mediated adaptive immunity, are lost from the circulation due to the process of thymic involution, which begins very early in life [44] . in mouse models of iav infection, aged mice exhibit slower antiviral and adaptive immune responses, and more severe disease [45] . expansion of clonal t cell populations, driven by cytomegalovirus (cmv), occurs in older adults and may impair t cell responses to new pathogens [46] . in contrast, in the young, a multi-omic systems analysis demonstrated that cmv infection is associated with an enhanced t cell mediated response to iav vaccination [47] . integrating systems studies of host response to iav infection with markers for genetic susceptibility (see below) may in the future reveal new biological pathways and patterns of disease [48] . as with pregnancy and obesity, ageing is associated with both an increase in the basal activation of the innate immune system (sometimes referred to as "inflammaging") and a decrease in cell-mediated immunity. this combination of mechanisms may explain the particularly strong effects on susceptibility. susceptibility to death from any infection is strongly inherited by children from their parents [49] . in iav, numerous genetic studies in humans and animal models have revealed specific genes associated with susceptibility, which are extensively reviewed elsewhere [50] [51] [52] . in addition to the specific genetic variants discussed below, there is direct evidence, from a study of death records in utah, that susceptibility to iav is heritable at a population level [53] . much of what is known about human genes associated with iav susceptibility has been discovered from lossof-function mutations in the immune system, which lead to loss of the gene product or a substantial reduction in gene function. these often lead to severe defects that are likely to present in childhood. such variants can reveal key components of the immune response to a specific infection. in considering the biological lessons from such discoveries, it is important to consider that, in most people, these components of the immune system function perfectly well and may not be suitable targets for therapy. secondly, there is little that can be inferred from the absence of any particular gene, or immune process, from the list of loss-of-function defects associated with susceptibility to iav. the conditions that must be met for such a gene to be discovered are not restricted to disease susceptibility. many variants that confer susceptibility to iav have broader pleiotropic effects that may be terminal in utero or in early life, or may lead to susceptibility to other infections or autoimmune conditions that obscure the clinical picture. alternatively, some variants may lead to strain-specific susceptibility and will only be detected following exposure to the right virus. the full range of genetic defects associated with susceptibility to iav in animal models is reviewed elsewhere [54, 55] . so far, three known human genes, all transcription factors active primarily in myeloid cells, have been found to have loss-of-function variants that increase susceptibility to iav. since transcription factors function as master regulators of large numbers of genes, functional deficiencies are expected to have broad, nonspecific effects. in 2015, ciancanelli et al. identified a patient with a mutation in the transcription factor interferon regulatory factor 7 (irf7) that led to severe infection and ards when she was 2.5 years old [56] . irf7 is a transcription factor and a key regulator of the type i interferon response. this was the first published example of a singlegene inborn error of immunity that was specific to iav. both parents were heterozygous for different loss-offunction alleles, but each had sufficient functional irf7 activity allowing them to avoid severe iav. the patient inherited these two different loss-of-function alleles (compound heterozygosity) leading to complete loss of functional irf7. leukocytes and plasmacytoid dendritic cells from this patient produced very little interferon type i (α/β) and iii (γ) in vitro indicating that expression and production of these interferons in these cell types is specifically irf7-dependent in iav infection in humans. whole exome sequencing of 20 children identified a variant in the gene encoding interferon regulatory factor 9 (irf9) in a 2-year-old child who had previously suffered from bronchitis and biliary perforation [57] . the child inherited a mutation on both alleles from consanguineous parents leading to a single change in the dna sequence (single nucleotide polymorphism, snp) in the irf9 gene. this snp occurs at an essential site leading to aberrant processing of the gene transcript and thus the expression of a truncated, functionally defective, protein product. in this case, irf9 was only partially defective. activation of interferon-stimulated gene 3 (isg 3) was impaired in response to iav infection or interferon α stimulation, but other irf9-dependent pathways remained intact. the consequence of this appears to be a global reduction in type i interferon responses, a key mechanism of early mucosal resistance to infection, in all cell types. unrestricted viral replication was observed in cells from the patient and was also shown for parainfluenza virus and respiratory syncytial virus. gata2 is a zinc finger transcription factor, part of the gata family, so named because they bind a g-a-t-a pattern (also called a motif) in dna sequence. transcription factor binding at sites bearing this motif alters the probability that a given gene will be transcribed, and ultimately controls the amount of the encoded protein that is made. gata2 deficiency results in primary immune cell deficiency and affects a wide range of cell types. decreased circulating counts of b lymphocytes, nk cells, monocytes and plasmacytoid dendritic cells have been observed, along with reduced t cell thymic output. in 2018, sologuren et al. published a case study of a father and son who contracted and subsequently died from severe iav [58] . both patients were heterozygous for a novel mutation in gata2 that led to a dysfunctional protein. despite the known effects of gata2 deficiency on primary immune development, the first, older patient had suffered few health problems prior to his 30th year, after which frequent respiratory illnesses and a single incidence of viral pneumonia is reported prior to his severe illness. the second patient had been hospitalised with pneumonia at 16 without recurrence until hospitalisation with severe iav at 31. the authors attribute protection from viral and bacterial infection observed in the lifetime of these patients to long-living memory t and b cells. genetic variants with less drastic effects on susceptibility can be detected by comparing flu-susceptible populations with control populations (table 1) . these studies generally look for candidate genes or take a genomewide approach. candidate gene association studies have a long but troubled history in human genetics. genes are selected because of some underlying hypothesis; single variants within these genes are then chosen because they are believed to have an effect on the expression or function of the gene. genotype frequencies (that is, the proportion of a population who have a given variant) at these genomic positions are then compared between a case and control group. this has the advantage of economy, since only one or two variants need to be genotyped for each participant, and has the superficial appearance of statistical efficiency, since fewer comparisons are made. the fundamental limitation is that, in a human genome composed of 3 × 10 9 bases, of which 4 − 5 × 10 6 are different between any random pair of people [59] , the probability of choosing the right base is very low. in the event that a given variant meets a nominal level of significance, the evidence for an association is easily misinterpreted. looking backwards from a single small p value, it is common to focus on the fact that probability of seeing such an association by chance alone is very low. what is easy to forget is that the probability of such an association existing is also very low. an understanding of this methodology is important for the interpretation of such studies. many of the positive studies reflect more the biases of well-informed investigators in the choice of target genes. the additional value of an unreplicated genetic association on this background is often small. lgals1 galectin-1 cell-cell interactions rs4820294 [82] rs2899292 [82] rs4820294 [82] st3gal1 (*) st3 beta-galactoside alpha-2, [85, 88] gene: gene symbol. gene name: gene name and alternate name. function: summary function of gene product. snp: snps associated with host susceptibility to influenza a associated with gene. (*) represents genes not addressed in the text nonetheless, candidate gene approaches in various forms detected numerous real and informative associations with disease before the advent of genome-wide genotyping technology [60] . we focus here on larger studies, those that have been replicated, and studies with particular relevance to the pathogenesis of severe iav. genome-wide approaches seek to eliminate the aforementioned bias. in the most widely used design, the genome-wide association study (gwas), hundreds of thousands of common variants are genotyped in each patient. this is expensive and requires correction for multiple comparisons. a widely used convention is to correct for 1 × 10 6 independent comparisons in each study, requiring a p value <5 × 10 −8 for significance. large numbers of patients are needed to detect associations at this level above the background noise of variation in human populations. however, genome-wide approaches use no preconceptions about the pathogenesis of disease. hence, such methods have the potential to teach us something that we did not already know. because of the stringent threshold for statistical significance, and the burden of multiple testing, statistical power to detect small effects is usually lacking unless many tens of thousands of patients are included. for this reason, the expected outcome is false-negatives. hence, we would caution against drawing any conclusion from the absence of significant associations within a given gene. genome-wide in vitro knockdown screens can also be used to limit bias and enable genome-wide discovery. in this approach, although a candidate gene is often selected from cell culture results and tested for genetic associations in patients, there is an important difference from single-gene candidate studies: the pool of genes from which the candidate is chosen comprises the entire protein-coding part of the genome. a role for interferon-induced transmembrane protein 3 (ifitm3) in iav replication was discovered in an in vitro genome-wide knockdown screen in cultured cells [61] . the protein product of this gene restricts iav entry by blocking the fusion of host and viral membranes [62] and acts as a restriction factor in viral infections, along with family members ifitm1 and ifitm2 [61] . ifitm proteins were also shown to inhibit the early replication of other virus types, for example the west nile virus [63] . based on this genome-wide knockdown screen, a candidate gene sequencing approach was conducted to test for an association with severe illness. the 2009/2010 pandemic provided a colossal natural experiment-a large proportion of the population were exposed to a new pathogen, but only a small minority developed lifethreatening illness requiring critical care. focusing on these previously healthy adults with life-threatening iav (in the genisis and mosaic studies) may have increased the effect size seen [64] . genotypes at every variant within the ifitm3 gene were compared with population controls, identifying a single variant (rs12252-c) associated with severe iav. this variant is rare in the european cohorts in which it was discovered, but is frequent in the han chinese cohorts hospitalised with severe h1n1pdm09 infection [65] . the association has been replicated in independent studies in different populations [66] . a second snp associated has been shown in populationlevel studies to regulate ifitm3 expression. rs34481144-a encourages the transcription factor ctcf to bind to the regulatory region of ifitm3 and repress gene expression in response to iav infection [67] . this snp can also disrupt the methylation pattern (a key modification of dna that usually silences genes) in the regulatory region leading to cell type-specific effects. ifitm3 expression in memory cd8(+) t cells in response to viral infection has been found to protect and encourage survival of these cells allowing for the establishment of adaptive immunity. loss of methylation at this site prevents ctcf from binding to the dna and inducing expression of ifitm3 in response to the pathogen, thus reducing cell survival. this is estimated to lead to a 2.6-fold increased risk of a severe outcome upon iav virus infection. ifitm3 has also been recently shown to have a protective effect on the heart during severe iav infection. myocarditis has been associated with iav infection since the 1918 pandemic [68] , and iav has been shown to lead to a sixfold increased risk of myocardial infarction in the 7 days post infection [69] . so far, ifitm3 is the only gene for which snps have been identified and independently confirmed in vivo and in vitro to restrict iav replication [70] . however, this gene is not specific to iav replication and the full extent of the antiviral actions remains to be discovered. unbound complement is rapidly inactivated in plasma. where this process is defective, uncontrolled complement activation can damage host cells. cd55 prevents the formation and accelerates the decay of c3 and c5 convertases. these proteases are part of the complement system and have roles in opsonisation and the release of inflammatory molecules. cd55 polymorphisms were associated with severe h1n1pdm09 infection (defined as requiring supplementary oxygen, admission to intensive care or death) [71] . this study found carriers of the rs2564978-t/t polymorphism had significantly lower levels of surface cd55 on their circulating monocytes compared to the more common c allele. further work identified a deletion in a nearby regulatory region as the element responsible for the specific effect on both protein and mrna levels of cd55 in monocytes. a more recent study of han chinese individuals that looked at several genes confirmed an association between cd55 rs2564978 t/t and death from severe iav infection [72] . the cumulative effects of multiple snps (ifitm3, cd55, and the immune cell receptors tlr3 and tlr4) on iav susceptibility have been examined in a targeted study [72] . this independently confirmed the association of the cd55 rs2564978 polymorphism with severity, and the ifitm3 rs12252-c and tlr3 rs5743313-cc genotypes were both over represented in fatal cases. in a small-scale pilot study, genome-wide genotypes of 42 patients with severe iav were compared to 42 controls with mild iav. the rs2070788-g allele of tmprs22 was significantly overrepresented in severe compared with mild cases of h1n1pdm09, with a > 2fold higher risk of severe infection. there was higher tmprs22 expression in human lung tissues with the high-risk gg genotype [73] . this was replicated in a targeted study of 162 severe and 247 mild iav patients. this genetic association in humans is highly biologically plausible: tmprs22 has been shown to play a role in haemagluttinin cleavage, an important step in iav replication. additionally, mice lacking this gene are strongly protected from iav infection [74] [75] [76] . this genome-wide array also identified a snp in pulmonary-surfactant-associated protein b (sp-b), rs1130866, as a potential association. this snp was genotyped in a targeted study of 111 severe and 185 mild iav patients to replicate the finding [77] . again, this is a plausible association with severe disease: sp-b forms a key part of pulmonary surfactant and is essential for lung function. a subset of the same protein family, sp-a and sp-d, have been shown to initiate and enhance immune cell ingestion and killing (opsonisation) of pathogens and play a role in the progression of iav in mice [78] . a polymorphism associated with sp-b, rs1130866 [77] , has also been associated with copd in several cohorts [79] . susceptibility to severe h1n1 infection was analysed in a recent genome-wide study (integrated with data on genetic variants associated with altered gene expression) which implicated an intronic snp of gldc, rs1755609-g [80] . the gldc gene encodes glycine decarboxylase, also known as the p protein of the glycine cleavage system, a pathway in glycine metabolism [81] . the association was replicated by targeted genotyping in a larger cohort of 174 patients suffering severe iav infection and 258 mildly infected controls. the risk variant corresponds to higher gldc expression in lymphoblastoid cell lines and human lung tissues. consistent with this effect, inhibition of gldc in cultured bronchial epithelium using sirna or a specific inhibitor, aminooxyacetic acid (aoaa), leads to an increased type i ifn response and a restriction of viral replication in vitro. this effect on viral restriction was seen with both h1n1 and h7n9, and the allele genotype was replicated in susceptibility cohorts for both viruses. the protective effect of aoaa against h1n1 was shown in mice to be comparable with that of zanamivir. susceptibility to severe h7n9 was examined in a gwas performed with 102 patients and 106 controls who worked with poultry. this study identified rs13057866, associated with galectin-1 (lgals1), as a potential susceptibility factor. lgals1 is a lectin that may have a role in modulating cell-cell and cell-matrix interactions. the study further demonstrated that genetic variants of lgals1, including rs4820294 and rs13057866, lead to higher expression of lgals1 protein in human cells, possibly leading to a protective effect. carriers of the rs4820294/rs2899292 gg haplotype were found to have higher lgals1 protein in lymphoblastoid cells and expression levels of lgals1 in human lung correlated with the rs4820294 snp [82] . the role of host factors in susceptibility suggests a clinically important conclusion: there is something unusual about the small minority of patients who develop critical illness following iav. therefore, extrapolating from human challenge and primary care studies of viral clearance is very likely to lead to error. viral clearance among critically ill patients is slow and incomplete [83] . hence, the critically ill population should be regarded-by 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otherwise healthy children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no competing interests. key: cord-003498-4ct0ywnw authors: bdeir, najat; arora, prerna; gärtner, sabine; hoffmann, markus; reichl, udo; pöhlmann, stefan; winkler, michael title: a system for production of defective interfering particles in the absence of infectious influenza a virus date: 2019-03-01 journal: plos one doi: 10.1371/journal.pone.0212757 sha: doc_id: 3498 cord_uid: 4ct0ywnw influenza a virus (iav) infection poses a serious health threat and novel antiviral strategies are needed. defective interfering particles (dips) can be generated in iav infected cells due to errors of the viral polymerase and may suppress spread of wild type (wt) virus. the antiviral activity of dips is exerted by a di genomic rna segment that usually contains a large deletion and suppresses amplification of wt segments, potentially by competing for cellular and viral resources. di-244 is a naturally occurring prototypic segment 1-derived di rna in which most of the pb2 open reading frame has been deleted and which is currently developed for antiviral therapy. at present, coinfection with wt virus is required for production of di-244 particles which raises concerns regarding biosafety and may complicate interpretation of research results. here, we show that cocultures of 293t and mdck cell lines stably expressing codon optimized pb2 allow production of di-244 particles solely from plasmids and in the absence of helper virus. moreover, we demonstrate that infectivity of these particles can be quantified using mdck-pb2 cells. finally, we report that the di-244 particles produced in this novel system exert potent antiviral activity against h1n1 and h3n2 iav but not against the unrelated vesicular stomatitis virus. this is the first report of dip production in the absence of infectious iav and may spur efforts to develop dips for antiviral therapy. influenza a virus infection is responsible for annual influenza epidemics and intermittent pandemics that are associated with significant morbidity and mortality [1] . the ability of iav to constantly change in response to immune pressure or antiviral treatment limits the effectiveness of currently used antiviral interventions. thus, vaccines against seasonal influenza need to be annually reformulated and will provide little if any protection against pandemic influenza [1] . moreover, the effectiveness of antivirals targeting the viral proteins m2 and neuraminidase a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 is compromised by the frequent emergence and transmission of resistance mutations [1, 2] . therefore, novel approaches to combat influenza are urgently needed. iavs are enveloped and harbor eight segments of genomic viral rna. defective interfering (di) genomic segments can be generated in iav infected cells due to errors of the viral polymerase [3, 4] . di segments usually harbor a large deletion which inactivates the open reading frame encoded by the segment [3, 4] . the di segments can interfere with amplification of wild type (wt) segments, potentially by competing for viral and cellular resources required for segment replication. moreover, di rnas can be packaged into progeny virions, termed defective interfering particles (dips), and coinfection of target cells with dips and iav will result in preferential amplification of dips and suppression of iav spread [3, 4] . this effect has been observed in cell culture [5] [6] [7] [8] and in experimentally infected animals [5, [9] [10] [11] [12] [13] [14] [15] and may extend to unrelated viruses [14, 16] , due to the activation of the interferon system [15, 16] . moreover, dip application in a therapeutic or preventive setting prevents or ameliorates influenza in animal models [3] [4] [5] [10] [11] [12] [13] [14] [15] [16] . in sum, dips can be considered natural antivirals produced in the context of infection with iav and many other viruses and may provide a basis for the development of new strategies for antiviral intervention. at present, amplification of dips requires coinfection of cells with dips and wt virus, termed standard or helper virus, which subsequently needs to be inactivated by uv light [3, 4, 17, 18] . the presence of standard virus poses a safety concern when products for animal and human use are generated and complicates the interpretation of experimental data. plasmid systems encoding for wt and di segments along with cell lines expressing the iav proteins for which the genomic information has been lost upon di rna formation might circumvent this issue [4, 19] . however, expression of the viral polymerase subunit pb2 in trans was found to be insufficient for robust amplification of iav variants harboring temperature sensitive mutations [20, 21] and it has been speculated that similar limitations might apply to the production of dips [4] . moreover, it has been suggested that pb2 expression might be toxic to cells [4] . therefore, it is currently unknown whether the strategy outlined above might allow for production of segment 1-derived dips and at present no system for generation of dips in the absence of standard virus has been reported. di-244 is a naturally occurring di-rna found in hen's eggs [22] . di-244 is derived from segment 1, which encodes pb2, and harbors a 1,946 nucleotides comprising deletion [4, 22] . this deletion removes most of the pb2 orf but leaves the 3' 244 nucleotides and 5' 151 nucleotides of segment 1 intact which are sufficient for segment replication and packaging [4, 22] . here, we investigated whether coexpression of wt segments 2-8, pb2 protein and di-244 rna allows for production of dips. employing a novel di-244 variant encoding mscarlet-i, we show that di-244-based dips are efficiently produced in cells expressing a codon optimized version of pb2 and that these dips exert potent antiviral activity. plasmids for rescue of the a/pr/8/34 (h1n1) strain, phw191-phw198, were used throughout this study and have been previously described [23] . to generate a retroviral vector encoding pb2, the pb2 open reading frame was amplified from phw191 using primers pb2-qcx ip-5n (5-ccgcggccgcaccatggaaagaataaaagaactac-3) and pb2-3xbgl (5-gg agatctcgagctaattgatggccatccgaat-3) and cloned into the retroviral vector pqcxip-mcs using noti and xhoi [24] . this self-inactivating vector allows constitutive expression of pb2 and puromycin resistance genes coupled by an internal ribosome entry site (ires). an optimized sequence of pb2 was generated by hand to maximize sequence deviation from pb2 and optimizing codon usage for influenza a virus and humans (s1 fig). this sequence was synthesized and cloned by geneart (regensburg, germany) and subcloned using noti and xhoi sites into pqcxip-mcs. a plasmid for di-244 rescue was generated by splice overlap pcr, using phw191 as template and primer pairs flua aari-pb2-1g (5-cga tcacctgctcgagggagcgaaagcaggtc-3)/iavseg1-di244rep-rev (5-aatgaggaa tcccctcagttaagcggccgctgcggtaccagatctcttctcctgtcttcctga-3) and iavseg1-di244rep-for (5-tcaggaagacaggagaagagatctggtaccgcagcggccgct taactgaggggattcctcatt-3)/flua aari-pb1-2341r (5-cgatcacctgc tctctat tagtagaaacaaggcattt-3). the product of the splice overlap pcr was then purified and amplified with the segment specific primer pair flua aari-pb2-1g/flua aari-pb1-2341r and cloned into phw2000-ggaari, using golden gate cloning, generating phw2000-di244-mcs [25] . in addition, a construct containing a multiple cloning site (mcs) was generated for later insertion of reporter genes. for this, the pcr fragments were amplified using phw191 as template and primer pairs flua aari-pb2-1g/iavseg1-di244rep-rev (5-aatgaggaatcccct cagttaagcggccgctgcggtaccagatctcttctcctgtcttcc tga-3) and iavseg1-di244rep-for (5-tcaggaagacaggagaagagatctggtaccgca gcggccgcttaactg aggggattcctcatt-3)/ flua aari-pb1-2341r followed by splice overlap joining and golden gate cloning. as reporter gene, mscarlet-i without internal sali and noti sites and fused to the porcine teschovirus-1 (ptv1) 2a sequence (gatnfsllkqagdveenpgp) was cloned into the mcs as a bglii/noti fragment. in this way, a pb2 (aa 1-41)-2a-mscarlet-i orf was generated, which allows the detection of the presence of di-244 via mscarlet-i fluorescence. the template for mscarlet-i, pmscarlet-i_c1, was a gift from dorus gadella (addgene plasmid # 85044) [26] . the integrity of pcr-amplified, cloned sequences was verified by sequence analysis. all cells were cultured at 37˚c and 5% co 2 . 293t human embryonic kidney cells and vero cells were maintained in dulbecco's modified eagle medium (dmem; gibco) containing 10% fetal bovine serum (fbs, gibco), penicillin (pen, 100 iu/ml) and streptomycin (strep, 100 μg/ ml). 293t cell lines stably expressing pb2 were grown in the presence of 1 μg/ml puromycin. madin-darby canine kidney cells (mdck) were cultured in glasgow's mem (gmem) with 10% fetal bovine serum (fbs, gibco) and pen/strep. all cell lines were obtained from collaborators and were regularly checked for mycoplasma contamination. mdck cells stably expressing pb2 or pb2opt were cultivated in the presence of 1.5 μg/ml puromycin. influenza a viruses a/panama/2007/99 (h3n2) [24] and a/pr/8/34 (h1n1) produced in embryonated chicken eggs were used to assess the antiviral activity of dips. we further employed a recombinant vesicular stomatitis virus (vsv) that expresses a dual reporter consisting of egfp and firefly luciferase from an additional transcription unit located between the open reading frames for the viral glycoprotein and polymerase [27] . the production of mlv particles for transduction of cells followed an established protocol [25, 28] . briefly, 293t cells seeded in t25 flasks were transfected with 6 μg of retroviral vector (e.g. pqcxip-pb2), 3 μg mlv-gag-pol plasmid and 3 μg vsv-g expression plasmid, employing the calcium phosphate transfection method. the culture medium was exchanged at 8 h after transfection. after 48 h, mlv particle-containing supernatant was harvested, cleared by passing through a 0.45 μm filter, aliquoted and then stored at -80˚c. for retroviral transduction, cells were seeded in 96-well plates at 5,000 (mdck) or 10,000 (293t) cells/well in 50 μl cell culture medium. on the next day, 50 μl of supernatant containing mlv particles was added per well followed by spinoculation at 4,000 × g for 30 min for enhancement of transduction [29] . two days after transduction, the cells were detached and transferred into 24-well plates containing cell culture medium supplemented with 1 μg/ml (293t) and 1.5 μg/ml (mdck) puromycin. in parallel, non-transduced cells were treated similarly to control for effective cell killing by the antibiotics. 293t were seeded at a cell density of 2 × 10 5 cells/well in 12-well plates. the following day, the cells were transfected using the calcium phosphate method. the concentrations of plasmids to be transfected were largely adapted from published work [30] : 10 ng of pcaggs plasmids encoding viral rna polymerase proteins (pb2, pb, pa) and 100 ng of plasmid encoding np were cotransfected with 50 ng of plasmid ppoli-luc, which encodes the firefly luciferase reporter gene flanked by the noncoding regions of segment 8 of a/wsn/33. empty plasmid was used to ensure that all transfections were conducted with the same total amount of plasmid dna. for analysis of functionality of pb2 in 293t cells stably expressing this protein, transfection was carried out as described above but the plasmid encoding pb2 was omitted. as control, the plasmid encoding pb1 was omitted. the cells were washed at 6-8 h after transfection and harvested at 24 h post transfection. luciferase activities in cell lysates were measured using the plate chameleon v plate reader (hidex) and microwin 2000 software. for analysis of pb2 expression in 293t and mdck cells, the cells were seeded in 6-well plates, incubated for 24 h, harvested and lysed in 200 μl of laemmli sds-page sample buffer (5% glycerine, 1% sds, 2.5% ß-mercaptoethanol, 0.5% bromophenol blue, 0.5 mm edta,0.5m tris ph 6.8). samples were heated to 95˚c for 10 min and separated via sds-page using 12.5% polyacrylamide gels. proteins were then transferred onto a nitrocellulose membrane (ge health care) using a mini-protean tetra cell (biorad) powered at 110 v for 90 minutes. membranes were blocked with 5% skimmed milk diluted in pbs-tween and incubated with primary rabbit polyclonal antibodies against pb2 (1:1,000, gentex, irvine, usa) overnight at 4˚c. subsequently, membranes were washed and incubated with anti-rabbit hrp (horseradish peroxidase)-conjugated secondary antibodies (1:10,000, dianova) for one hour. finally, chemiluminescent substrate hrp juice plus (p.j.k.) was added onto the membrane and bands were visualized using a chemocam imager (intas). in order to detect ß-actin, the membrane was subsequently stripped using stripping buffer (62.5 mm tris hcl ph 6.8, 2% sds, 100 mm ß-mercaptoethanol) for 30 min at 5˚c, washed three times with pbs-tween, and incubated with anti ß-actin mouse (1:500 sigma-aldrich) overnight. the membrane was then washed and incubated with antimouse hrp-conjugated secondary antibody (1:10,000, dianova) for one hour. hrp juice plus was added and bands were visualized as previously described. quantification of pb2 and pb2opt expression was carried out using the program imagej (fiji distribution) [31] . in order to normalize data, signals measured for pb2/pb2opt were divided by those measured for beta-actin. for dip production, a coculture of 200,000 mdck cells and 700,000 293t cells stably expressing pb2 was seeded in t25 flasks. the next day, cells were cotransfected via the calcium phosphate method with 1 μg each of plasmids encoding di-244-mscarlet-i and wt iav genomic segments 2-8. culture medium was changed at 8 h post transfection. at 48 h post transfection, cells were washed with phosphate buffered saline (pbs) without calcium and magnesium and dmem medium supplemented with 0.2% bsa (macs bsa), 0.5 μg/ml tosyl-phenylalanychloromethyl-ketone (tpck)-trypsin (sigma), penicillin (100 iu/ml) and streptomycin (100 μg/ ml) was added. as negative control, transfection of parental mdck and 293t cells was analyzed. supernatants were harvested from all cultures at 4, 6, 8 and 10 days post transfection, cleared by centrifugation at 4,000 rpm for 10 min to remove debris, aliquoted and stored at -80˚c. infectivity of supernatants was analyzed by focus formation assay as described [25, 32] but using mdck cells expressing pb2 or pb2opt as targets. in brief, mdck-pb2/pb2opt cells seeded in 96-well plates were washed and incubated for 1 h with serial dilutions of dip-containing supernatants. thereafter, supernatants were removed and infection medium (gmem with 0.2% bsa and pen/ strep) supplemented with 0.5% methylcellulose and 0.5 μg/ml tpck-trypsin was added. plates were incubated for 72 h and then stained using anti iav polyclonal antibody (millipore). images were taken on a zeiss lsm800 equipped with a 10x/0.45 plan-apochromat objective, 488 nm and 561 nm diode lasers and zen imaging software (zeiss). fluorescent signals (red channel, 561 nm laser) were detected with gaasp detector employing the same sensitivity for all images of a series, while bright field signals were recorded with an esid detector (photodiode) with individually adjusted sensitivity. to test antiviral activity of dips against iav and unrelated vsv, we performed infection experiments in the presence of dip-containing or dip-free supernatants and subsequently compared viral titers in the culture supernatants. for this, mdck cells were seeded in 96-well plates at a density of 10,000 cells/well. on the next day, dip-containing supernatants or dip-free control supernatants were 10-fold serially diluted. subsequently, mdck cells were washed twice with pbs and 50 μl of the respective supernatants were mixed with 50 μl of virus and the mixture inoculated onto the mdck cells. after a 1 h incubation, 100 μl of fresh infection medium supplemented with 0.5 μg/ml tpck-trypsin was added and the cells were further incubated for 24 h (vsv) or 72 h (iav) before viral titers in the culture supernatants were determined. virus titration was performed on confluent monolayers of mdck (iav) or vero (vsv) cells that were grown in 96-well plates. after aspiration of the culture medium, cells were washed twice with pbs and inoculated with 50 μl of 10-fold serial dilutions of the culture supernatants of iav or vsv infected mdck cells. after 1 h of incubation with iav containing supernatants, the medium was removed and 100 μl infection medium supplemented with 1% avicel and 0.5 μg/ml tpck-trypsin (iav/mdck) was added per well. after 1h incubation with vsv-containing supernatants, 200 μl infection medium supplemented with 0.5% methylcellulose (vsv/vero) were added on top, and the cells were further incubated for 24 h. iav titers were quantified by antibody staining, using the focus formation assay as previously described [25, 32] . in order to quantify vsv titers, egfp-positive foci were counted under the fluorescence microscope. all titers are given as focus forming units per ml (ffu/ml). we sought to determine whether di-244 particles can be amplified in the absence of standard virus if producer cells are engineered to express pb2. for this, we first used retroviral transduction and selection antibiotics to generate 293t and mdck cell lines stably expressing pb2. immunoblot revealed that the cell lines obtained by selection expressed robust levels of pb2 (fig 1a and fig 1b) . in order to analyze whether pb2 is functional in these cells, we employed a mini-replicon system, which measures the amplification of a firefly luciferase encoding iav reporter segment upon coexpression of pb2, pb1, pa and np [30] . we found that transfection of 293t-pb2 cells with a plasmid encoding the reporter segment alone yielded luciferase activity in the background range while cotransfection of pb2, pb1, pa and np expression plasmids increased luciferase activity more than 1,000-fold (fig 1c) . importantly, this increase was not observed when the pb1 plasmid was omitted while omission of the pb2 plasmid had no impact on reporter activity (fig 1c) . thus, the pb2 protein stably expressed in 293t cells was functional. unfortunately, similar studies in mdck cells were not feasible due to the low transfectability of these cells. we next investigated whether the 293t-pb2 and mdck-pb2 cells allowed the generation of di-244 particles, using the experimental setup depicted in fig 2a. in order to be able to visually inspect di-244 production and spread, we generated a di-244 variant that encodes for mscarlet-i, a red fluorescent protein [26] . transfection of a mixture of 293t/mdck cells with plasmids encoding iav wt segments 2-8 jointly with a plasmid encoding di-244-mscarlet-i resulted in occasional and moderate red fluorescence (fig 3a) . in contrast, frequent and prominent red fluorescence was observed in 293t-pb2/mdck-pb2 cocultures (fig 3a) , indicating that the stably expressed pb2 promoted amplification of the di-244-mscarlet-i di rna. in order to examine whether amplification of the di-244-mscarlet-i di rna resulted in the production of infectious dips, the supernatants of the transfected 293t-pb2/mdck-pb2 cells were inoculated onto mdck-pb2 cells ( fig 2b) . as controls, the supernatants were also added to mdck wt cells. inoculation of mdck-pb2 cells with supernatants from 293t-pb2/ mdck-pb2 cells resulted in infection of the target cells, as determined by expression of mscarlet-i (fig 3b) . the number of mscarlet-i-positive cells was concentration dependent and supernatants taken at 6 days post transfection from dip producing cells contained the highest amount of infectivity ( fig 3b) . finally, no cells with prominent red fluorescence were detected under control conditions, indicating that dips were only infectious for mdck-pb2 but not mdck wt cells. we next asked whether di-244 production could be quantified by focus formation assay, which is based on detection of iav antigens by antibody staining and is frequently employed to measure iav infectivity. moreover, we examined whether results obtained in the focus formation assay would match those obtained upon counting of foci based upon red fluorescence. foci were observed in mdck-pb2 but not in mdck control cells, confirming that dip infectivity requires pb2 expression in target cells. quantification of dip infectivity by focus formation assay revealed that maximum titers of roughly 1 x 10 3 dips per ml were obtained and counting red fluorescent foci yielded roughly comparable results (fig 3b and fig 3c) . thus, expression of pb2 is sufficient for di-244 production in the absence of helper virus but production efficiency is moderate. dip titers of 1 x 10 3 particles per ml are low and may limit experimentation. therefore, we next asked whether alteration of codon usage for pb2 expression might increase pb2 expression efficiency and dip production. for this, we modified the codons in the pb2 expression plasmid (s1 fig) to reflect codon preferences of human genes and iav. as a second criterion for codon choice, we opted for maximal sequence difference between the a/pr/8/ 34-based sequence previously used for pb2 expression and the newly generated, optimized pb2 sequence (pb2opt), in order to prevent potential recombination events. 293t and mdck cells were engineered to stably express pb2opt and immunoblot revealed that expression levels of pb2opt in mdck but not 293t cells were higher than those obtained upon expression of non-codon-optimized pb2 (fig 1a and fig 1b) . moreover, growth of pb2opt cells was comparable to that of control cells and pb2opt expression was readily detectable after multiple passages, suggesting that expression was not associated with overt cytotoxicity. finally, analysis of the average of five experiments conducted as described for panel a and quantified via the imagej program is shown. signals measured for pb2 or pb2opt were normalized against those measured for beta-actin. error bars indicate standard error of the mean (sem). two tailed paired students t-test was used to assess statistical significance. (c) 293t cells stably expressing pb2 were cotransfected with plasmids encoding an iav luciferase reporter segment and the indicated iav proteins. luciferase activities in cell lysates were determined at 24 h post transfection. the results of a representative experiment carried out with triplicate samples are shown. error bars indicate standard deviation. two tailed paired students t-test was used to assess statistical significance. similar results were obtained in three separate experiments. c.p.s., counts per second. https://doi.org/10.1371/journal.pone.0212757.g001 new system for production of defective interfering particles 293t-pb2opt cells in the mini-replicon assay showed that pb2opt supported iav segment replication (fig 4) . next, we examined whether pb2opt supports dip production with higher efficiency than unmodified pb2. efficient di-244-mscarlet-i di rna amplification was observed in transfected pb2opt cells (not shown) and supernatants obtained from these cells were highly infectious for mdck-pb2opt cells even when diluted 1:1,000 (fig 5a) . in contrast, the supernatants were not infectious for mdck cells (fig 5a) . moreover, a direct comparison of 293t-pb2/mdck-pb2 and 293t-pb2opt/mdck-pb2opt cells for production of infectious dips and for dip amplification upon infection revealed that the pb2opt cells were more efficient. thus, more red fluorescent cells were observed when supernatants from pb2 expressing cells were added to mdck-pb2opt as compared to mdck-pb2 cells (fig 5b) . similarly, supernatants from pb2opt cells were more infectious for target mdck-pb2opt cells as compared to mdck-pb2 cells. in keeping with this observation, quantification of production of infectious dips by focus formation assay and counting of red fluorescent cells revealed that at least 80% of foci (identified by antibody staining) were positive for mscarlet-i, as expected, and that pb2opt cells produced up to 4 x 10 6 infectious dips per ml and thereby exceeded titers obtained with pb2 cells (2,5 x 10 3 ) by~1,500-fold (fig 5c and fig 5d) . di-244 can inhibit spread of diverse iavs and, likely via induction of interferon (ifn), may also inhibit spread of unrelated viruses [3, 4] . in order to investigate the antiviral activity of di-244-mscarlet-i, we first analyzed whether di-244-mscarlet-i produced in pb2opt cells interfered with the spread of a homologous iav, a/pr/8/34, in mdck cells (fig 2c) . for this, mdck cells were coinfected with the indicated dilutions of di-244 containing supernatants and a/pr/8/34 at an moi of 0.1, 0.01 and 0.001 (fig 6a) . this resulted in iav/dip ratios of approximately 1:10 (undiluted dip containing supernatants, iav at moi 0.1), 1:100 (undiluted dip containing supernatants, iav at moi 0.01) and 1:1,000 (undiluted dip containing supernatants, iav at moi 0.001), respectively. the supernatants from 293t/mdck wt cells transfected with plasmids for di-244 production were used as negative control. the control supernatants did not appreciably interfere with a/pr/8/34 infection while supernatants from pb2opt cells efficiently blocked iav infection in a concentration dependent manner, with highest antiviral activity observed at an iav/dip ratio of 1:1,000 (fig 6a) . specifically, infection efficiency relative to untreated virus (set as 100%) was 1 ± 0.5% in the presence of dip containing supernatants at a dilution of 10 0 and 93 ± 13% in the presence of control supernatants (average of six independent experiments). moreover, di-244 containing supernatants also inhibited infection by a/panama/2007/99 (h3n2) in a concentration dependent manner (fig 6b) , in keeping with the concept that di-244 exerts broad anti-iav activity [3, 4] . finally, di-244 containing supernatants did not inhibit vsv infection (fig 6c) , indicating that di-244 neither interfered with vsv genome replication nor altered viral control by a potential ifn response in mdck cells. these results show that di-244 produced in pb2opt expressing cells exerts potent anti-iav activity. for production of dips (di-244-mscarlet-i), a coculture of 293t-pb2 and mdck-pb2 cells was cotransfected with plasmids harboring di-244-mscarlet-i and the wt iav genomic segments two to eight. subsequently, trypsin was added for ha activation and supernatants were harvested at the indicated time points. (b) for quantification of dip production, mdck-pb2 cells were inoculated with dip containing supernatants and the number of red cells was counted or the number of foci was determined using focus formation assay. (c) for analysis of antiviral activity of dips, mdck cells were coinfected with iav wt and dips followed by focus formation assay. https://doi.org/10.1371/journal.pone.0212757.g002 the generation of dips in iav infected cells has been recognized by von magnus several decades ago [33] and dips hold promise as novel antiviral agents [3, 4] . however, exploitation of dips for antiviral therapy requires efficient production systems that do not depend on the presence of standard virus. here, we report a di-244 variant encoding a fluorescent protein that permits monitoring of dip production. moreover, we demonstrate that cells expressing microscopy. (c) the number of infected cells (as determined by red fluorescence) in panel b was quantified. in parallel, infection of cells was analyzed by focus formation assay and the number of foci quantified. the results of a representative experiment are shown in panels a-c and were confirmed in two separate experiments. https://doi.org/10.1371/journal.pone.0212757.g003 two tailed paired students t-test was used to assess statistical significance. c.p.s., counts per second. https://doi.org/10.1371/journal.pone.0212757.g004 new system for production of defective interfering particles pb2 allow generation of infectious di-244 particles solely from plasmids and in the absence of standard virus. finally, our study shows that dips produced in this system suppress spread of different iav subtypes but not vsv in cell culture. di-244 particles and other dips have so far been amplified in cell culture or hen's eggs in the presence of standard virus [3, 4, 17] . in addition, production of di-244 particles from a plasmid system has been described [34, 35] . this approach relies on the transfection of plasmids for production of infectious iav in conjunction with a plasmid containing the di-244 segment and results in the co-production of dips and standard virus [34, 35] . before dip preparations produced in these systems can be used for experimentation, the remaining standard virus needs to be inactivated by uv light [18] . this approach builds on the preferential inactivation of standard virus relative to dips. thus, a mutation in a gene essential for viral spread will abrogate infectivity of standard virus but may have no effect on dip infectivity since the missing proteins will be complemented in trans in cells coinfected with dips and standard virus. however, controlling the efficiency of uv inactivation of standard virus is technically challenging. moreover, the effect of uv light on dip infectivity is difficult to determine and both issues may complicate large scale production of dips as well as interpretation of experimental data and animal trials. thus, establishment of novel cell culture systems for dip production in the absence of standard virus is an important task. our results show that cell lines expressing pb2 allow production and quantification of di-244 particles solely from plasmids and in the absence of standard virus. this finding was not expected given that several reports indicate that pb2 expression alone is insufficient to allow robust spread of iav variants with temperature sensitive mutations in the pb2 gene at nonpermissive temperatures [20, 21] . moreover, it has been suggested that pb2 expression might be associated with unwanted cytotoxic effects [4] . the present study suggests that up to 4 x 10 6 di-244 particles/ml can be produced in cells expressing codon optimized pb2, which roughly translates into production of 10 infectious dips per cell, and it can be speculated that efficiency of dip production can be further increased by employing cell lines stably coexpressing pb1, pb2 and pa. occasionally, weak fluorescence has been observed in dip inoculated control cells. this is most likely attributable to low levels of di-244 mrna expression facilitated by pb2 protein associated with di-244 vrna present in the infecting dips. in contrast, no evidence for production of infectious iav due to recombination between the di-244 rna and the rna encoding for pb2 was obtained, as judged by bright field microscopy, immunofluorescence, focus formation assay and rt-pcr analysis, indicating that the dip production system reported here is safe. quantification of dip production so far relied on quantitative rt-pcr and hemagglutination assay [4, 8, 17] , which do not provide information on particle infectivity. this limitation has been overcome by the present study which demonstrates that infectivity of di-244 particles can be quantified using a standard technique, focus formation assay. the availability of this new system for production of defective interfering particles method should help comparing results obtained with different di-244 preparations or other segment 1 dips and should thus advance the development of dips as antiviral agents. in this context, it is noteworthy that a iav/dip ratio of 1:1,000 resulted in the most prominent antiviral activity in our hands and a very similar ratio, 1:3,400 (as determined by estimations based on quantitative rt-pcr (dip) and infectious units (iav)), was previously reported to be the average of three (moi 0.1, moi 0.01) and six (moi 0.001), respectively, independent experiments is shown. infection in the absence of supernatants was set as 100%. error bars indicate standard error of the mean (sem). two tailed paired students t-test was used to assess statistical significance. (b) the experiment was carried out as described for panel a but a/panama/2007/99 (h3n2) was used for infection. the average of three independent experiments is shown. infection in the absence of supernatants was set as 100%. error bars indicate sem. two tailed paired students t-test was used to assess statistical significance. (c) the experiment was carried out as for panel a but cells were infected with gfp-encoding vsv and supernatants were harvested for titration at 24 h post infection. the results of a single representative experiment conducted with triplicate samples are shown and were confirmed in two separate experiments. https://doi.org/10.1371/journal.pone.0212757.g006 minimally required to protect mice from severe influenza [4] . thus, our study confirms and extends published work indicating that dips have to be provided in vast excess to exert antiviral activity. whether sufficient numbers of dips can be delivered to the human respiratory tract and remain stable to provide protection against influenza for a prolonged time remains to be determined. in this context, one can speculate that an iav:dip ratio of less than 1:1,000 might be sufficient for antiviral activity in humans, since dips might exert direct antiviral activity by inhibiting iav genome replication and induce the ifn system. moreover, dips were reported to have a long residence time in the respiratory tract of mice and dip-treated animals were found to still be protected at one week after treatment [4, 35] . thus, dip stability in the respiratory tract might not pose a major hurdle to the use of dips for influenza prevention and therapy in humans. finally, it should be stated that reassortment of dips with iav in coinfected cells is likely to occur. however, if dips based on the low pathogenic a/pr/8/34 or related viruses are used (like in the present study), such reassortment events should not result in viruses with increased transmissibility or virulence as compared to the wt virus. it is believed that di-244 can interfere with spread of diverse iav in cell culture due to genome competition [3, 4] . indeed, di-244 produced in pb2opt cells exerted comparable antiviral activity against h1n1 and h3n2 iav (no statistically significant differences), in keeping with h3n2 polymerase complexes being fully functional on h1n1 genomic segments [36] . this matches data published for di-244 generated by use of standard virus [35] and demonstrates that dips produced in pb2 expressing cells are fully functional, although the activity of purified dips remains to be examined. di-244 can also interfere with the spread of influenza b virus (ibv) and unrelated respiratory viruses in the infected host and this is thought to be due to induction of innate immune responses, particularly the ifn response [14, 16] . in contrast, dip-mediated inhibition of ibv infection in cell culture is not observed, due to absence of genome competition [13, 14] . the absence of antiviral activity of dips against vsv confirms lack of genome competition. moreover, it suggests that dips might not have modulated a potential ifn response in mdck cells, although it should be noted that such a response might have been impeded due to the presence of trypsin in the culture medium [37] . collectively, we report, to our knowledge, the first 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influenza a virus protects in vivo against disease caused by a heterologous influenza b virus cell culture-based production of defective interfering particles for influenza antiviral therapy homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus replication-incompetent influenza a viruses that stably express a foreign gene expression of a functional influenza viral cap-recognizing protein by using a bovine papilloma virus vector expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants characterization of putative defective interfering (di) a/wsn rnas isolated from the lungs of mice protected from an otherwise lethal respiratory infection with influenza virus a/ wsn (h1n1): a subset of the inoculum di rnas eight-plasmid system for rapid generation of influenza virus vaccines tetherin sensitivity of influenza a viruses is strain specific: role of hemagglutinin and neuraminidase influenza a virus encoding secreted gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins mscarlet: a bright monomeric red fluorescent protein for cellular imaging a gxxxa motif in the transmembrane domain of the ebola virus glycoprotein is required for tetherin antagonism ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms human immunodeficiency virus type 1 spinoculation enhances infection through virus binding the viral nucleoprotein determines mx sensitivity of influenza a viruses fiji: an open-source platform for biological-image analysis influenza a virus does not encode a tetherin antagonist with vpu-like activity and induces ifn-dependent tetherin expression in infected cells incomplete forms of influenza virus defective segment 1 rnas that interfere with production of infectious influenza a virus require at least 150 nucleotides of 5' sequence: evidence from a plasmid-driven system influenza virus protecting rna: an effective prophylactic and therapeutic antiviral seasonal h3n2 and 2009 pandemic h1n1 influenza a viruses reassort efficiently but produce attenuated progeny trypsin promotes efficient influenza vaccine production in mdck cells by interfering with the antiviral host response we thank robert webster for the 8-plasmid system for pr8 (phw191-phw198) and georg kochs and martin schwemmle for plasmids for the replicon assay and defense advanced research projects agency (darpa, intercept program) for support. conceptualization: stefan pöhlmann, michael winkler. key: cord-332725-2oc1yrzx authors: kosmider, beata; messier, elise m; janssen, william j; nahreini, piruz; wang, jieru; hartshorn, kevan l; mason, robert j title: nrf2 protects human alveolar epithelial cells against injury induced by influenza a virus date: 2012-06-06 journal: respir res doi: 10.1186/1465-9921-13-43 sha: doc_id: 332725 cord_uid: 2oc1yrzx background: influenza a virus (iav) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. recent studies have shown the importance of lung antioxidant defense systems against injury by iav. nuclear factor-erythroid 2 related factor 2 (nrf2) activates the majority of antioxidant genes. methods: alveolar type ii (atii) cells and alveolar macrophages (am) were isolated from human lungs not suitable for transplantation and donated for medical research. in some studies atii cells were transdifferentiated to alveolar type i-like (ati-like) cells. alveolar epithelial cells were infected with a/pr/8/34 (pr8) virus. we analyzed pr8 virus production, influenza a nucleoprotein levels, ros generation and expression of antiviral genes. immunocytofluorescence was used to determine nrf2 translocation and western blotting to detect nrf2, ho-1 and caspase 1 and 3 cleavage. we also analyzed ingestion of pr8 virus infected apoptotic atii cells by am, cytokine levels by elisa, glutathione levels, necrosis and apoptosis by tunel assay. moreover, we determined the critical importance of nrf2 using adenovirus nrf2 (adnrf2) or nrf2 sirna to overexpress or knockdown nrf2, respectively. results: we found that iav induced oxidative stress, cytotoxicity and apoptosis in ati-like and atii cells. we also found that am can ingest pr8 virus-induced apoptotic atii cells (efferocytosis) but not viable cells, whereas atii cells did not ingest these apoptotic cells. pr8 virus increased ros production, nrf2, ho-1, mx1 and oas1 expression and nrf2 translocation to the nucleus. nrf2 knockdown with sirna sensitized ati-like cells and atii cells to injury induced by iav and overexpression of nrf2 with adnrf2 protected these cells. furthermore, nrf2 overexpression followed by infection with pr8 virus decreased virus replication, influenza a nucleoprotein expression, antiviral response and oxidative stress. however, adnrf2 did not increase ifn-λ1 (il-29) levels. conclusions: our results indicate that iav induces alveolar epithelial injury and that nrf2 protects these cells from the cytopathic effects of iav likely by increasing the expression of antioxidant genes. identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies. influenza a virus (iav) targets the lung epithelial cells for infection and produces clinical outcomes ranging from a mild upper respiratory infection to severe pneumonia [1, 2] . influenza viruses cause oxidative stress and acute respiratory inflammation [3, 4] . recent studies have focused on the role of lung antioxidant defense systems against injury induced by this virus because they likely play a role in virus-associated inflammation, viral susceptibility and immune clearance [5, 6] . it has been shown that antioxidant compounds inhibit influenza virus replication and diminish the release of inflammatory and apoptotic mediators during virus infection [7] . moreover, the combination of antioxidants with antiviral drugs synergistically reduces the lethal effects of influenza virus infections. this suggests that agents with antiviral and antioxidant activities could be a strategy for the treatment of patients with severe influenza-associated complications [8] . epithelial cells are the primary site of viral replication for influenza virus. upon viral entry, iav inhibits host cell protein synthesis and initiates fast and efficient viral replication. the end result of this process is host cell apoptosis and cytotoxicity [9] . apparently, neighboring cells sense the presence of apoptotic cells and actively extrude them from the monolayer [10] [11] [12] . nuclear factor-erythroid 2 related factor 2 (nrf2) is a member of the family of cap'n'collar basic leucine zipper transcription factors [6] and, although it is ubiquitously expressed throughout the lung, it is found predominantly in the epithelium and alveolar macrophages (am) [13] . the activation of the majority of antioxidant and defense genes are regulated by nrf2 through binding to antioxidant response elements (ares) [6] . it has been recently reported that the antioxidant pathway controlled by nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and lung injury in mice in vivo under oxidative conditions [6] . nrf2 also plays a key role in host defense against respiratory syncytial virus (rsv) in vivo [14] , and rsv infection induces down-regulation of airway antioxidant systems in mice [15] . moreover, cho et al. reported that nrf2 has antiviral activity in murine models of rsv [14] , and nrf2 activation by epigallocatechin gallate decreased viral replication in response to influenza a/bangkok/1/79 infection in human nasal epithelial cells [5] . these results suggest that attenuation of oxidative stress, inflammation and apoptosis may lessen influenza virus-induced lung injury and exacerbation of existing respiratory diseases. children are particularly susceptible to influenza, they account for numerous outpatient visits, and have a central role for spreading infection within the community [16, 17] . it has also been reported that neonatal mice are more susceptible to a/pr/8/34 (pr8) virus than adult mice [18] . therefore in this study, we chose to use alveolar type ii (atii) cells from pediatric lung donors to focus on the response of lung cells isolated from children. we have recently reported the effects of influenza on alveolar epithelial cells from adults [19] . in the current study, we used human primary alveolar type i-like (ati-like) and atii cells. alveolar type i (ati) cells are large flat cells that cover~95% of the alveolar surface and through which gas exchange takes place. atii cells make and secrete pulmonary surfactant, and they proliferate to restore the epithelium after damage to the more sensitive ati cells [20] . human ati cells have not been isolated and cultured. we chose to use ati-like cells, which are type ii cells cultured to transdifferentiate into a ati cell phenotype in vitro [20] [21] [22] . we analyzed clearance of pr8 virus-induced apoptotic cells by human primary am and atii cells. furthermore, to improve our knowledge on pathways involved in lessening the cellular injury associated with influenza infection, we focused on the role of nrf2 in human primary alveolar epithelial cells infected with iav. this is the first study on the effect of iav on ati-like cells and to our knowledge there is no study of the role of nrf2 in influenza a virus infection in cells obtained from children. our hypothesis is that nrf2 protects alveolar cells against injury induced by pr8 virus by activation of antioxidant defense genes and decreasing oxidative stress and viral replication. to test this hypothesis we used adenovirus nrf2 (adnrf2) and nrf2 sirna strategies to study the effect of nrf2 overexpression and knockdown, respectively in these cells infected by pr8 virus. deidentified human lungs not suitable for transplantation were donated for medical research from the national disease research interchange (philadelphia, pa) and the international institute for the advancement of medicine (edison, nj). for this study we selected non-smoker lung donors (n = 6, 2-18 years old). the committee for the protection of human subjects at national jewish health approved this research. to our knowledge this is the first report using alveolar epithelial cells from children to study the effect of iav. the atii cell isolation method has been published previously [23, 24] . briefly, the right middle lobe was perfused and lavaged, and then instilled with elastase (roche diagnostics, indianapolis, in). subsequently, the lung was minced and the cells were filtrated and purified by centrifugation on a density gradient made of optiprep (accurate chemical scientific corp., westbury, ny) and by negative selection with cd14-coated magnetic beads (dynal biotech asa, oslo, norway) and binding to iggcoated (sigma chemicals inc., st. louis, mo) dishes. the purity of atii cells was~80% before plating and over 95% after adherence in culture [20] . the isolated atii cells were cultured as we described previously [19, 23] . briefly, they were resuspended in dmem supplemented with 10% fetal bovine serum (fbs), 2 mm glutamine, 100 μg/ml streptomycin, 100 u/ml penicillin (all from thermo scientific, franklin, ma), 2.5 μg/ml amphotericin b (mediatech inc., manassas, va) and 10 μg/ml gentamicin (sigma chemicals inc., st. louis, mo). to maintain their differentiated state, atii cells were plated for 2 d with 10% fbs on millicell inserts (millipore corp., bedford, ma) coated with a mixture of 20% engelbreth-holm-swarm tumor matrix (bd biosciences, san jose, ca) and 80% rat-tail collagen (rtc) in dmem with additives as mentioned above and then cultured for 2 d with 1% charcoal-stripped fbs along with 10 ng/ml keratinocyte growth factor (kgf, r&d systems inc., minneapolis, mn), and for an additional 2 d with 10 ng/ml kgf, 0.1 mm isobutylmethylxanthine, 0.1 mm 8-br-camp, and 10 nm dexamethasone (all from sigma chemicals inc., st. louis, mo) [19] . to transdifferentiate atii cells into ati-like cells, atii cells were plated on rtc-coated plates or glass coverslips in dmem with 10% fbs for 2 d and then cultured in dmem with 5% fbs for 4 d [20] in addition to glutamine, amphotericin b, streptomycin, penicillin, and gentamicin as mentioned above. am were isolated as we previously described [25] . briefly, the lung was lavaged with hepes-buffered saline and 2 mm edta and the lavage fluid was centrifuged at 4°c for 10 min. the resulting pellet was resuspended and plated in dmem supplemented with 10% fbs in addition to glutamine, amphotericin b, streptomycin, penicillin, and gentamicin as mentioned above. after 24 h, am were cultured for 2 d in dmem with 5% fbs. the h1n1 strain a/pr/8/34 (pr8) (used interchangeably with iav throughout the manuscript) was an original gift from dr. j. abramson (bowman gray school of medicine, winston-salem, nc). pr8 virus was grown in 10-day-old chicken eggs and virus-containing allantoic fluid was processed as previously reported [26] . ati-like and atii cells were infected with pr8 virus as we described [25] . briefly, cells were inoculated with pbs or pr8 virus at a moi of 0.05, 0.5 and 1 pfu/cell. after 1 h cells were washed twice with dmem and incubated for 24 h or 48 h. adenovirus nrf2 (adnrf2) with green fluorescent protein (gfp) and adenovirus gfp (adgfp) were obtained from dr. timothy h. murphy [27] . for adenovirus infection in ati-like or atii cells we used virus diluted to a moi of 200 pfu/cell in pbs. cells were allowed to express transgenes for 24 h before usage. all infected cell cultures were examined for adequate infection efficiency as assessed by gfp fluorescence (88% for ati-like cells and 85% for atii cells) and by western blotting for nrf2. nrf2 sirna duplex showing maximum knockdown in a549 cells (sense: 5' cagcagaacuguaccuguuuu 3'; antisense: 3' uugucgucuugacauggacaa 5') [28] was purchased from dharmacon research, inc (lafayette, co). to confirm the specificity of the inhibition, the control, nontargeting (nt) sirna was used as negative control (sense: 5' uagcgacuaaacacauca auu 3'; antisense 3' uuaucgcugauuuguguag uu 5') [28] . cells were transfected with 100 nmol of sirna duplexes by using genomone hvj envelope vector kit (cosmo bio co. ltd. carlsbad, ca) according to the manufacturer's instructions. after 24 h, cells were infected with pr8 virus as described above. knockdown of the target gene was quantified by western blotting with gapdh for normalization. to detect viral antigen, ati-like cells were fixed with methanol and blocked with 3% normal donkey serum (jackson immunoresearch; west grove, pa) in pbs. the cells were incubated with an antibody specific to influenza a nucleoprotein (millipore corp., billerica, ma) and anticytokeratin mfn116 (dako, carpinteria, ca). the secondary antibody, alexa fluor 594 igg (invitrogen corp., carlsbad, ca) and alexa fluor 488 igg were applied for 1 h. cells were mounted with vectashield medium containing dapi (vector laboratories, burlingame, ca). the same protocol was used to detect nrf2 translocation in ati-like cells, atii cells and am infected with pr8 virus. cells were incubated with rabbit anti-nrf2 antibody (santa cruz biotechnology inc., santa cruz, ca) and subsequently with alexa fluor 594 anti-rabbit igg. to detect oxidative stress induced by pr8 virus we applied rabbit anti-4-hydroxynonenal (4-hne) antibody (abcam, cambridge, ma). 4-hne is a product of lipid peroxidation and hence a marker of oxidative stress. we used alexa fluor 594 anti-rabbit igg as described above. floating atii cells were collected from control cells and cells infected at a moi of 1 pfu/cell pr8 virus for 48 h. the latter ones contained around 70% apoptotic cells as detected by ethidium bromide and acridine orange double staining (data not shown). this method is more sensitive than tunel (tdt-mediated dutp nick-end labeling) assay and allows distinguishing early and late apoptotic cells from necrotic or alive cells [23] . to study efferocytosis we used pkh26 red fluorescent cell linker kit and pkh2 green fluorescent phagocytic cell linker kit (both from sigma, st. louis, mo) according to the manufacturer's instructions. pkh2 labeled am were incubated with pkh26 labeled floating atii cells (ratio 1:10) for 3 h. cells were mounted with vectashield medium containing dapi. a minimum 200 am were counted. the phagocytic index was calculated using the following formula: ((number of apoptotic bodies)/(200 total macrophages)) x 100 [29] . to distinguish between live and necrotic cells 10 mg/ml hoechst 33342 and 1 mg/ml propidium iodide (both from sigma chemicals inc., st. louis, mo) were used for a double staining. three hundred cells were analyzed in each of three independent experiments [30] . expression of proteins was measured by western blotting according to the protocol described previously [23] . protein loading was normalized to gapdh. we used mouse anti-gapdh, rabbit anti-caspase 3 and rabbit anti-caspase 1 (all from abcam, cambridge, ma), mouse anti-ho-1 (assay designs, ann arbor, mi), rabbit anti-nrf2 (santa cruz biotechnology, santa cruz, ca) and mouse anti-influenza a nucleoprotein (millipore, corp., bedford, ma). the blots were then developed using an enhanced chemiluminesence (ecl) western blotting kit according to the manufacturer's instructions (amersham pharmacia biotech, piscataway, nj). images obtained were quantitated using nih image 1.62 software. total rna was isolated from cells using the rneasy mini kit (qiagen, valencia, ca) according to the manufacturer's recommendations. taqman qpcr was performed on a cfx c1000 cfx96 thermocycler (biorad, hercules, ca). probes and cycling condition were optimized in accordance with miqe guidelines for pcr [31] . gene expression levels were calculated as a ratio to the expression of the reference gene, gapdh and data were analyzed using the δδct method. the probes for nrf2, ho-1, mx1 and oas1 were designed by the manufacturer and purchased from applied biosystems (carlsbad, ca). plaque assay was performed as we previously described [32] . briefly, medium from pr8 virus infected ati-like cells was serially diluted in dmem and used to inoculate madin-darby canine kidney (mdck) cells. confluent mdck cells were infected with pr8-infected ati-like cell supernatant for 1 h at 37°c. the inoculum was removed and the cells were overlaid with mem, fbs, antibiotics and seakem le agarose (cambrex, rockland, me). plaques were stained after 72 h incubation at 37°c, with the agarose overlay medium containing 6% neutral red (sigma, st. louis, mo). il-8 and il-29 (ifn-λ1) were measured by elisa (elisa tech., aurora, co) in the ati-like and atii cell culture supernatant according to the manufacturer's recommendations. we used a microquant microplate spectrophotometer (biotek instruments, winooski, vt) and analyzed with kcjunior data analysis software. we compared ros production in ati-like and atii cells transfected with nrf2 sirna or adnrf2 followed by infection with pr8 virus at a moi of 0.5 pfu/cell for 24 h. we used the amplex red hydrogen peroxide assay kit (invitrogen corp., carlsbad, ca) as a quantititative index of ros generation [33] . because h 2 o 2 is one of the most stable forms of ros, this detection method allows observation of oxidation processes in real time. amplex red reacts with hydrogen peroxide in the presence of horseradish peroxidase (hrp) with a 1:1 stoichiometry to form resorufin. briefly, 50 μl of samples and standards were mixed with 50 μl of 100 μm amplex red and 0.2 u/ml hrp solution and incubated for 30 min at room temperature. absorbance was measured at 560 nm and calculated concentrations were normalized to protein content. ati-like cells and atii cells were cultured and infected at a moi of 0.5 pfu/cell pr8 virus for 24 h. total glutathione (gsh) was analyzed as previously described [34, 35] . briefly, gsh was measured by mixing 100 μl of 1:1 3 u/ml glutathione reductase with 0.67 mg/ml 5,5'-dithiobis (2-nitrobenzoic acid, dtnb) with sample or standard. the reaction was initiated by the addition of 50 μl of 0.67 mg/ml nadph (all from sigma, st. louis, ma) absorbance was measured spectrophotometrically at 412 nm and obtained values were normalized to protein content. one-way anova by graphpad prism 4 was used to evaluate statistical differences among experimental groups. a dunnett's test was applied and a value of p < 0.05 was considered significant. data are shown here as the mean ± sem from three independent experiments. we wanted to study the role of nrf2 and oxidative injury during influenza infection. in this report we focused mostly on ati-like cells. the alveolar wall is covered primarily by ati cells and these cells are more sensitive to oxidative injury than atii cells. our initial study was simply to document that ati-like cells could be infected at comparable levels to our previous observations with atii cells [19] . we used cytokeratin mfn 116 as a marker of ati-like cells, and we found that pr8 virus readily infected these cells (figure 1 ) similar to what we have observed with atii cells. iav induces apoptosis and secondary necrosis in vitro because of the lack of phagocytes. we wanted to study the role of nrf2 in preventing cell injury, therefore, we determined the extent of cytotoxicity of pr8 virus, which can result in apoptosis and/or necrosis. we observed necrosis in cells infected with pr8 virus and a higher percentage of necrotic ati-like cells than atii cells (additional file 1: figure s1 ). we used tunel assay to determine whether moi of 0.05, 0.5 or 1 pfu/ cell pr8 virus induces apoptosis after 24 h or 48 h. we observed morphological characteristics of apoptosis (additional file 2: figure s2 , panel i) and a higher percentage of apoptotic cells in the floating cell population than attached ati-like and atii cells (additional file 2: figure s2 , panel ii). we also found a statistically significant higher percentage of apoptotic ati-like cells than atii cells, which suggests that these cells are more sensitive to injury induced by pr8 virus. subsequently, we wanted to document that apoptosis induced by pr8 virus in alveolar cells is associated with caspase activation. we observed caspase 1 and caspase 3 cleavage ( figure 2 ) and also parp cleavage (data not shown) in a concentration-dependent manner after atilike and atii cell infection with pr8 virus at 24 hpi and 48 hpi. hence, these results indicate that pr8 virus induces apoptosis and necrosis. one of the major questions related to apoptosis is what happens to the viral-induced apoptotic cells. virusinfected cells undergo apoptosis and ingestion of apoptotic cells leads to inhibition of virus spread in vivo [36] . when epithelial cells undergo apoptosis, the non-apoptotic epithelial cells actively extrude the apoptotic cells from the monolayer in an actin-dependent process [11, 12] . we wanted to determine potential clearance routes for the apoptotic cells [10] . we analyzed uptake of viral infected apoptotic or viable atii cells by am or atii cells. we found significant ingestion of apoptotic atii cells by am (figure 3 ). in addition, under these same conditions atii cells did not ingest apoptotic atii cells (data not shown). removal of apoptotic cells by am avoids secondary necrosis and the release of cell contents that may promote further inflammation [37] . our observation that am ingest influenza a virus-induced apoptotic cells may further improve strategies aimed on the increasing efferocytosis by these cells that may prevent excessive inflammation and pneumonia. we found that pr8 virus at a moi of 0.5 pfu/cell significantly increased nrf2 and downstream ho-1 mrna levels in ati-like cells, atii cells and am after 48 h (figure 4 , panel i). we also verified these results on the protein level and observed by immunoblotting higher nrf2 and ho-1 expression in these cells after treatment with iav ( figure 4 , panel ii). we also found nrf2 translocation to the nucleus in ati-like cells, atii cells and am infected at a moi of 0.5 pfu/cell pr8 virus for 48 h in comparison with controls ( figure 5 ). our results indicate induction of the nrf2 pathway in these cells in response to iav infection. nrf2 translocation to the nucleus indicates activation of the antioxidant defense system mediated by nrf2. to further study the role of the nrf2 pathway, we overexpressed nrf2 in ati-like cells ( figure 6 , panel i). we infected ati-like cells with adnrf2 followed by pr8 virus (figure 6 , panel ii). we found a significantly higher percentage of necrotic cells after cell infection with pr8 virus alone. the highest percentage of necrotic cells (14.1%) was observed at a moi of 1 pfu/cell. furthermore, we observed a significant decrease in the percentage of necrotic cells after ati-like cell infection with adnrf2 followed by a moi of 0.5 or 1 pfu/cell pr8 virus in comparison with pr8 virus alone. to determine the potential mechanisms mediating the decreased cell injury after pr8 infection due to nrf2 overexpression, we assessed oxidative stress. to do this we analyzed the level of 4-hne. pr8 virus induced oxidative stress and 4-hne immunostaining was decreased after cell infection with adnrf2 followed by pr8 virus (figure 7 , panel i). to further investigate the mechanisms responsible for ati-like cell protection after nrf2 overexpression, we evaluated the expression of ho-1, which is a well-defined target of nrf2 and induced by oxidative stress (figure 7 , panel ii). we found that pr8 virus significantly increases ho-1 level, and this expression was decreased after infection with adnrf2 followed by pr8 virus. these results suggest that infection of ati-like cells with pr8 virus induces oxidative stress, which was diminished by nrf2 overexpression. next we wanted to determine whether adnrf2 affects iav replication. culture media from ati-like cells inoculated with pr8 virus were collected and titrated by plaque assay. we found that infection with adnrf2 followed by iav slightly decreased pr8 virus titer released into the media (figure 7 , panel iii). we also observed a significant increase in influenza a nucleoprotein expression in ati-like cells infected with pr8 virus at a moi of 1 pfu/cell and a slight decrease after infection with adnrf2 followed by iav (figure 7 , panel ii). these results suggest that nrf2 overexpression decreased viral replication in ati-like cells. to determine the potential mechanisms mediating decreased iav replication after infection with adnrf2, we assessed antiviral immune response mediators. specifically, we analyzed the effect of adnrf2 alone or nrf2 overexpression followed by infection with pr8 virus on the interferon-induced mx1 and the oas1 genes, which are involved in the innate immune response to viral infection [25] . nrf2 overexpression did not increase mx1 and oas1 mrna (figure 7 , panel iv). however, infection with adnrf2 followed by pr8 virus significantly reduced expression of these genes in comparison with iav alone which indicates cytoprotective mechanisms orchestrated by nrf2 against cell injury by iav. in summary, our data demonstrate that expression level of nrf2 plays a role in decreasing infection of iav in atilike cells by antiviral activity of nrf2, reducing oxidative stress and induction of cellular defense systems. to further investigate the mechanism of nrf2 protection against iav we analyzed cytokine levels. pr8 virus significantly increased il-8 secretion in comparison with untreated controls (figure 8, panel i) . unexpectedly, we observed a much higher il-8 secretion by adnrf2 in comparison with cells infected with adgfp in ati-like we also found that pr8 virus significantly increased il-29 secretion in ati-like cells (figure 8 , panel ii, a) and atii cells (figure 8, panel ii, b) . iav induced higher il-29 expression in ati-like than atii cells. however, we did not observe higher il-29 levels after cell infection with adnrf2 or adgfp. these results confirm our previous observations that the interferon response to pr8 virus is not altered by nrf2 levels. to verify our results of the protective role of the nrf2 pathway in cells infected with pr8 virus, we knocked down nrf2 and then infected cells with pr8 virus. we were able to knockdown nrf2 in ati-like cells using nrf2 sirna (figure 9 , panel i). we found that nrf2 knockdown followed by infection with pr8 virus increased the cytotoxicity for all applied virus concentrations in comparison with pr8 virus alone (figure 9 , panel ii). this indicates that nrf2 knockdown sensitizes ati-like cells to injury induced by pr8 virus and nrf2 level modulates cell injury by iav. among the methods commonly used to measure h 2 o 2 , amplexred has several advantages. the ability to simply and accurately calibrate signals to peroxide concentrations offers the opportunity to carefully quantitate the production of oxidants by biological systems [33] . we transfected ati-like cells and atii cells with nrf2 sirna or adnrf2 followed by infection at a moi of 0.5 pfu/cell pr8 virus for 24 h. we found significantly higher ros generation in cells transfected with nrf2 sirna followed by infection with pr8 virus in comparison with iav alone (figure 10 , panel i). to complete our results we also infected these cells with adnrf2 followed by pr8 virus and we found lower ros generation in comparison with iav alone. our results indicate the protective role of nrf2 against ros generation by pr8 virus and are in agreement with a positive staining using 4-hne (figure 7) . to determine the protective mechanism of nrf2 against oxidative stress induced by iav we measured gsh level in ati-like and atii cells transfected with nrf2 sirna or adnrf2 followed by infection at a moi of 0.5 pfu/cell pr8 virus for 24 h. we found significantly lower levels of gsh after cell tranfection with nrf2 sirna followed by infection with pr8 virus (figure 10 , panel ii). moreover, adnrf2 increased gsh levels, which protected cells against injury by iav. these results indicate that the protective role of nrf2 against cell injury induced by pr8 virus is in part by increasing glutathione levels. to date, whether or to what extent oxidative stress contributes to the highly virulent property of influenza virus is not fully known. we observed that pr8 virus induced oxidative stress, cell injury, apoptosis, and proinflammatory cytokine secretion in ati-like cells and atii cells. in order to protect cells from injury induced by the pr8 virus, host cells must activate some defense against oxidative stress. nrf2 is critical factor and can activate antioxidant response genes. ours is a novel approach in that first, we studied the response of ati-like cells infected with iav, second, compared the effect of pr8 virus on ati-like cells, atii cells and am obtained from the same donors and third, to our knowledge this is the first report on the role of nrf2 in alveolar cells infected with influenza a virus. our results clearly show that nrf2 regulates alveolar cells' susceptibility to infection and injury induced by pr8 virus. we confirmed our hypotheses and demonstrated the protective role of nrf2 against pr8 virus infection. to our knowledge there are only two reports on the role of nrf2 and influenza virus. these studies were performed in human nasal epithelial cells in vitro [5] and in mice in vivo [6] . cigarette smoke-exposed nrf2 -/mice showed higher rates of mortality than did wild-type mice after iav infection, with higher peribronchial inflammation, lung permeability damage, and mucus hypersecretion. it has been recently reported that supplementation with the potent nrf2 activator epigallocatechin gallate significantly decreased influenza a/bangkok/1/79 virus entry and replication in nasal epithelial cells [5] . the suppressive effect of this compound on viral replication was abolished in cells with knocked down nrf2 expression. this suggests a relationship between induction of the nrf2 pathway and the ability to protect against viral infection. our results are in agreement with these findings. we found nrf2 translocated to the nucleus in ati-like cells, atii cells and am infected with pr8 virus. we also showed significant induction of nrf2 and downstream ho-1. this suggests activation of the nrf2 pathway in response to iav infection. we also observed the protective effect of nrf2 against iav in human alveolar cells. we found that nrf2 overexpression using adnrf2 decreased iav replication, influenza a nucleoprotein expression, antiviral gene expression, ros generation and oxidative stress in comparison with pr8 virus alone. furthermore, adnrf2 also increased gsh level in ati-like and atii cells, which protected against injury by pr8 virus. shih et al. [27] also observed higher intracellular gsh levels in neurons infected with adnrf2, which protected against oxidative stress and gsh has been reported to be an inhibitor of iav infection [38] . moreover, we found that nrf2 knockdown followed by infection with pr8 virus decreased cell viability, increased ros generation and decreased gsh levels. kesic et al. [5] observed that epigallocatechin gallate increased expression of antiviral genes rig-1, ifn-β and mx1. however, these authors suggested that their expression can be nrf2independent or that the effects are species and/or cell-type specific. we found that adnrf2 did not induce mrna levels of the antiviral genes mx1 and oas1. our results can be explained by a different strategy to overexpress nrf2 and/or the cell type studied. moreover, to our knowledge, essential nrf2 binding sites have not been identified in promoters of any of these genes [5] . our results are also in agreement with studies of gene expression profiling nrf2 in mice showing that there are no differences in the antiviral or interferon responsive genes [5, 39, 40] . our results suggest that the activation of antioxidant and cytoprotective mechanisms orchestrated by nrf2 (e.g., ho-1 activation) are responsible for cell protection against iav. to our knowledge, this is the first observation on the antiviral role of nrf2 in human alveolar epithelial cells. we found that moi of 1 pfu/cell pr8 virus induces il-8 and il-29 secretion in ati-like and atii cells. surprisingly, we observed that adnrf2 increased il-8 levels but not il-29. it was postulated that the nrf2/antioxidant response pathway regulates il-8 expression and nrf2-dependent rna binding protein may directly stabilize il-8 mrna [41, 42] . the protective effect of nrf2 also suggests that under certain circumstances, il-8 might have a protective function and serves an anti-inflammatory role in remodeling during the resolution of inflammation. apoptosis induced by influenza virus has been shown in a variety of cell lines [43] [44] [45] . we observed both necrosis and apoptosis induced by pr8 virus in primary human ati-like and atii cells. we found chromatin fragmentation and condensation in attached cells and floating apoptotic cells after infection with pr8 as detected by tunel assay. floating murine primary apoptotic macrophages were observed after treatment with pr8 virus [46] . furthermore, eckardt-michel et al. [47] found that the fusion protein of rsv also induced floating cells that have characteristics of the apoptotic dna ladder, which suggests that they are extruded from the monolayer before late apoptotic events became apparent. in our studies these floating viral infected apoptotic cells were ingested by am but not by atii cells. these results suggest that inflammatory macrophages not the resident epithelial cells are likely responsible for clearing iav-induced apoptotic cells. this is different from the involutional mammary gland where the epithelium itself is responsible for clearing the apoptotic epithelial cells [10, 11] . to investigate the characteristics of the onset of apoptosis ati-like and atii cells infected with pr8 virus, we measured caspase 1 and 3 cleavage. our results are in agreement with our previous study showing caspase 3 and parp cleavage in atii cells isolated from adult lung donors and infected at a moi of 0.5 pfu/cell pr8 for 24 h [19] . this is the first study on the effect of iav on ati-like cells and the results are in agreement with studies showing involvement of caspases in apoptosis upon influenza virus infection in cell lines and human monocyte-derived macrophages [43, 48, 49] . in summary, the effects of nrf2 activation during influenza infections are complex. there is inhibition of viral replication, which might be due to reduced viral entry [5] . there is apparently no alteration in the antiviral genes examined or in the interferon response. there is a significant antioxidant pro-survival response typical for the nrf2 pathway in oxidative stress. we used ati-like, atii cells and am to study a response to iav and to show for the first time, the protective role of nrf2 in human alveolar cells. our results suggest that nrf2 is involved in the cellular antioxidant defense system, is activated upon infection with pr8 virus, and protects the host from the cytopathic effects of oxidative stress induced by iav in interferonindependent manner. taken together, our results indicate that nrf2 is an important factor that can modify the response to pr8 virus. identifying the pathways involved in the cell response to this infection are particularly important for new therapeutic strategies. nevertheless, this study will need to be compared with cells from other vulnerable populations, such as cigarette smokers, and patients with chronic obstructive pulmonary disease. additional studies will be necessary to fully understand the role of nrf2 in the pathogenesis of viral pneumonia (additional file 3). additional file 1: figure s1 . ati-like cells are more sensitive to pr8 virus. ati-like (panel i) and atii (panel ii) cells were infected with pr8 virus at a moi of 0.05, 0.5 or 1 pfu/cell and cell viability was assessed 24 h and 48 h after cell inoculation. the percent of cells that were injured as measured by hoechst 33342 and propidium iodide double staining is shown. there was much more injury in the floating cells (b) than the attached cells (a). * -statistically significant increase in percentage of necrotic cells induced by pr8 virus in comparison with control. figure 10 adnrf2 protects cells and nrf2 sirna sensitizes cells to injury induced by pr8 virus. ati-like cells (a) and atii cells (b) were grown as described in the methods section and infected with pr8 virus at a moi of 0.5 pfu/cell for 24 h. panel i, cell infection with adnrf2 followed by infection with pr8 virus decreased ros generation in comparison with pr8 alone as measured by amplex red kit. cell transfection with nrf2 sirna followed by infection with pr8 virus increased ros production in comparison with pr8 alone. * statistically significant difference in comparison with control. # statistically significant decrease in comparison with pr8 virus alone. & -statistically significant increase in comparison with pr8 virus alone. data represent results from three independent experiments. panel ii, adnrf2 increased gsh level in comparison with control. cell transfection with nrf2 sirna followed by infection with pr8 virus decreased gsh level in comparison with pr8 virus alone. * statistically significant difference in comparison with control. # statistically significant increase in comparison with pr8 virus alone. & -statistically significant decrease in comparison with pr8 virus alone. data represent results from three independent experiments (mean ± sem, * p < 0.05). reading pc: responses of mouse airway epithelial cells and alveolar macrophages to virulent and avirulent strains of influenza a virus the functional impairment of natural killer cells during influenza virus infection the pathogenesis of influenza virus infections: the contributions of virus and host factors effect of influenza vaccination on oxidative stress products in breath nrf2 expression modifies influenza a entry and replication in nasal epithelial cells role of nrf2 in host defense against influenza virus in cigarette smoke-exposed mice n-acetyl-l-cysteine (nac) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type ii epithelial cells infected with influenza virus a and b and with respiratory syncytial virus (rsv) antioxidant therapy as a potential approach to severe influenza-associated complications innate immune response to h3n2 and h1n1 influenza virus infection in a human lung organ culture model apoptosis in the lung: induction, clearance and detection epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release an epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin-and myosin-dependent mechanism oxidative stress targets in pulmonary emphysema: focus on the nrf2 pathway antiviral activity of nrf2 in a murine model of respiratory syncytial virus disease viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis heikkinen t: influenza in children the underrecognized burden of influenza in young children the migration of t cells in response to influenza virus is altered in neonatal mice innate immune response to influenza a virus in differentiated human alveolar type ii cells differentiated human alveolar epithelial cells and reversibility of their phenotype in vitro alveolar epithelial cells secrete chemokines in response to il-1beta and lipopolysaccharide but not to ozone freshly isolated rat alveolar type i cells, type ii cells, and cultured type ii cells have distinct molecular phenotypes apoptosis induced by ozone and oxysterols in human alveolar epithelial cells sars-cov replicates in primary human alveolar type ii cell cultures but not in type i-like cells differentiated human alveolar type ii cells secrete antiviral il-29 (ifn-lambda 1) in response to influenza a infection effects of influenza a virus on human neutrophil calcium metabolism coordinate regulation of glutathione biosynthesis and release by nrf2-expressing glia potently protects neurons from oxidative stress glutathione peroxidase 2, the major cigarette smoke-inducible isoform of gpx in lungs, is regulated by nrf2 tnf-alpha inhibits macrophage clearance of apoptotic cells via cytosolic phospholipase a2 and oxidant-dependent mechanisms human alveolar epithelial cell injury induced by cigarette smoke the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments rat coronaviruses infect rat alveolar type i epithelial cells and induce expression of cxc chemokines detection of hydrogen peroxide with amplex red: interference by nadh and reduced glutathione auto-oxidation lung glutathione adaptive responses to cigarette smoke exposure assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method augmentation of fatality of influenza in mice by inhibition of phagocytosis azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages inhibition of influenza infection by glutathione nrf2 defends the lung from oxidative stress gene expression profiling of nrf2-mediated protection against oxidative injury activation of the nrf2/ antioxidant response pathway increases il-8 expression linking stress, oxidation and the chemokine system avian influenza virus a/hk/483/97(h5n1) ns1 protein induces apoptosis in human airway epithelial cells differential induction of cytotoxicity and apoptosis by influenza virus strains of differing virulence influenza virus ns1 protein induces apoptosis in cultured cells reading pc: critical role of airway macrophages in modulating disease severity during influenza virus infection of mice the fusion protein of respiratory syncytial virus triggers p53-dependent apoptosis differential onset of apoptosis in influenza a virus h5n1-and h1n1-infected human blood macrophages ask1 regulates influenza virus infection-induced apoptotic cell death nrf2 protects human alveolar epithelial cells against injury induced by influenza a virus acknowledgements b.k. received a children's environmental health center faculty development investigator award as part of the environmental determinants of airway disease in children (nih/niehs and epa) 5p01eso18181 (d. schwartz). this work was also supported by 5uo1ai082982 (r.j.m.) and usamramc w81xwh-07-1-0550 (r.j.m.). we thank yoko ito, emily a. travanty and karen e. edeen for assistance with human type ii cell isolations, brian day and joshua chandler for helpful discussions. we also thank boyd jacobson, lydia orth and teneke m. warren for help with manuscript preparation. the authors declare that they have no competing interests. key: cord-339392-2ocz784l authors: sharma, kulbhushan; tripathi, shashank; ranjan, priya; kumar, purnima; garten, rebecca; deyde, varough; katz, jacqueline m.; cox, nancy j.; lal, renu b.; sambhara, suryaprakash; lal, sunil k. title: influenza a virus nucleoprotein exploits hsp40 to inhibit pkr activation date: 2011-06-15 journal: plos one doi: 10.1371/journal.pone.0020215 sha: doc_id: 339392 cord_uid: 2ocz784l background: double-stranded rna dependent protein kinase (pkr) is a key regulator of the anti-viral innate immune response in mammalian cells. pkr activity is regulated by a 58 kilo dalton cellular inhibitor (p58(ipk)), which is present in inactive state as a complex with hsp40 under normal conditions. in case of influenza a virus (iav) infection, p58(ipk) is known to dissociate from hsp40 and inhibit pkr activation. however the influenza virus component responsible for pkr inhibition through p58(ipk) activation was hitherto unknown. principal findings: human heat shock 40 protein (hsp40) was identified as an interacting partner of influenza a virus nucleoprotein (iav np) using a yeast two-hybrid screen. this interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with iav np expressing plasmid. further, the iav np-hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. cellular localization studies showed that np and hsp40 co-localize primarily in the nucleus. during iav infection in mammalian cells, expression of np coincided with the dissociation of p58(ipk) from hsp40 and decrease pkr phosphorylation. we observed that, plasmid based expression of np in mammalian cells leads to decrease in pkr phosphorylation. furthermore, inhibition of np expression during influenza virus replication led to pkr activation and concomitant increase in eif2α phosphorylation. inhibition of np expression also led to reduced irf3 phosphorylation, enhanced ifn β production and concomitant reduction of virus replication. taken together our data suggest that np is the viral factor responsible for p58(ipk) activation and subsequent inhibition of pkr-mediated host response during iav infection. significance: our findings demonstrate a novel role of iav np in inhibiting pkr-mediated anti-viral host response and help us understand p58(ipk) mediated inhibition of pkr activity during iav infection. influenza a viruses (iav) are negative sense segmented rna genome viruses [1, 2] which can rapidly develop resistance to the drugs available against them [3] . these viruses pose a continuing threat of pandemics, thus it is imperative to develop novel strategies to prevent their infection and spread [4] . interactions between viral proteins and host factors are often crucial for successful replication of the virus in host cells [5] . many of these interactions are aimed at overcoming the early innate immune response of infected cells against the virus [6] . mammalian cells respond to viral infections through several innate immune mechanisms [7] . one such crucial antiviral mechanism is activation of pkr (a dsrna dependent protein kinase) which is phosphorylated upon encountering viral dsrna [8] . activated pkr has several downstream substrates, one of which is the eukaryotic translation initiation factor 2 alpha subunit (eif2a) [9] [10] [11] . phosphorylation of eif2a by activated pkr renders it unable to participate in translation initiation leading to translation arrest and inhibition of protein synthesis from viral mrnas [12, 13] . another effector function of pkr is activation of transcription factor irf3, which leads to the expression of ifn b and inhibition of virus replication [14, 15] . being such a crucial component of the host innate immune system, pkr is tightly regulated by cellular inhibitors [16] and very often targeted by viral proteins [17] [18] [19] [20] . for example, the non-structural protein 1 (ns1) of influenza virus directly binds to pkr and prevents its activation [21, 22] . apart from pkr inhibition, ns1 is also involved in the inhibition of other cellular signaling cascades, which lead to the activation of anti-viral interferon response [23, 24] . pkr activity is also inhibited by a cellular 58 kda protein, p58 ipk , which promotes influenza viral replication [25, 26] . in naïve cells, p58 ipk exists in an inactive state in a complex with heat shock protein 40 (hsp40), which becomes active upon release from this complex [27] . influenza virus infection leads to the dissociation of p58 ipk from hsp40 and suppression of the pkr response [28, 29] . however, neither the viral component nor the mechanism responsible for this event is known to-date. segment 5 of the influenza virus genome encodes for 498 amino acids nucleoprotein (np) whose primary function is viral genome encapsidation [30] . apart from that, np is also known to interact with several viral and host factors and play additional roles in the viral life cycle [31] [32] [33] [34] [35] [36] [37] . we were interested in identifying new cellular interactors of np from a highly virulent a/h5n1 bird-flu isolate {a/hatay/2004(h5n1)}, which may facilitate viral replication. for this a h5n1 np was used as bait to search for novel interactors in a yeast two-hybrid system based screen of human lung cdna library. in the screen, we identified that iav np interacts with human chaperone heat shock protein 40 (hsp40) [38] . considering the known role of hsp40 in regulation of pkr activity during influenza a virus infection [27] , we explored the possibility of np playing a regulatory role in this process. we observed that expression of iav np in mammalian cells lead to reduced phosphorylation of pkr and its substrate eif2a. we thus hypothesize that influenza np is the viral factor that facilitates the inhibition of pkr activation by releasing p58 ipk from hsp40-p58 ipk complex. consistent with this hypothesis, we observed that during iav infection, the association of np with hsp40 coincided with the release of p58 ipk from hsp40. also, rnai-mediated inhibition of np expression in iav infected cells enhanced the phosphorylation of pkr and its downstream substrate eif2a. np inhibition also led to enhanced irf3 phosphorylation and ifn b production which may be mediated by pkr activation. collectively, these findings identify a novel role for influenza a virus np in blocking the pkr-dependent antiviral response in influenza a virus infected cells. a human lung cdna library was screened using iav np as bait, in gal4 based matchmaker yeast two-hybrid system (clontech). yeast cells (ah-109) were co-transformed with bait and prey plasmids, and selected for growth on selective l -t -hplates supplemented with 50 mm aminotriazole. b-galactosidase positive colonies were further analyzed (fig. s1a ). plasmids from positive colonies were isolated and subjected to dna sequencing followed by blast analysis to identify their cdna insert. the mammalian chaperone heat shock protein 40 (hsp40/ dnajb11) was thus identified as an interacting partner of np. the strength of the np-hsp40 interaction was determined using a quantitative b-galactosidase assay, and was found to be comparable to the positive control used in the assay (fig. s1b , bars 6 and 7 respectively, p-value = 0.0668). the np-hsp40 interaction in mammalian cells was ascertained using co-transfection of plasmids coding h5n1 np and hsp40 in the hek293t cells. transfected cells were metabolically labeled with s 35 , and co-immunoprecipitation was performed using the lysates with either np-or hsp40-specific antibodies. these results showed that np co-precipitated with hsp40 and vice-versa (fig. 1a , lane 2 and 4). these results were confirmed using a549 lung epithelial cells, which were transfected with np expressing plasmid, followed by immunoprecipitation. it was observed that ectopically expressed np could immunoprecipitate endogenous cellular hsp40 and vice-versa (fig. 1b, panel 1 and 2) . a direct interaction between np-hsp40 was further confirmed using a co-immunoprecipitation assay in which 35 s labeled np and hsp40 proteins were expressed from plasmids using in-vitro coupled transcription-translation rabbit reticulocyte lysate system (tnt, promega, inc) (data not shown). collectively, these results showed that iav np directly interacts with hsp40. to validate the interaction between iav-np and hsp40 in virus-infected cells, we investigated the kinetics of expression of np and hsp40 in a549 cells. a549 cells were infected with a figure 1 . detection of iav np-hsp40 interaction in mammalian cells tranfected with np expressing plasmid by co-immunoprecipitation. a. hek293t cells were pcdna3.1-np and pcdna3.1-hsp40 plasmids alone or in combination, followed by metabolic labeling with s 35 . 48 hours post-transfection cells were harvested and ip was setup using anti-np-specific antibody and anti-hsp40-specific antibody followed by autoradiography. lanes 2 and 4 show co-ip of hsp40 with np and vice-versa. lanes 1 and 3 show anti-np and anti-hsp40 antibodies were not cross reacting with hsp40 and np, respectively. b. a549 cells were transfected with pcdna3.1-np plasmid or control pcdna3.1 plasmid. cells were harvested 48 hours post-transfection and immunoprecipitation was setup using anti-myc tag antibody and anti-hsp40 antibody, followed by western blotting. although np expression was maximal at 24 h post-infection, hsp40 expression levels remained unchanged throughout the course of infection ( fig. 2a) . therefore, in subsequent experiments, a549 cells were infected with pr8 at an moi of 1 for 24 h and lysates were prepared. a co-immunoprecipitation assay was performed using infected and control cell extracts. pr8 np was able to co-precipitate endogenous hsp40 (fig. 2b, panel 2 ). conversely, hsp40 was able to co-precipitate np (fig. 2b, panel 1) . the np-hsp40 interaction was also observed when a549 cells were infected with influenza virus isolates belonging to various subtypes (table 1) . co-immunoprecipitation of proteins from a549 cells infected with these select viral isolates showed that np co-precipitated with hsp40 in all cases without exception (fig. 2c , panel 1). a phylogenetic analysis of influenza np genes used in the experiment was constructed using the neighbor-joining method, nucleotide model tamura-nei, in mega version 4 (fig. s2) . the diversity of changes in np amino-acid sequences of the strains used in this study are shown in fig. s3 . the np genes of iav used in the study had amino-acid sequence divergence in the range of 1% to 10% from hatay/h5n1/2004 isolate. these results clearly indicated that the np-hsp40 interaction was conserved among seasonal human, avian h5n1 and the 2009 h1n1 pandemic influenza a viruses. it is known that under stress conditions the expression level of hsp40 is enhanced and its cellular localization changes from cytoplasmic to nuclear [38] , however its distribution in influenza virus infected cells was not studied. thus we investigated the cellular localization pattern of iav np in context to hsp40 in mammalian cells. we transfected a549 cells with iav np expressing plasmid for 24 h, and an immunofluorescence staining was performed with specific antibodies. results showed that np and cellular hsp40 colocalize primarily in the nucleus (fig. 3a , lower right panel). similar results were obtained with a549 cells infected with pr8 virus. confocal microscopy revealed that np and hsp40 were present primarily in the nucleus. however there was significant amount of np present in the cytoplasm at 24 h post-infection (fig. 3c , lower right panel). we also observed that figure 3 . co-localization of iav np and hsp40 in nucleus of mammalian cells. a and b. a549 cells were transfected with pcdna3.1-np or control pcdna3.1 plasmid for 24 hours, and cells were fixed and processed for immunostaining. np was stained using anti-myc tag specific primary antibody and alexa488 conjugated secondary antibody (green). hsp40 was stained using hsp40 specific primary antibody and alexa 594 conjugated secondary antibody (red). nuclei were stained with dapi. a shows pcdna3.1-np transfected cells whereas b shows control pcdna3.1 transfected cells. panels are labeled for their respective staining. lower right panel shows nuclear colocalization of np and hsp40. c and d. a549 cells were infected with pr8 influenza a virus at 1 moi for 24 hours, and cells were fixed and processed for immunostaining. np was stained using anti-np monoclonal primary antibody and alexa488 conjugated secondary antibody (green). hsp40 was stained using hsp40 specific primary antibody and alexa 594 conjugated secondary antibody (red). nuclei were stained with dapi. panels are labeled for their respective staining. c shows pr8 infected cells whereas d shows control uninfected cells. lower right panel in c shows primarily nuclear colocalization of np and hsp40. doi:10.1371/journal.pone.0020215.g003 hsp40 cellular levels were elevated after iav infection, as compared to uninfected cells ( fig. 3 c and d, upper right panels). hsp40 is known to negatively regulate eif2a phosphorylation through pkr (27) . therefore, we next assessed if changes in pkr and eif2a phosphorylation occurred during the course of iav infection of a549 cells. we found that the phosphorylation of both pkr and eif2a increased initially between 1-2 h post-infection and subsequently declined between 4-8 h post-infection ( during iav infection p58 ipk activity was increased as it was released from hsp40 binding [27] . we hypothesized that np might disrupt the p58 ipk -hsp40 complex too and liberate p58 ipk . to investigate this possibility, we monitored changes in the cellular levels of p58 ipk and np associated with hsp40 during iav infection. a549 cells were infected with the pr8 virus, harvested at different time points after infection and immunoprecipitation was conducted using equal amounts of total protein and anti-hsp40 antibody. western blot analysis revealed that between 4 and 8 h post-infection, the levels of np associated with hsp40 continued to rise (fig. 4b , panel 1) with a concomitant decline in p58 ipk associated with hsp40 (fig. 4b, panel 2) . during this period, total amounts of hsp40 and p58 ipk remained constant (fig. 4b , panel 5, 6). these results were consistent with the hypothesis of replacement of p58 ipk from hsp40, by np. these findings indicate that the dissociation of p58 ipk -hsp40 complex occurs around 4 to 8 h post-infection, and is associated with downregulation of eif2a phosphorylation. taken together, these results suggest that during iav infection, np induces the dissociation of the p58 ipk -hsp40 complex leading to an inhibition of pkr activation and downregulation of eif2a phosphorylation. during iav infection, the ns1 protein inhibits pkr activation by directly interacting with it, and thereby ensuring continued viral mrna translation [21, 22] . results from our study indicated that np may also play a role in inhibiting pkr activity by intercepting this pathway at the level of hsp40. to examine this aspect, we performed a time course study in hek293t cells transfected with the plasmid expressing iav h5n1 np by monitoring p-eif2a and p-pkr levels. western blot analysis of transfected cell lysate showed downregulation of both pkr and eif2a phosphorylation, which began as early as 12 h and was most prominent at 36 h post-transfection ( fig. s4 a fig. 5d ). to confirm the pkr inhibitory action of np, we transfected hek293t cells with np-gfp expressing plasmid and inhibited np expression using specific sirna ( table 2) against it (fig. s5a) . inhibition of np expression led to an increase in p-pkr and p-eif2a levels as compared to control np-gfp transfected cells (fig. s5b) . these results suggest that the expression of np leads to inhibition of pkr activity in a ns1 independent manner. this action of iav np is likely to be mediated through its interaction with hsp40. we suppressed np expression pr8 iav infected a549 cells, using a pool of gene specific sirnas (table 2) , and determined its effect on pkr activity. expression of ns1 was also blocked using sirna against ns1 alone and in combination with np sirna to establish their exclusive and combined contribution to pkrinhibition. a549 cells were first transfected with the indicated sirnas, and 6 hours later were infected with pr8 influenza virus. infected cells were harvested 24 h post-infection and cell lysates were subjected to western blot analysis. we observed that inhibition of np expression led to upregulation of pkr phosphorylation, as compared to the control (fig. 6a, panel 1, fig. 6b ). increased pkr activity resulted in enhanced phosphorylation of eif2a and irf3 (fig. 6a, panel 2, 4, fig. 6c, d) . inhibition of ns1 had a similar effect, whereas inhibition of both np and ns1 had a cumulative effect on upregulation of pkr, eif2a, and irf3 phosphorylation (fig. 6b, c, d) . pkr mediated activation of irf3 should lead to an increased ifn response. previous results confirmed the involvement of np in the inhibition of pkr and irf3. to check the further downstream effect of np, we suppressed the expression of np using sirnas as mentioned earlier. 24 hours post-infection, the cells were harvested and rna was isolated to determine ifnb and viral rna levels by real-time pcr using gene-specific primers. the inhibition of np expression led to increased ifnb production as compared to control (fig. 7a, bar 2, 3) . furthermore, the inhibition of ns1 had greater impact on ifnb production as compared to that of np, and there was a synergistic effect when both ns1 and np were inhibited (fig. 7a, bar 4, 5) . increased ifnb production should lead to reduced virus replication and reduced production of influenza vrna. to confirm this, influenza vrna levels in the above mentioned samples were measured by real-time pcr. inhibition of np or ns1 led to reduced vrna production as compared to control (fig. 7a, bar 2, 3, 4) , and inhibition of both np and ns1 together had greater synergistic effect (fig. 7b, bar 5 ). heat shock proteins are stress response factors which also regulate several cellular processes [39] . the hsp40 family chaperones are known to play important roles in protein folding, translocation, cell signaling and apoptosis [40] [41] [42] . very often they are targeted by viral components for successful virus replication. for example, hsp40 is known to interact with hiv type 2 vpx protein and facilitate nuclear import of the pre-integration complex [43] . hiv type 1 nef protein interacts with hsp40 to enhance viral gene expression [44] . hsp40 is also known to interact with the hbv core protein and affect viral turnover [45] . heat shock proteins are known to affect the viral replication of influenza viruses also. for example hsp90 is known to interact with influenza virus polymerase components and aid in viral rna synthesis [46] . hsp70 is also known to be involved in the nuclear export of the rnp complex and play a role in temperature dependence of iav replication [47, 48] . likewise, hsp40 is also known to regulate pkr signaling in influenza virus infected cells [25] . similarly, the iav np is also a multifunctional protein that interacts with a wide variety of viral and cellular macromolecules, including rna, pb1 and pb2 subunits of the viral rnadependent rna polymerase and the viral matrix protein [30] [31] [32] [33] . it also binds to several host factors which include crm1, uap56, alpha-importin 1 and nf90 [33] [34] [35] [36] [37] . through these interactions, iav-np is known to encapsidate the viral genome, regulate virus transcription and replication, contribute towards pathogenicity of virus, and help in interspecies transmission of the virus [30] . however, so far iav np is not reported to play any role in modulating the host antiviral response. a key component of mammalian antiviral response mechanism is dsrna dependent protein kinase pkr, which is activated by viral dsrna [8] . upon activation, pkr gets dimerized and autophosphorylated at multiple serine and threonine residues. activated pkr phosphorylates eukaryotic translation initiation factor eif2a, which in phosphorylated state cannot participate in mrna translation [12] . this is an important strategy of the host to arrest translation of viral mrnas thereby limiting viral replication [9, 13] . another crucial host pathway which is activated by pkr is irf3mediated ifnb production. activation of pkr is known to enhance irf3 phosphorylation and nuclear movement where it drives expression of interferon b production and built up of antiviral host response [14] . similarly, pkr also has other substrates such as mapk and ikkß which upon phosphorylation trigger various signaling pathways leading to apoptosis or interferon response [10, 11] . being such a crucial molecule, pkr is very often the target of viral factors [15] [16] [17] [18] . in case of influenza virus infection, viral ns1 protein is known to bind directly to pkr and inhibit its activation [20, 21] . ns1 also inhibits the function of retinoic acid inducible gene-i (rig-i), a cytosolic pathogen sensor involved in the antiviral response [49] . apart from that, pkr activity is controlled by another mechanism where p58 ipk , the cellular inhibitor of pkr is activated in influenza virus infected cells [25, 28] . further, p58 ipk itself is inhibited by hsp40 and is present as p58 ipk -hsp40 complex under normal conditions. however upon influenza virus infection, it is released from the hsp40 complex and inhibits pkr activation [24] . in a recent report, it was shown that m2 protein of influenza a virus stabilizes the p58 ipk -hsp40 complex and activates pkr phosphorylation, probably during later stage of infection [50] . however the mechanism of dissociation of hsp40-p58 ipk complex and concomitant pkr inhibition during influenza virus infection remain unknown. here, we report that iav np interacts with the human chaperone hsp40 and employs this interaction to mitigate pkrmediated antiviral response of the host. np-hsp40 interaction was identified through a yeast two-hybrid screen and confirmed in a cell-free translation system, in transfected cells and in influenza virus infected cells. the interaction was found to be conserved across different influenza a viruses, ranging from seasonal, avian h5n1 virus and the 2009 h1n1 pandemic virus despite substantial amino acid differences that range from 0-5% within a subtype/group and 6-10% between the subgroups in np amino-acid sequence. our findings demonstrate that iav np is the viral component that dissociates p58 ipk from the p58 ipk -hsp40 complex during influenza virus infection in mammalian cells. it was observed that during the course of influenza virus infection in lung epithelial cells, a gradual increase in the association of np with hsp40 coincided with a concomitant decrease in p58 ipk association with hsp40. increased activity of p58 ipk , promoted by np, should lead to the inhibition of pkr activation and subsequent downstream effects (fig. 8 ). in accordance with the above hypothesis, we observed that ectopic expression of iav np in mammalian cells substantially reduced the phosphorylation levels of pkr and eif2a. furthermore, sirna-mediated inhibition of np expression during influenza virus infection led to increased phosphorylation of pkr and eif2a, confirming the role of np in the negative regulation of pkr. although eif2a is phosphorylated by other kinases also, namely, hri, gcn2 and perk which are activated during stress condition, only pkr is known to be targeted by viral inhibitors [12] . in line with this, np and ns1 had similar effects on pkr mediated eif2a phosphorylation; however their synergistic effect was higher than their individual effects (fig. 6 b) . activation of pkr signaling during virus infections is known to result in irf3 phosphorylation and concomitant ifnb production. however irf3 is not a direct substrate of pkr and it can get activated by the rig i pathway, nfkb pathway and other unknown mechanisms [14, 15] . influenza ns1 protein is known to inhibit pkr, rig i and nfkb pathways, thus it is expected to have greater impact on irf3 phosphorylation as compared to np, which may inhibit only pkr mediated irf3 phosphorylation [23, 24] . in line with this, we observed that np inhibition during iav infection led to enhanced irf3 phosphorylation, ifnb production and reduced viral replication; however inhibition of ns1 had greater impact on these events. as expected the synergistic effect of np and ns1 inhibition on irf3 activity was higher than their individual effects. the effect of np on ifnb production is also reflected on virus replication as sirna-mediated inhibition of np led to reduced vrna production. this effect may also be attributed to the essential requirement of np for proper functioning of influenza virus polymerase. however the inhibitory action of np on pkr-mediated host response may also contribute to the reduced virus replication in case of sirna-mediated inhibition of np. based on our findings, we proposed a model for pkr inhibition by influenza virus nucleoprotein as shown in fig. 7 . according to this model, iav np interacts with hsp40 and facilitates the release of p58 ipk from it, which in turn inhibits pkr activation (fig. 8) . reduced pkr activity, on one hand leads to reduced eif2a phosphorylation and ensures continued translation from viral mrnas and on the other hand leads to reduced irf3 mediated ifnb production. therefore, apart from the ns1 protein which is already known to inhibit pkr activation and irf3 phosphorylation [21, 24] , np also participates in this process, but through a different mechanism involving hsp40. with structure information of both np and hsp40 being available [30, 42] , it would be interesting to see which domains and key amino-acid residues are involved in this interaction. since the np-hsp40 interaction is conserved across influenza viruses of various subtypes including the 2009 pandemic h1n1 virus, it serves as an important target for developing anti-viral strategies. yeast two-hybrid screening gal4 based matchmaker (clontech) yeast two-hybrid system was used for screening human lung cdna library, as described previously [51] . h5n1 np (a/hatay/2004) gene cloned in pgbk vector (clontech) was used as bait and a mammalian cdna library cloned in pgad (clontech) vector was used as prey. the ah109 strain of yeast was used for co-transformation of bait and prey plasmids. the full-length hsp40 gene was cloned into pgad vector and used in yeast two-hybrid assays. colonies which grew on l -t -hplates (leucine, tyrosine and histidine dropout standard dextrose media) supplemented with 50 mm aminotriazole were considered positive. ß-gal assays (liquid and filter) were performed as per manufacturer's protocols. all dna transfections were done using lipofectamine 2000 (invitrogen) and cells were maintained in dmem medium devoid of serum and antibiotics. six hours post-transfection, culture medium was supplemented with 5% fcs and 24 h post-transfection the medium was replaced with fresh culture medium. all virus infections were done at multiplicity of infection (moi) of 1 for 1 h in dmem medium supplemented with 2% bsa (gibco). after 1 h incubation, the cells were washed with dmem once and then grown with dmem supplemented with 0.2% bsa and 1 mg/ml n-p-tosyl-1phenyl alanine chloromethyl ketone (tpck) (sigma aldrich). the virus strains used in infection experiments are listed in table 1 . cells were lysed using a buffer (20 mm hepes, ph 7.5, 150 mm nacl, 1 mm edta, 10% glycerol, 1% triton x-100) supplemented with protease-inhibitors (roche diagnostics) and the lysates were subject to sds page. anti-np antibodies were obtained from abcam and the immunology and pathogenesis branch, influenza division, centres for disease control and prevention, atlanta, ga, usa. antibodies against pkr, p-pkr, eif2a, p-eif2a, p58 ipk and hsp40 were obtained from cell signaling. anti-ß-actin antibody was purchased from sigma-aldrich. anti-myc tag and anti-ns1 antibodies were purchased from santa cruz. cellular lysates were incubated with primary antibody overnight followed by incubation with protein a dyna beads (invitrogen) for 2 hours. beads were washed thrice and the ip products were subjected to western blotting. np was immunoprecipitated using anti-np monoclonal antibody (immunology and pathogenesis branch/ipb, cdc, atlanta) in case of infection or anti-myc tag antibody in case of transfection. hsp40 was immunoprecipitated using anti-hsp40 monoclonal antibody (cell signaling). after infection or transfection for 24 h, a549 cells were fixed with 2% paraformaldehyde for 30 min at room temperature. they were permeabilized with 0.5% triton x-100 for 5 min at room temperature and blocked with pbs containing 2% bovine albumin. immunostaining was performed using rabbit anti-hsp40 (cell signaling) and mouse anti-np (ipb, cdc, atlanta) antibodies. unbound antibodies were washed away with pbs and cells were incubated with alexa488 tagged goat anti-rabbit antibodies and alexa594 tagged goat anti-mouse. nuclei were stained with dapi. photomicrographs were captured at 1006 magnification using a leica dm6000b confocal microscope. images were processed using nis elements ar 3.0 software (nikon). control (non-targeting) and np-and ns1-specific sirnas of pr8 were purchased from dharmacon and the cells were transfected using the dharmafect 1 transfection reagent (dharmacon). in each case, a pool of three specific sirnas capable of targeting different regions of np or ns1 were used (table 2) . a549 cells at a density of 10 6 /well of a 6-well plate were transfected with 90 nm of the indicated sirna for 6 h prior to infection with a/ pr/8/34 at a moi of 1. lysates were prepared 24 h postinfection and analyzed for the expression of np, ns1 and other cellular proteins by western blotting. total rna was isolated from cells using the rnaeasy kit (qiagen, valencia, ca, usa) and real-time rt-pcr was conducted using a stratagene q3005 pcr machine for expression of ifnb, b-actin mrna and np vrna. for each sample, 2 mg of rna was reverse transcribed using superscript ii reverse transcriptase (invitrogen, carlsbad, ca, usa) according to the manufacturer's directions. oligo dt primers were used for ifnb and b-actin cdna synthesis. for np vrna, cdna was synthesized as described by ge et al [52] . (parallel reactions without reverse transcriptase were included as negative controls. reverse transcription reactions (1/50 th of each reaction) were analyzed in using syber green q-pcr reagents (stratagene, la jolla, ca, usa). interacts with hsp40, thereby displacing p58 ipk from the hsp40-p58 ipk complex. as a result, there is an increased amount of free p58 ipk available in the cell which prevents pkr activation. downregulation in pkr activity ensures less eif2a phosphorylation and continued translation from viral mrnas. on the other hand reduced pkr activity also leads to reduced irf3 activation and subsequent ifnb production. doi:10.1371/journal.pone.0020215.g008 pcr condition was kept as 94uc for 15 s, annealing at 56uc for 30 s, and extension at 72uc for 30 s for a total of 45 cycles. the threshold cycle number for cdna was normalized to that of b-actin mrna, and the resulting value was converted to a linear scale. data from three independent experiments were taken account for analysis. all data points fell into a normal distribution and there were no outliers. primer sets used for these studies are provided in table 3 . figure s1 human heat shock protein 40 was found to interact with influenza a nucleoprotein in yeast twohybrid system. a. yeast two-hybrid screen was performed to find the host interacting partners for h5n1 iav np. results with one of the positive co-transformants (later found to be hsp40 by blast analysis) are shown. ah109 yeast strain cotransformed with np-gbk bait plasmid and hsp40-gad prey plasmid grew in minimal synthetic ypd media devoid of leucine, tryptophan and histidine amino-acids. positive colonies grew on plates supplemented with up to 50 mm aminotriazole (at). a filter b-gal assay was performed to confirm the interaction. blue colored colonies indicate positive clones. b. np-hsp40 interaction was confirmed by liquid ß-gal assay and was found to be statistically comparable to the positive control used (p-value = 0.0668). in the bar-graph, bar 1 represents untransformed ah109 yeast cells; bars 2 and 3 represent control prey plasmids, bars 4 and 5 represent prey plasmids expressing full-length hsp40 and np, respectively; bar 6 represents the co-transformation of hsp40 and np plasmids; bar 7 is a positive control (sars coronavirus np both as bait and prey self-associating to form oligomers) [53] . (tif) figure s2 phylogenetic analysis of np sequence used in the study. a phylogenetic tree was constructed using neighbor-joining method, nucleotide model tamura-nei, in mega version 4 [54] . np gene sequences from selected human seasonal, avian, swine and 2009 pandemic influenza viral isolates were used. the tree shows evolutionary distances between various strains of influenza. the 2009 pandemic h1n1 np belongs to the classical swine lineage which had previous limited introductions into humans and is more distantly related to the np of seasonal or h5 influenza viruses. the h5n1 virus used in this study is shown in red and other iavs used in infection assays are boxed. (tif) figure s3 amino acid sequence comparison of np sequence used in the study. the number and percent difference in amino acids of the iav subtypes used in the infection assays including seasonal h1n1 and h3n2, avian h5n1 and 2009 h1n1 pandemic are compared to a/puerto rico/8/1934 (h1n1) virus. analyses were conducted using the dayhoff matrix based method in mega4 [48] . orthomyxoviridae: the viruses and their replication fields virology fourth edition structures of influenza a proteins and insights into antiviral drug targets h5n1 avian influenza: preventive and therapeutic strategies against a pandemic cellular networks involved in the influenza virus life cycle inverse interference: how viruses fight the interferon system cytoplasmic nucleic acid sensors in antiviral immunity molecular cloning and characterization of the human double-stranded rna-activated protein kinase induced by interferon constitutive expression of human double-stranded rna-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth protein kinase r (pkr) interacts with and activates mitogen-activated protein kinase kinase 6 (mkk6) in response to double-stranded rna stimulation pkr stimulates nf-kappab irrespective of its kinase function by interacting with the ikappab kinase complex coping with stress: eif2a kinases and translational control inhibition of host and viral translation during vesicular stomatitis virus infection. eif2a is responsible for the inhibition of viral but not host translation multiple signaling pathways leading to activation of interferon regulatory factor 3 irf3 and irf7 phosphorylation in virus-infected cells does not require double-stranded rnadependent protein kinase r or ikb kinase but is blocked by vaccinia virus e3l protein cellular inhibitors of the interferon induced, dsrnaactivated protein kinase molecular mechanisms of interferon resistance mediated by viral-directed inhibition of pkr, the interferon-induced protein kinase inhibition of pkr by rna and dna viruses the dsrna protein kinase pkr: virus and cell control influenza virus regulates protein synthesis during infection by repressing autophosphorylation and activity of the cellular 68,000-mr protein kinase binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor influenza virus ns1 protein counteracts pkr-mediated inhibition of replication influenza a virus ns1 protein prevents activation of nf-kappab and induction of alpha/beta interferon the ns1 protein of a human influenza virus inhibits type i interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells the p58 cellular inhibitor complexes with the interferon-induced, double-stranded rnadependent protein kinase, pkr, to regulate its autophosphorylation and activity double-stranded rna-independent dimerization of interferon-induced protein kinase pkr and inhibition of dimerization by the cellular p58ipk inhibitor the molecular chaperone hsp40 regulates the activity of p58ipk, the cellular inhibitor of pkr the cellular protein p58ipk regulates influenza virus mrna translation and replication through a pkr-mediated mechanism p58ipk: a novel ''cihd'' member of the host innate defense response against pathogenic virus infection the mechanism by which influenza a virus nucleoprotein forms oligomers and binds rna the influenza virus nucleoprotein: a multifunctional rna-binding protein pivotal to virus replication structure and sequence analysis of influenza a virus nucleoprotein influenza virus nucleoprotein interacts with influenza virus polymerase proteins interaction of the influenza virus nucleoprotein with the cellular crm1-mediated nuclear export pathway cellular splicing factor raf-2p48/npi-5/bat1/uap56 interacts with the influenza virus nucleoprotein and enhances viral rna synthesis interaction of polymerase subunit pb2 and np with importin alpha1 is a determinant of host range of influenza a virus nuclear factor 90 negatively regulates influenza virus replication by interacting with viral nucleoprotein a stressinducible 40 kda protein (hsp40): purification by modified two-dimensional gel electrophoresis and co-localization with hsc70(p73) in heat-shocked hela cells dual role of heat shock proteins as regulators of apoptosis and innate immunity structure, function and evolution of dnaj: conservation and adaptation of chaperone function the diversity of the dnaj/hsp40 family, the crucial partners for hsp70 chaperones heat shock protein 40: structural studies and their functional implications hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 vpx-mediated preintegration complex heat shock protein 40 is necessary for human immunodeficiency virus-1 nef-mediated enhancement of viral gene expression and replication turnover of hepatitis b virus x protein is facilitated by hdj1, a human hsp40/dnaj protein involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus ribonucleoprotein complex molecular chaperone function of mammalian hsp70 and hsp40-a review ns1 protein of influenza a virus inhibits the function of intracytoplasmic pathogen sensor, rig-i interaction of hsp40 with influenza virus m2 protein: implications for pkr signaling pathway the orf3 protein of hepatitis e virus interacts with hemopexin by means of its 26 amino acid n-terminal hydrophobic domain ii rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription the nucleocapsid protein of the sars coronavirus is capable of self-association through a c-terminal 209 amino acid interaction domain mega4: molecular evolutionary genetics analysis (mega) software version 4.0 key: cord-329680-ekxsv91t authors: yu, yunjia; zhang, yang; wang, shuyao; liu, wei; hao, cui; wang, wei title: inhibition effects of patchouli alcohol against influenza a virus through targeting cellular pi3k/akt and erk/mapk signaling pathways date: 2019-12-23 journal: virol j doi: 10.1186/s12985-019-1266-x sha: doc_id: 329680 cord_uid: ekxsv91t background: patchouli alcohol (pa) is a tricyclic sesquiterpene extracted from pogostemonis herba, which is a traditional chinese medicine used for therapy of inflammatory diseases. recent studies have shown that pa has various pharmacological activities, including anti-bacterial and anti-viral effects. methods: in this study, the anti-influenza virus (iav) activities and mechanisms were investigated both in vitro and in vivo. the inhibitory effects of pa against iav in vitro were evaluated by plaque assay and immunofluorescence assay. the neuraminidase inhibition assay, hemagglutination inhibition (hi) assay, and western blot assay were used to explore the anti-viral mechanisms. the anti-iav activities in vivo were determined by mice pneumonia model and he staining. results: the results showed that pa significantly inhibited different iav strains multiplication in vitro, and may block iav infection through inactivating virus particles directly and interfering with some early stages after virus adsorption. cellular pi3k/akt and erk/mapk signaling pathways may be involved in the anti-iav actions of pa. intranasal administration of pa markedly improved mice survival and attenuated pneumonia symptoms in iav infected mice, comparable to the effects of oseltamivir. conclusions: therefore, patchouli alcohol has the potential to be developed into a novel anti-iav agent in the future. influenza a virus (iav) belongs to the orthomyxoviridae family, being segmented, single stranded, negative sense rna viruses [1] . iav caused at least three large-scale influenza outbreaks in the twentieth century, the most serious of which was the spanish influenza outbreak in 1918, which caused more than 40 million deaths [2, 3] . more recently, the emergence and global spread of h1n1 influenza from the 2009 pandemic and recent lethal cases of h5n1 and h7n9 influenza demonstrate the limitations of currently available strategies to control influenza infection [4] . currently, there are three main types of anti-iav drugs approved for clinical use: (1) m2 ion channel inhibitors such as amantadine and rimantadine; (2) neuraminidase inhibitors such as oseltamivir, zanamivir, and peramivir [5, 6] ; (3) polymerase inhibitors such as baloxavir. however, the emergence of drugresistant influenza variants such as amantadine and oseltamivir resistant iav strains has led to a decline in the efficacy of these drugs. in addition, most of these anti-iav drugs also have some side effects such as nervous system damage [7] [8] [9] . therefore, new influenza therapeutics with novel mechanisms of action are urgently required to combat the persistent threat of influenza viruses. patchouli alcohol is a tricyclic sesquiterpene extracted from pogostemonis herba, which has long been used in the treatment of inflammatory diseases as a traditional chinese medicine [10] . recent studies have shown that patchouli oil has various pharmacological activities, including anti-emetic [11] , anti-inflammatory [12] , antibacterial [13] , and anti-viral effects [14] . li et al. found that oral administration of pa appeared to be able to augment protection against influenza virus infection in mice via enhancement of host immune responses, and attenuation of systemic and pulmonary inflammatory responses [15] . wu and co-workers reported that patchouli alcohol inhibited influenza a (h2n2) virus mainly through interfering with the functions of virus neuraminidase [16] . therefore, pa has the potential to be developed into a novel anti-viral agent in the future. to further correlate the potential anti-iav applications of pa with its underlying molecular mechanisms, the anti-iav (h1n1) effects and mechanisms of pa were investigated in vitro and in vivo in this study. the results showed that pa may block iav infection through inactivating iav directly and interfering with some early steps after virus adsorption. cellular pi3k/akt and erk/ mapk signaling pathways may be involved in the anti-iav actions of pa. in addition, intranasal administration of pa markedly improved mice survival and attenuated pneumonia symptoms in iav infected mice. patchouli alcohol (pa) (with purity > 98%) was purchased from targetmol (shanghai, china). dulbecco's modified eagle's medium (dmem), penicillin, and streptomycin were purchased from gibco (grand island, ny, usa). fetal bovine serum (fbs) was obtained from excell (suzhou, china). mouse anti-influenza a virus np antibody and alkaline phosphatase (ap)-labeled secondary antibodies were obtained from santa cruz biotechnology (usa). dylight 649 conjugated secondary antibody was obtained from abbkine (california, usa). ribavirin injection (50 mg/ml) was purchased from lukang cisen (jining, china). oseltamivir carboxylate was purchased from santa cruz biotechnology (santa cruz, ca, usa). oseltamivir phosphate was obtained from roche (shanghai, china). the influenza neuraminidase inhibitor detection kit was purchased from beyotime (shanghai, china). the anti-np protein was provided by abcam (ab128193), and other antibody, such as anti-phosphorylated pi3k(4228 s), akt (9271 s), mtor (5536 s), erk1/2 (4370 s), and nf-κb (3033 s) antibodies, or anti-gapdh (2118 s) and α-tubulin antibodies (2125 s), were obtained from cell signaling technology (danvers, usa). madin-darby canine kidney (mdck) cells were grown in dmem medium supplemented with 10% fbs, 100 u/ml of penicillin and 100 μg/ml of streptomycin. a549 cells were cultivated in f12 medium containing 10% fbs and 2 mm l-glutamine. influenza a virus h1n1 (a/puerto rico/8/34), h1n1 (a/nws/33), and h1n1 (a/virginia/ atcc1/2009) were propagated in 10-day-old embryonated eggs for 3 days at 36.5°c. for infection, virus propagation solution was diluted in pbs containing 0.2% bovine serum albumin (bsa) and was added to cells at the indicated multiplicity of infection (moi). virus was allowed to adsorb 60 min at 37°c. after removing the virus inoculum, cells were maintained in infecting media (dmem, 4 μg/ml trypsin) at 37°c in 5% co 2 . the cytotoxicity of compounds was measured by the mtt (sigma-aldrich, usa) assay. confluent mdck, a549 and 293ft cell cultures in 96-well plates were exposed to different concentrations of pa (6.25, 12.5, 25, 50 , 100 μg/ml) in triplicate for 24 h. after that, 10 ul of pbs containing mtt (final concentration: 0.5 mg/ml) was added to each well. after 4 h incubation at 37°c, the supernatant was removed and 200 ul of dmso was added to each well to solubilize the formazan crystals. after vigorous shaking, absorbance values were measured in a microplate reader (bio-rad, usa) at 570 nm. the cc 50 was calculated as the compound concentration necessary to reduce cell viability by 50%. different concentrations (50, 25, 12.5, 6.25, 3.125 or 1.56 μg/ml) of pa in 500 μl dmem media were mixed with an equal volume of infectious iav (50-100 pfu/ well) in dmem, and incubated at 37°c for 1 h. the virus-pa mixtures were then transferred to confluent mdck cell monolayers in 12-well plates, and incubated at 37°c for 1 h with gentle shaking every 15 min. after that, the inoculum was removed and each well was overlaid with 1 ml of agar overlay medium (1.5% agarose, 0.02% deae dextran, 1 mm l-glutamine, 0.1 mm nonessential amino acids, 100 u/ml penicillin, 100 mg/ml streptomycin and 1 mg/ml tpck treated trypsin). after incubation for 3 days at 37°c in 5% co 2 , cells were fixed with 4% para-formaldehyde (pfa), followed by staining with 1% crystal violet for plaque counting. mdck cells were infected with h1n1 (vir09, moi = 1.0) under four different treatment conditions. i) pretreatment of virus: iav was pretreated with 25 μg/ml of pa at 37°c for 1 h before infection. ii) pretreatment of cells: mdck cells were pretreated with 25 μg/ml of pa at 37°c for 1 h before infection. iii) adsorption: mdck cells were infected in media containing 25 μg/ml of pa and, after 1 h adsorption at 37°c, were overlaid with compound-free media. iv) after adsorption: after 1 h adsorption at 37°c, the inoculum was removed and the infecting media containing 25 μg/ml of pa were added to cells. at 24 h p.i., the antiviral activity was determined by plaque assay. mean percentage virus titers were calculated as a percentage of plaque titers from untreated control group. iav virus (moi = 3.0) infected a549 cells were treated with or without pa (10, 20 μg/ml) after adsorption. at 2 h post infection, mdck cells were fixed with 4% pfa for 15 min. then cells were permeabilized and incubated sequentially with primary antibodies against iav np protein and dylight 649 conjugated secondary antibody. then after washing, the cell nucleus was stained with dapi for 20 min before confocal imaging. images were recorded using a nikon confocal microscope, and analyzed by imagej (nih) version 1.33 u (usa). the hemagglutination (ha) assay was performed as previously reported [17] . standardized chicken red blood cell (crbc) solutions were prepared according to the who manual. virus propagation solutions were serially diluted 2-fold in round bottomed 96-well plate and 1% crbcs were then added at an equal volume. after 60 min incubation at 4°c, rbcs in negative wells sedimented and formed red buttons, whereas positive wells had an opaque appearance with no sedimentation. 293ft cells were cultured in 24 well plates at 37°c for 24 h before transfected with plasmids (pb2/pcdna3.1, pb1/pcdna3.1, pa/pcdna3.1, or np/pcdna3.1) encoding pr8 virus polymerase subunits (pa, pb1, and pb2 protein) and virus nucleoprotein (np), and a luciferase rna expression vector (vns1-luc/phh21). then the cells were treated with or without pa (3.125, 6.25, 12.5, 25 and 50 μg/ml) or nucleozin (10 μm). the effect of vrna transcription was then evaluated by measuring luciferase activity according to manufacturer's instructions after incubation at 37°c for 48 h. the influenza neuraminidase inhibitor detection kit was used to measure the inhibition of na activity [17] . briefly, inactivated pr8 virus supernatants was added to a 96-well plate and then mixed with different compounds (diluted in 33 mm mes buffer (ph 3.5), 4 mm cacl 2 ) at 37°c for 30 min. then munana (20 μm) was added as the substrate and incubated at 37°c for 30 min. the reaction was stopped by the addition of stop solution (25% ethanol, 0.1 m glycine, ph 10.7). fluorescence was measured using a spectramax m5 plate reader with excitation and emission wavelengths of 360 and 440 nm, respectively. total rna was extracted from vir09 virus (moi = 1.0) infected mdck cells using an rnaiso™ plus kit (takara, japan), and analysed by using the one step sybr prime-script rt-pcr kit (takara, japan). the real-time rt-pcr was performed using the following primers: virus ha mrna, 5′-aagtcctcgtgctatggg-3′ and 5′-tgggaggctggtgtttat-3′; β-actin mrna, 5′-ctccatcctggcctcgctgt-3′ and 5′-gctgtc accttcaccgttcc-3′. the real-time rt-pcr was performed at 42°c 5 min, 95°c 10 s, 40 cycles of 95°c 5 s, 60°c 34 s, followed by melting curve analysis, according to the instrument documentation (abi prism 7500, applied biosystems, usa). the relative amounts of virus ha mrna molecules were determined using the comparative (2 -δδct ) method, as previously described (18) . after drug treatment, the cell lysate was separated by sds-page and transferred to nitrocellulose membrane. after being blocked in tris-buffered saline (tbs) containing 0.1% tween 20 (v/v) and 5% bsa (w/v) at room temperature for 2 h, the membranes were rinsed and incubated at 4°c overnight with anti-np protein (santa cruz, usa), anti-phosphorylated pi3k, akt, erk1/2, and nf-κb antibodies, or anti-gapdh, β-actin, and αtubulin antibodies (cell signaling technology, danvers, usa) as control. the membranes were washed and incubated with ap-labeled secondary antibody (1:2000 dilutions) at rt for 2 h. the protein bands were then visualized by incubating with the developing solution (pnitro blue tetrazolium chloride (nbt) and 5-bromo-4chloro-3-indolyl phosphate toluidine (bcip) at rt for 30 min. the relative densities of proteins were all determined by using imagej (nih) v.1.33 u (usa). four-week-old female kunming mice (average weight, 14.0 ± 1.0 g) were housed and studied under protocols approved by the animal care and use committee of ocean university of china (oucyy-2018003). mice received humane care in accordance with the guidelines provided by the national institutes of health for the use of animals in laboratory experiments. 10 mice per group were inoculated intranasally with pr8 (2ld 50 /mouse) diluted in 40 ul of 1× pbs. all the mice were randomly divided into experimental groups. four hours after inoculation, mice received intranasal therapy of pa (20 or 40 μg/day) or oral therapy of oseltamivir phosphate (10 mg/kg/day), and the treatments were repeated once daily for 7 days. mice were weighed and killed on day 4 after inoculation, and the lungs were then removed, weighed, and homogenized in 1 × pbs for determination of viral titers by plaque assay. histopathological analysis was performed using h&e staining on samples collected on 4 days post infection (dpi) as described previously [18] . in the survival experiments, 10 mice per group were intranasally infected with pr/8 virus (4 ld 50 /mouse) at day 0. the drugs administration was repeated once daily during the experiment, and survival was assessed in all groups for 14 days after infection. mice were monitored daily for weight loss and clinical signs. if a mouse lost body weight over 25% of its pre-infection weight, it was defined as dead and humanely euthanized immediately; the rest of the mice were sacrificed at the end of experiment on 14 dpi. all data are representative of at least three independent experiments. data are presented as means ± standard deviations (sd). statistical significance was calculated by graphpad prism 7 software using one-way anova with turkey's test, with p values < 0.05 considered significant. the cytotoxicity of patchouli alcohol (pa) was firstly evaluated by mtt assay in mdck, a549 and 293ft cells [19] . the results showed that pa exhibited no significant cytotoxicity at the concentrations from 6.25 to 100 μg/ml (fig. 1a) . the cc 50 (50% cytotoxicity concentration) values for pa in mdck, 293ft and a549 cells were about 550.8, 914.8, and 454.5 μg/ml, respectively. these results were used to determine the dose range of pa for the subsequent experiments. pa was then assayed for its ability to inhibit iav multiplication in vitro using plaque assay [20] . firstly, the inhibition of pa on the virus yields from mdck cells infected with vir09 (a/virginia/atcc1/2009), nws (a/nws/33) or pr8 (a/puerto rico/8/34) at high moi (≈1.0 pfu/cell) were examined by plaque assay. as shown in fig. 1b and c, pa treatment reduced the virus titers of vir09, nws, and pr8 in a dose-dependent manner when used at the concentrations of 6.25-50 μg/ ml. the 50% inhibitory concentration (ic 50 value) of pa for vir09, nws, and pr8 was about 6.3 ± 1.3, 3.5 ± 1.4, and 6.1 ± 1.7 μg/ml, respectively (table 1) . at the concentration of 12.5 μg/ml, the virus titers reduced about 30 fold of that in the untreated control group for vir09, 3.0 fold of that for nws, and 2.5 fold of that for pr8 virus (fig. 1b and c) . to further explore whether pa had direct inhibition actions on viral particles, the plaque reduction assay was performed as previously described [21] . in brief, vir09 virus (50-100 pfu/well) was pre-incubated with or without pa for 60 min at 37°c before infection. ten the virus-pa mixture was transferred to confluent cell monolayers in 6-well plates incubated at 37°c for 1 h and subjected to plaque assay. as shown in fig. 1d and e, pre-incubation of pr8 with pa at the concentrations of 12.5 and 25 μg/ml markedly reduced the number of plaques and protected mdck cells, suggesting that pa may be able to inactivate viral particles directly. furthermore, the inhibition effects of pa on iav infection was also examined over multiple cycles of infection using plaque reduction assay [17] . briefly, mdck cells were infected with pa pretreated virus (vir09, nws, and pr8) at an moi of 0.001 pfu for 1 h at 37°c, and then subjected to plaque assay. as shown in fig. 1f , pa also significantly inhibited the plaque formation in vir09, nws and pr8 (moi = 0.001) infected cells when used at the concentration > 3.125 μg/ml (fig. 1f) . the ic 50 values of pa for vir09, nws, and pr8 was about 2.2 ± 0.2, 3.2 ± 0.2, and 2.9 ± 0.4 μg/ml, respectively (table 1) . however, ribavirin could not significantly inhibit the plaque formation of vir09 with ic 50 value > 120 μg/ml (table 1) . thus, pa possessed anti-iav effects in vitro, and the pandemic h1n1 virus (vir09) was most susceptible to pa treatment. various time-points were assessed to determine the stage(s) at which pa exerted its inhibitory effects in vitro. briefly, mdck cells were infected with vir09 virus (h1n1) (moi = 1.0) under four different treatment conditions: pre-treatment of viruses, pre-treatment of cells, during adsorption, or after adsorption. at 24 h p.i., the antiviral activity was determined by plaque assay. as shown in fig. 2a , pretreatment of vir09 virus with 25 μg/ml pa for 1 h before infection markedly reduced virus titers, suggesting that pa may have direct interaction with iav particles. however, either the addition of pa during adsorption or pretreatment of cells only weakly inhibited virus multiplication (fig. 2a) , suggesting that pa may not interact with mdck cells directly. interestingly, treatment of pa after adsorption also significantly reduced virus titers as compared to the nontreated virus control group (fig. 2a) . thus, pa may be able to inactivate virus particles directly and block some stages after virus adsorption. moreover, another time course study was also performed to explore which viral stage after adsorption is inhibited by pa as described previously [17] . briefly, (fig. 2b ). since pa may be able to inactivate virus particles directly, we then explored whether pa had direct interaction with virus surface na and ha protein by using the neuraminidase inhibition assay and hemagglutination inhibition (hi) assay. as shown in fig. 2c , pa could not significantly inhibit the na activities of vir09 virus at the concentrations of 12.5-100 μg/ml, while zanamivir possessed high inhibition percentage (> 80%) at 10 μg/ ml, suggesting that pa may have no direct interaction with virus na protein. moreover, the results of the hi assay showed that the anti-ha antibodies significantly inhibited the pr8 virus-induced aggregation of chicken erythrocytes at the concentrations of 0.3125-5 μg/ml (fig. 2d) , suggesting that the anti-ha antibody can block the virus attachment to red blood cells through binding to ha. however, pa did not obviously inhibit virusinduced aggregation of chicken erythrocytes even at a concentration of 50 μg/ml (fig. 2d) , suggesting that pa may have no direct interaction with viral ha protein. furthermore, we also performed mini-genome assay to evaluate the influence of pa on viral genome replication, which occur during the early stages in viral life cycle. briefly, 293ft cells were transfected with four expression plasmids encoding pr8 virus pb2, pb1, pa, and np proteins, and the luciferase-containing plasmid vns1luc/phh21, which encodes a viral-like genome in the absence or presence of pa (3.125, 6.25, 12.5, 25 and 50 μg/ml). the effect of vrna transcription was then evaluated by measuring luciferase activity at 48 h p.i. the results showed that the positive drug nucleozin caused a notable reduction in luciferase activity at 10 μm as compared with control (dmso) treatment (fig. 2e) . by contrast, treatment with pa (3.125, 6.25, 12.5, 25 and 50 μg/ml) did not significantly inhibit luciferase activity, suggesting that np protein may be not the direct target of pa. in summary, virus ha, na and np proteins may not be the main targets of pa in vitro. since pa may inhibit some steps after virus adsorption ( fig. 2a and b) , the effects of pa on viral protein synthesis and rna replication were evaluated by using immunofluorescence assay and real-time rt-pcr assay as described previously [19] . firstly, vir09 virus (moi = 1.0) infected a549 cells were added with 10 or 20 μg/ml of pa after virus adsorption and then incubated at 37°c for 2 h. after that, viral np protein expression was detected by immunofluorescence assay. as shown in fig. 3a , in virus-infected cells without drug treatment, the fluorescence of viral np proteins could be obviously found in both the cell nucleus and cytoplasm (fig. 3a) , while nearly non fluorescence could be found in the noninfected cells (fig. 3a) . however, after treatment with pa for 2 h, the number of virus antigen-expressing cells was drastically reduced, and only very few fluorescence could be found in the cytoplasm (fig. 3a) . quantitation of data of the fluorescence intensity in iav infected cells showed that pa treatment (5, 10 μg/ml) significantly reduced the fluorescence intensity of np in a549 cells, suggesting that pa may block some steps of iav life cycle after adsorption to interfering with nuclear import and expression of np protein (fig. 3b) . moreover, the inhibition effect of pa on virus mrna expression was then evaluated by real-time rt-pcr assay. iav (moi = 1.0) infected cells were added with pa (10, 20, 40 μg/ml) after virus adsorption and then incubated at 37°c for 8 h. after that, total rna was extracted for real-time rt-pcr. as shown in fig. 3c , after treatment with pa (20, 40 μg/ml) for 8 h, the iav np mrna levels decreased to about 36.6 and 14.2% of that of untreated cells after pa treatment, respectively, consistent to the results of immunofluorescence assay. furthermore, western blot assay was also performed to verify the inhibition of pa on viral protein production. mdck cells were firstly infected with iav (moi = 1.0), and then treated with or without pa at indicated concentrations after adsorption. after incubation for 8 h, viral np protein production was detected by western blot assay. as shown in fig. 3d and e, the level of viral np protein was significantly reduced by pa in a dosedependent manner as compared to that of the nontreated virus control group (pr8) (p < 0.01). the treatment with pa at 40 μg/ml reduced the production of iav np protein by more than 80% (fig. 3e ). therefore, pa may also be able to inhibit iav protein and mrna expression through interfering with some early steps of virus life cycle. since pa may inhibit some steps after virus adsorption to reduce iav mrna and protein expression in vitro, so we further explored if pa could influence some cellular signaling pathways required for iav infection. the cellular pi3k/akt signaling pathway was reported to be required for virus endocytosis and replication, and the inhibitors of pi3k/akt signaling could inhibit both entry and replication of virus [21, 22] . in this study, after iav infection for 2 h, the levels of phosphorylated pi3k proteins were significantly increased to about 1.3 fold higher than normal control group in iav infected cells (p < 0.01) (fig. 4a and e) . however, after treatment with pa (6.25, 12.5, 25, 50 μg/ml) for 2 h, the expression level of phosphorylated pi3k significantly decreased from about 1.3 to about 1.1, 0.9, 0.6, and 0.3-fold of normal control group, respectively (p < 0.05) (fig. 4a and e) . moreover, the activation of pi3k can induce the activation of some downstream signals such as akt, and the level of phosphorylated akt was truly significantly increased in virus-control group to about 4.5 fold higher than normal control group at 2 h p.i. (p < 0.01) (fig. 4b and f ). but treatment with pa (25, 50 μg/ml) for 2 h could significantly reduce the activation of akt from about 4.5 to about 3.4 and 2.8-fold of normal control group, respectively ( fig. 4b and f) . thus, the pi3k/akt signaling pathway may be involved in the anti-iav mechanisms of pa in vitro. furthermore, the mapk signaling pathway was reported to be required for efficient vrnp export from nucleus, and the inhibitors of mapk pathway could reduce iav replication and inflammatory symptoms [23] [24] [25] . in this study, erk1/2 protein was significantly activated in virus-control group to approximately 9.2 fold higher than normal control group at 2 h p.i. (p < 0.01) ( fig. 4c and g). however, after treatment with pa (6.25, 12.5, 25 and 50 μg/ml) for 2 h, the expression level of phosphorylated erk1/2 protein significantly decreased from about 9.2 to about 6.0, 6.3, 5.9, and 5.3-fold of normal control group, respectively (p < 0.01) ( fig. 4c and g) . however, treatment with pa (6.25, 12.5, 25 and 50 μg/ml) for 2 h could not significantly reduce the expression level of phosphorylated nf-κb protein as compared to the virus control group (fig. 4d and h) . thus, pa may inhibit erk/mapk rather than nf-κb pathway to interfere with iav replication. moreover, the pi3k/akt pathway was reported to be associated with host antiviral response [26, 27] , so we further explored the influence of pa on immune response by using western blot and elisa assay. we first evaluate the direct actions of pa on cellular pi3k/akt pathway in non-infected a549 cells using western blotting. the results showed that pa treatment (6.25, 12.5, 25, 50 μg/ml) could not significantly influence the activation of pi3k and akt proteins in the non-infected a549 cells ( fig. 5a and b) , suggesting that the inhibition of pi3k/akt pathway by pa may be related to its inhibition of iav infection. treatment of pa for different time intervals within 24 h showed no significant cytotoxicity to noninfected a549 cells (fig. 5c ). in addition, iav infection significantly increased the production of cellular interferon-β (ifn-β) in vir09 virus infected a549 cells, however, pa treatment (6.25, 12.5, 25, 50 μg/ml) could not significantly influence the production of ifn-β as compared to the virus control group (fig. 5d) , suggesting that pa had no direct action on cellular antiviral response. furthermore, we also evaluated the influence of pa on the production of interferon-γ (ifn-γ) and interleukin 2 (il-2) in mice with or without pr8 virus infection. as shown in fig. 5e , intranasal treatment of pa (20 or 40 μg/day) for four days had no significant influence on the production of ifn-γ and il-2 in non-infected mice. however, pa treatment could significantly reverse the reduction of ifn-γ and il-2 in iav infected mice (fig. 5f) , suggesting that the enhancement of pa on type-ii interferon system may be related to its inhibition of iav inhibition in vivo. thus, the inhibition of pi3k/akt pathway by pa may be related to its inhibition of iav infection rather than direct actions on host antiviral response. the anti-iav effects of pa in vivo were further explored using a mouse pneumonia model [18] . in brief, iav-infected mice received intranasal administration of pa (20 or 40 μg/day) or placebo (pbs) once daily for the entire experiment, and the selected subset of treated, infected mice were then sacrificed on day 4 and the tissue samples were removed for further analysis. subsequently, the pulmonary viral titers were determined by plaque assay [22] . as shown in fig. 6a , after treatment of pa (20, 40 μg/day) for 4 days, the pulmonary viral titers significantly decreased compared to that of the virus control group (p < 0.01), suggesting that intranasal therapy with pa could inhibit iav multiplication in mice lungs. oral therapy of oseltamivir (10 mg/kg/day) also showed significant reduction of virus titers in mice lungs (p < 0.05) (fig. 6a) . moreover, the survival experiments were also performed to evaluate the effects of pa on the survival of iav-infected mice. as shown in fig. 6b , intranasal administration with pa (40 μg/day) significantly increased survival rates as compared to the placebo-treated control group (p < 0.05). by day 14 post infection, only 30% of the individuals in the placebo group survived whereas 100% of animals in the pa (40 μg/day)-treated group survived, superior to that in oseltamivir (10 mg/kg/day)treated group (90%). pa treatment at 20 μg/day also increased the survival rate of iav infected mice (50%) but without significance (fig. 6b) . to further evaluate the effects of pa on viral pneumonia in mice, histopathology analysis was also performed (see figure on previous page.) fig. 3 the influence of patchouli alcohol on virus protein and mrna expression. a immunofluorescence assay of virus np protein in h1n1 (vir09) infected a549 cells at 2 h p.i. scale bar represents 50 μm. b the average fluorescence intensity of np proteins in (a) was measured by imagej (nih) version 1.33u (usa) to calculate the average intensity per unit area of cells of different images (n = 30). significance: * p < 0.05, * * p < 0.01 vs virus control group. c vir09 (moi = 1.0) infected mdck cells were treated with different concentrations of pa (10-40 μg/ml), and incubated at 37°c for 8 h. after that, total rna was extracted for real-time rt-pcr assay of iav ha mrna and cellular β-actin mrna. the relative amounts of virus ha mrna were determined using the comparative ( 2-δδct ) method. rna levels for non-drug treated cells (virus control) were assigned values of 1. values are means ± sd (n = 3). significance: *p < 0.05 vs. virus control group. d mdck cells were firstly infected with iav (moi = 1.0), and then treated with or without pa at indicated concentrations after adsorption. at 8 h p.i., the virus np protein expression was evaluated by western blotting. blots were also probed for β-actin protein as loading controls. e quantification of immunoblot for the ratio of iav np protein to actin. the ratio for non-treated virus control group (pr8) were assigned values of 1 and the data presented as mean ± sd (n = 3). significance: **p < 0.01 vs. virus control group (pr8) as described previously [18] . as shown in fig. 6c , lung tissues in virus-control group showed marked infiltration of inflammatory cells in the alveolar walls and the presence of massive serocellular exudates in the lumen. however, after treatment with pa (20 or 40 μg/day) for 7 days, the lung tissues showed intact columnar epithelium in the bronchiole even in the presence of some serocellular exudates in the lumen (fig. 6c ). mice treated with oseltamivir (10 mg/kg/day) also had intact columnar epithelium (fig. 6c) . thus, pa may be able to attenuate pneumonia symptoms in iav infected mice. natural products from chinese medicine have been attracting more and more attention of pharmacists. patchouli alcohol (pa), a tricyclic sesquiterpene extracted from pogostemonis herba, was reported to possess anti-viral activities against different viruses especially influenza virus [14] [15] [16] . in the current study, we found that pa could inhibit different influenza a virus replication in vitro, and the pandemic h1n1 virus (vir09) was most susceptible to pa treatment (ic 50 < 6.5 μg/ml). intranasal administration of pa significantly promoted the survival rate of mice and attenuated pneumonia symptoms in iav infected mice, comparable to the positive control drug oseltamivir. thus, patchouli alcohol merits further investigation as a novel anti-iav agent in the future. the time-of-addition assay indicated that pretreatment of iav with pa before infection or addition of pa during adsorption markedly reduced virus multiplication (fig. 2) , suggesting that pa may have direct inactivation effects on iav particles. pa was reported to inhibit h2n2 virus replication mainly through inhibition of the functions of virus neuraminidase [16] . however, in contrast to the previous studies, we found that pa could not significantly inhibit the na activity of h1n1 virus, and did not significantly block ha mediated aggregation of chicken red blood cells. thus, pa may be not able to directly bind to virus surface proteins but may interfere with the interaction between iav and cell receptors. interestingly, post-treatment of cells with pa after adsorption also dramatically inhibited virus multiplication, suggesting that pa may also block some stages after virus adsorption. cellular pi3k/akt signaling pathway is known to be able to augment replication of several viruses, and may be associated with lytic infections of both rna and dna viruses, including influenza a virus [21] . some inhibitors of pi3k or its downstream signal akt could significantly block virus entry and replication [20] [21] [22] . herein, patchouli alcohol was found to be able to significantly inhibit the phosphorylation of pi3k and akt proteins in iav-infected cells (fig. 5) , suggesting that pa may inhibit the activation of pi3k/akt signaling pathway to block virus infection and replication. however, pa could not influence the activation of pi3k/akt pathway in non-infected a549 cells and could not directly enhance the interferon system in vitro, suggesting that the inhibition of pi3k/akt pathway by pa may be related to its inhibition of iav infection rather than direct actions on host antiviral response. moreover, the mapk and nf-κb signaling pathways were reported to be required for efficient vrnp export from nucleus and virus rna synthesis, and the inhibitors of mapk pathway could reduce both iav replication and inflammatory symptoms [23] [24] [25] . in this study, pa significantly reduced the activation of erk1/2 rather than nf-κb in iav infected cells, suggesting that erk/mapk rather than nf-κb pathway may be involved in the anti-iav actions of pa. considered that pa could significantly inhibit virus mrna and protein expression in iav infected cells, we posit that pa may interfere with the activation of pi3k/akt and erk/mapk signaling pathways, thus inhibiting the invasion and subsequent replication of iav. the in vivo anti-iav effects of pa were also explored in a murine pneumonia model of influenza. intranasal treatment of pr8-infected mice with pa markedly improved their survival and decreased the pulmonary virus titers (fig. 6) . moreover, the histopathological analysis indicated that pa treatment also (see figure on previous page.) fig. 5 the influence of patchouli alcohol on host antiviral response. a a549 cells were treated with or without pa (6.25, 12.5, 25, 50 μg/ml) for 2 h, and then the phosphorylation of pi3k and akt proteins was evaluated via western blotting. blots were also probed for β-actin and gapdh protein as loading controls. the result shown is a representative of three separate experiments. b plots quantifying the immunoblots (as ratios to β-actin or gapdh) for p-pi3k and p-akt proteins, respectively. the ratios for non-treated cells (mock) were assigned values of 1.0 and the data presented as mean ± s.d. (n = 3). c a549 cells were treated with patchouli alcohol (50, 25 μg/ml) for specified time period, and then the media were removed and cells were overlaid with compound-free media. then at 24 h p.i., the cell viability of a549 cells was measured by mtt assay. values are means ± s.d. (n = 3). d vir09 virus (moi = 0.1) infected cells were treated with or without pa (6.25, 12.5, 25, 50 μg/ml) for 24 h, then the content of ifn-β in the culture supernatants was detected using elisa kits. values are means ± s.d. (n = 3). ##p < 0.01vs. non-infected group (mock control). e and f after treatment of pa (20 or 40 μg/day) for four days in non-infected mice (e) or pr8 virus infected mice (f), the production of interferon-γ (ifn-γ) and interleukin 2 (il-2) in lung tissues was determined by using the elisa kits for ifn-γ and il-2. values are means ± s.d. (n = 3). significance: ##p < 0.01 vs. non-infected mock control group; **p < 0.01 vs. virus control group attenuated the pneumonia symptoms in iav-infected lungs, comparable to the effects of oseltamivir. however, pa treatment exerted obvious therapeutic effect only when starting earlier (4 h p.i.), which will restrict its clinical application to some extent. different to the oral administration of oseltamivir, pa was administrated through intranasal, and low dose therapy of pa (20 μg/day) had comparable anti-iav effects to fig. 6 the anti-iav effects of patchouli alcohol in vivo. a viral titers in lungs. after treatment with oseltamivir (10 mg/kg/day) or pa (20 or 40 μg/ day) for 4 days, the pulmonary viral titers were evaluated by plaque assay. values are the mean ± sd (n = 3). significance: *p < 0.05, **p < 0.01 vs virus control group. b survival rate. iav infected mice received therapy with oseltamivir (10 mg/kg/day) or pa (20 or 40 μg/day) for the entire experiment. results are expressed as percentage of survival, evaluated daily for 14 days. significance: *p < 0.05 vs. virus control group (placebo). c histopathologic analyses of lung tissues on day 4 p.i. by he staining (× 10). the representative micrographs from each group were shown (n = 5 mice/group). mock: non-infected lungs; control: iav infected lungs without drugs; oseltamivir: iav infected lungs with oseltamivir (10 mg/kg/day) treatment; pa 20 μg/day: iav infected lungs with pa (20 μg/day) treatment; pa 40 μg/day: iav infected lungs with pa (40 μg/day) treatment. the red arrows indicate the presence of inflammatory cells in the alveolar walls and serocellular exudates in the lumen oseltamivir (10 mg/kg/day), suggesting that pa may be used alone or combined with oseltamivir for treatment of influenza by different administration. in summary, pa possesses anti-iav activities both in vitro and in vivo, and may block iav infection through targeting virus particles and cellular pi3k/akt and erk/mapk 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with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions ww and ch wrote the manuscript and designed the experiments. yjy, yz, sw and wl performed the experiments. ww, yy and yz analyzed the data. all authors read and approved the final manuscript. this work was supported by national natural science foundation of china (81874320, 81741146, and 31500646), nsfc-shandong joint fund (u1606403, u1706210), and shandong provincial natural science foundation (zr2017mh013). the datasets used during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate this work was approved by the ethics committee of ocean university of china. no applicable. the authors declare that they have no competing interests. key: cord-309010-tmfm5u5h authors: dietert, kristina; gutbier, birgitt; wienhold, sandra m.; reppe, katrin; jiang, xiaohui; yao, ling; chaput, catherine; naujoks, jan; brack, markus; kupke, alexandra; peteranderl, christin; becker, stephan; von lachner, carolin; baal, nelli; slevogt, hortense; hocke, andreas c.; witzenrath, martin; opitz, bastian; herold, susanne; hackstein, holger; sander, leif e.; suttorp, norbert; gruber, achim d. title: spectrum of pathogenand model-specific histopathologies in mouse models of acute pneumonia date: 2017-11-20 journal: plos one doi: 10.1371/journal.pone.0188251 sha: doc_id: 309010 cord_uid: tmfm5u5h pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by streptococcus (s.) pneumoniae, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, legionella pneumophila, escherichia coli, middle east respiratory syndrome (mers) coronavirus, influenza a virus (iav) and superinfection of iav-incuced pneumonia with s. pneumoniae. systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. we therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogenand model-specific patterns of pneumonia. other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. the substantial differences between the model-specific pathologies underscore the necessity of pathogenand model-adapted criteria for the comparative quantification of experimental outcomes. these criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 as one of the most frequent infectious diseases, pneumonia causes a tremendous socioeconomic burden in industrialized countries [1] and is the leading infectious cause of death in children worldwide [2] . numerous classes of pathogens can cause acute pneumonia [3] and the risk of pneumonia is greatly enhanced under conditions of impaired pulmonary host defense, including preceding viral infections [4] , mechanical ventilation [5] and sepsis [6] . the leading causative pathogen of community acquired pneumonia (cap) is the gram-positive bacterium streptococcus (s.) pneumoniae [7, 8] which accounts for the majority of bacterial upper and lower respiratory tract infections and is responsible for millions of deaths annually [9, 10] . as another cause of cap influenza a virus (iav) infection leads to rapid progression of lung failure with limited treatment options and frequent fatal outcome [3, 11, 12] . moreover, iav infections are commonly complicated by bacterial superinfection, mostly caused by s. pneumoniae, resulting in severe progressive pneumonia associated with increased mortality [13] . in contrast, the gram-negative and facultatively intracellular bacterium legionella (l.) pneumophila is the causative agent of the severe cap legionnaires' disease, and the second most commonly detected pathogen in pneumonia in patients admitted to intensive care units (icu) in industrialized countries [14, 15] . however, in addition to cap, ventilator-associated pneumonia (vap) is also a major cause of hospital morbidity and mortality in icus [16] and the spectrum of pathogens is shifted in these forms of pneumonia. here staphylococcus (s.) aureus, klebsiella (k.) pneumoniae, acinetobacter (a.) baumannii, and escherichia (e.) coli have been isolated with varying prevalences [17] [18] [19] . more specifically, the gram-negative k. pneumoniae is a significant opportunistic pathogen causing severe life-threatening hospitalacquired respiratory tract infections [20] [21] [22] while s. aureus, a gram-positive bacterium, is one of the most prevalent pathogens of community-and hospital-acquired lower respiratory tract infections in humans and accounts for a significant health and economic burden [23] [24] [25] . a. baumannii and e. coli are ubiquitous gram-negative bacteria which have recently emerged as major causes of community-associated, nosocomial [26, 27] and ventilator-associated pneumonia [19, 28] as well as septicemia induced acute lung injury (ali) [29, 30] . in addition, more recently discovered pulmonary pathogens indicate that novel emerging diseases may add to the list of highly relevant pneumonias that may also be of interest to be studied in animal models. for example, the middle east respiratory syndrome coronavirus (mers-cov) which is transmitted by dromedary camels as vectors [31] has emerged as the cause of severe human respiratory disease worldwide [32, 33] with elderly and immunocompromised individuals particularly in saudi arabia being at highest risk [34] [35] [36] . the various forms of pneumonia have been successfully reproduced in specific murine models of experimentally-induced acute pneumonia [37] [38] [39] . these models have substantially contributed to our understanding of the pathogenesis of community-and hospital-acquired pneumonia as well as emerging lung infections worldwide and are indispensable for the development of novel therapeutic strategies [40] [41] [42] . histopathology has been a powerful, reliable, and reproducible read-out tool for the evaluation of morphological changes in animal lung infection experiments for many decades [43, 44] . qualitative diagnoses are based on a summation of microscopically observable changes in the morphology and cellular composition of the tissue and cell types involved. for a more comparative inclusion of histopathologic information in biomedical research, scoring systems have been widely applied which allow for a first semiquantitative assessment of lesions compared to controls [44, 45] . moreover, all preclinical models used for the development of novel treatment strategies and acceptance by regulatory agencies need to be assessed histopathologically by board certified pathologists as gold standard for qualitative and semiquantitative evaluation of tissue alterations in experimental animals [46] [47] [48] . previous studies have revealed first fundamental differences in histopathologic lesions caused by different pathogens in mouse lungs [38, 41, 42] . however, scoring schemes for acute murine pneumonia existing to date are very superficial, addressing only a few, rather unspecific parameters [45, 49, 50] . more importantly, they hardly allow for a differentiating perspective between distinct pathogens or for group comparisons, e.g., infections of wild type versus genetically modified mice. clearly, there is a strong need for more precise and pathogen-as well as model-specific parameters to allow for an accurate description and semiquantification of the inflammatory phenotype for reliable and reproducible comparisons between experimental groups within each model. therefore, we have recently adapted more specific scoring criteria for s. pneumoniae and s. aureus-induced pneumonia [38, 42] . however, such pathogenspecific scoring criteria have not been employed for other lung pathogens in mice. here, we systematically describe and compare the histopathologies at their peaks of inflammation and injury of nine previously established acute lung infection models induced by s. pneumoniae, s. aureus, k. pneumoniae, a. baumannii, l. pneumophila, e. coli, mers-cov, iav and superinfection with iav and pneumococci. we provide model-specific criteria that can be used for appropriate histological quantitative comparisons, e.g., when different therapeutic interventions are evaluated within these established models. whole mouse lung sections were used to obtain complete overviews, particularly of the distributions of lesions and inflammatory patterns. on the basis of the different and oftentimes quite pathogen-and model-specific changes we identified the most suitable evaluation criteria for each model that will allow for more accurate semiquantitative assessments of the severities and distributions of pneumonic lesions. the lung tissues examined here were derived from experiments primarily conducted for purposes other than this study and most have been published elsewhere [38, 41, 42, [51] [52] [53] , except for the a. baumannii and e. coli experiments that will be published elsewhere. all animal procedures and protocols were approved by institutional ethics committees of charité-university of berlin, justus-liebig university of giessen, philipps university of marburg, university hospital of jena and local governmental authorities (landesamt für gesundheit und soziales (lageso) berlin, regierungspräsidium (rp) gießen and darmstadt, landesamt für verbraucherschutz (tlv) thüringen), respectively. permit numbers were g 0356/10, a-0050/15 (s. pneumoniae), g 0358/11 (s. aureus), g 75/2011, g 110/2014 (k. pneumoniae), a 0299/15 (a. baumannii), g 0175/12 (l. pneumophila), 02-049/12 (e. coli), 114/2012 (mers-cov), g 0152/ 12, and g 0044/11 (iav and superinfection). all animal studies were conducted in strict accordance with the federation of european laboratory animal science associations (felasa) guidelines and recommendations for the care and use of laboratory animals, and all efforts were made to minimize animal discomfort and suffering. all mice, except for mers-cov infected mice, were monitored at 12 hour intervals throughout the experiment to assess appearance, behavior, grooming, respiration, body weight, and rectal temperature. humane endpoints were defined (body temperature <30˚c, body weight loss = 20%, cumbersome breathing, accelerated breathing in combination with staggering, pain or paleness) but not reached by any of the mice at the indicated time points of termination of the experiments. mers-cov infected mice were monitored once daily and appearance, behavior, grooming, respiration and body weight were protocolled. here, a single humane endpoint (loss of body weight of >15%) was defined but not reached by any of the mice employed due to their favourable clinical outcome at the infection dose used. for all experimental infection models, except of k. pneumoniae, female mice (aged 8-12 weeks and weighing 17-22 g) were randomly assigned to groups (n = 2-4) per cage whereas in the k. pneumoniae model female and male mice (aged 23-25 weeks and weighing 22-24 g) were used for model specific reasons [41] . furthermore, for all experimental infection models, specificpathogen-free (spf) mice on c57bl/6 (all except for mers-cov) or balb/c (as previously used for the mers-cov model [52, 54] ) background were used and housed in individually ventilated cages under spf conditions with a room temperature of 22 ± 2˚c and a relative humidity of 55 ± 10%. a 12 hour light/ 12 hour dark cycle was maintained and the animals had unlimited access to standard pellet food and tap water. all experimental details of the infection models compared here were applied following previously published and well established protocols that partly vary in terms of infection doses, routes of infection and time point of examination due to pathogen-or model specific reasons, as given below. for bacterial infections, except for e. coli, mice were anesthetized intraperitoneally with ketamine (80 mg/kg) (ketavet; pfizer, berlin, germany) and xylazine (25 mg/kg) (rompun; bayer, leverkusen, germany). for experimental viral infections, mice were anesthetized using inhalation of isoflurane (forene; abbott, wiesbaden, germany). for lung histology, all mice except of the mers-cov model were humanely euthanized by exsanguination via the caudal vena cava after anesthesia by intraperitoneal injection of premixed ketamine (160 mg/kg) and xylazine (75 mg/kg). mers-cov infected mice were humanely euthanized by cervical dislocation after isoflurane anesthesia. s. pneumoniae (serotype 3 strain, nctc 7978), s. aureus newman (nctc 10833), k. pneumoniae (serotype 2, atcc 43816), a. baumannii (ruh 2037), l. pneumophila (serogroup 1 strain, jr 32) were cultured as described [37, 38, 40, 55, 56] and resuspended in sterile pbs. mice were anesthetized intraperitoneally (i.p.) with ketamine (80 mg/kg) and xylazine (25 mg/kg) and transnasally inoculated with 5 x 10 6 cfu s. pneumoniae (n = 14 mice), 5 x 10 7 cfu s. aureus (n = 4), 5 x 10 8 cfu a. baumannii (n = 8), in 20 μl pbs. mice transnasally infected with l. pneumophila (n = 8) received 1 × 10 6 cfu in 40 μl pbs. mice infected with k. pneumoniae (n = 16) received 3.5 x 10 5 cfu intratracheally in 50 μl nacl (0.9%) via microsprayer1 aerosolizer (model ia-1b, penn-century, inc., wyndmoor, pa) using intubation-mediated intratracheal instillation through intact airways [57] which has previously been optimized for this model [41, [57] [58] [59] . e. coli (atcc 25922) from -80˚c glycerol stock was added to lb broth (carl roth, karlsruhe, germany) and incubated for 12 hours at 200 rpm and 37˚c with 5% co 2 . optical density of 0.03 was adjusted in lb broth followed by incubation until midlogarithmic phase for 1.5 hours at 200 rpm and 37˚c. after centrifugation, the pellet was resuspended in sterile 0.9% nacl at 8 x 10 5 cfu e. coli / 200 μl and administered intraperitoneally (n = 10). for initial transduction of human dpp4 for subsequent infection of balb/c mice with mers-cov (hcov emc) viruses were cultured and prepared as described [52, 54, 60] . mice were transduced transnasally with 20 μl of an adenovirus vector encoding human dpp4 and mcherry with a final titer of 2.5 x 10 8 pfu per inoculum (adv-hdpp4; viraquest inc.), resulting in hdpp4 expression in the epithelial compartment of the lung [60] and transnasally infected with a final titer of 7 x 10 4 tcid 50 of mers-cov in 20 μl dmem (n = 17) under isoflurane anesthesia (forene; abbott, wiesbaden, germany). influenza a/pr/8/34 virus (h1n1; pr8) was grown as described [42] and mice were transnasally infected with 100 pfu pr8 in 50 μl pbs (n = 4) under isoflurane anesthesia. for superinfection experiments, the iav infection procedure was applied as described above with 40 pfu pr8 in 50 μl pbs. 8 days after viral infection, s. pneumoniae was cultured as described [37] and resuspended in sterile pbs. mice were anesthetized intraperitoneally and transnasally inoculated with 1 x 10 3 cfu s. pneumoniae in 20 μl pbs (n = 4). mice were humanely euthanized at model-specific time points as indicated ( table 1 ). between 2 and 6 repetitions of the entire experimental procedures were performed in each model with similar group sizes in each repetition. lungs were carefully removed after ligation of the trachea to prevent alveolar collapse, immersion-fixed in formalin ph 7.0 for 24 to 48 hours (mers-cov for 7 days), embedded in paraffin, cut in 2 μm sections and stained with hematoxylin and eosin (he) after dewaxing in xylene and rehydration in decreasing ethanol concentrations. bacteria were visualized using the giemsa and gram (modified by brown and brenn) stains. for the display of whole lung overviews, he stained slides of entire lung sections were automatically digitized using the aperio cs2 slide scanner (leica biosystems imaging inc., ca, usa) and image files were generated using the image scope software (leica biosystems imaging inc.). three evenly distributed whole-organ horizontal sections throughout the entire lungs were microscopically evaluated to assess the distribution and character of pathologic alterations, generating a modified panel of specific lung inflammation parameters adapted to each pathogen used (table 1 and table 2 ). all examinations were performed by trained veterinary experimental pathologists. for immunohistochemical detection of s. pneumoniae and iav (h1n1), antigen retrieval was performed with microwave heating (600 w) in 10 mm citric acid (750 ml, ph 6.0) for 12 minutes (min). lung sections were then incubated with a purified rabbit antibody polyclonal to s. pneumoniae (1:2,000, kindly provided by s. hammerschmidt) or with a purified goat antibody polyclonal to iav h1n1 (1:4,000, obt155, bio-rad, puchheim, germany) at 4˚c overnight. incubation with an immuno-purified rabbit or goat antibody at the same dilution served as negative controls. subsequently, slides were incubated with a secondary, alkaline phosphataseconjugated goat anti-rabbit (1:500, ap-1000, vector, burlingame, ca) antibody for 30 min at room temperature. the alkaline chromogen triamino-tritolyl-methanechloride (neufuchsin) was used as phosphatase substrate for color development. all slides were counterstained with hematoxylin, dehydrated through graded ethanols, cleared in xylene and coverslipped. transnasal infection of mice with s. pneumoniae, serotype 3 resulted in a broad spectrum of tissue lesions and immune cell infiltrations that are typical of aerogenic bacterial pneumonia. specific for this model, lesions widely expanded down to the periphery of the lung lobes (fig 1a) with inflammation closely surrounding the airways and blood vessels. pneumococcal spread led to an early immune response which was mainly characterized by predominantly intrabronchial ( fig 1b) and intraalveolar ( fig 1c) infiltrations of neutrophils provoking a lobular, suppurative bronchopneumonia with consolidation of affected lung areas. large areas of coagulation and liquefaction necrosis (fig 1d, arrowhead) as indicated by cellular fragmentation, decay, and loss of cellular details, accumulation of cellular and karyorrhectic debris as well as karyorrhexis, karyopyknosis, and karyolysis with consecutive hemorrhage were also present. the perivascular interstitium was widely expanded by edema due to vascular leakage [53] with massive extravasation of neutrophils recruited into perivascular spaces ( fig 1e) . furthermore, suppurative and necrotizing vasculitis accompanied by hyaline thrombi within small-sized blood vessels were occasionally present, indicating early histological evidence of incipient septicemia. increased pulmonary vascular permeability [53] also led to expanded areas of protein-rich alveolar edema which presented as homogenous, lightly pink material within the alveolar spaces in the he stain (fig 1f, asterisk) . a distinctive histopathological feature of pneumococcal pneumonia was the occurrence of massive suppurative to necrotizing pleuritis (fig 1g, arrowhead) and steatitis ( fig 1h) with widespread dispersion of bacteria into the thoracic cavity, likely accounting for the painful and morbid clinical behavior with rapid progression in affected mice. myriads of pneumococci were clearly visible as bluish to purple dots of approximately 1 μm in size in the standard he stain, mostly located on the pleural surface, in the mediastinal adipose tissue or within perivascular spaces in the lungs. + + ++ ++ ++ abscess formation ++ ++ + granuloma formation ++ alveolar edema ++ + ++ + + ++ ++ perivascular edema ++ ++ + + + + perivascular lymphocytic cuffing ++ + ++ ++ ++ + + ++ + vasculitis ++ + + + ++ + fibrinoid degeneration of vascular walls ++ vascular thrombosis +/++ ++ ++ ++ + + + ++ + + pleuritis ++ ++ ++ ++ ++ ++ in contrast, transnasal infection with s. aureus resulted in multifocally extensive but non-expansive bronchopneumonia predominantly located near the lung hilus (fig 2a) , affecting the bronchi, alveoli and interstitium. the main inflammatory cell population consisted of neutrophils, leading to mainly suppurative (fig 2b and 2c ) lesions with a tendency towards abscess formation. in contrast to pneumococci, macrophages were also present albeit at lower numbers than neutrophils. (fig 2c) . similar to klebsiella and streptococci, large areas of necrosis and hemorrhage ( fig 2d) were present. the perivascular areas were predominantly infiltrated by lymphocytes and fewer neutrophils (fig 2e) . compared to the s. pneumoniae model mentioned above [53] , vascular permeability seemed only slightly increased as reported before [38] and perivascular edema (fig 2e) as well as protein-rich alveolar edema (fig 2f, asterisk) were also present albeit to a lesser extent. neither pleuritis nor steatitis were observed consistent with a rather favorable clinical outcome under the conditions used. furthermore, staphylococci were largely undetectable by he stain which was possibly due to the low bacterial spread within the lungs. intratracheal infection of mice with k. pneumoniae resulted in severe widely expansive bronchopneumonia with increased lesion severity in the lung periphery ( fig 3a) . recruited immune cells predominantly consisted of neutrophils, leading to suppurative ( fig 3b) to abscessing ( fig 3c) bronchopneumonia with hemorrhage and necrosis as well as neutrophilic interstitial pneumonia (fig 3d) in less affected areas. increased vascular permeability as reported [40] was associated with massive alveolar (fig 3d, asterisk) and perivascular edema (fig 3e) , admixed with myriads of bacteria easily recognizable as purple dots in the he stain. suppurative to necrotizing vasculitis, pleuritis ( fig 3f, arrowhead) , and steatitis were also present and associated with marked bacterial spread and the rapid lethal clinical outcome. after transnasal infection with a. baumannii, mice developed a widely expansive ( fig 4a) bronchopneumonia with predominantly infiltrating neutrophils causing a suppurative ( fig 4b) to abscessing inflammation with areas of hemorrhage within alveoli and interstitium and large areas of parenchymal necrosis as well as alveolar edema. perivascular spaces had mild to moderate edema and infiltration of lymphocytes and neutrophils ( fig 4c) . vascular thrombosis was a common change (fig 4d, arrowhead) in small-sized blood vessels. similar to staphylococci, acinetobacter was invisible by he stain and neither pleuritis nor steatitis were present. transnasal infection of mice with l. pneumophila resulted in slightly different lesion patterns depending on the time point of examination after infection. at 48 hours after infection, nonexpansive interstitial pneumonia was found in close proximity to the hilus (fig 5a) with comparative histopathology of mouse models of acute pneumonia prominent alveolar wall necrosis (fig 5b) . at the 6 day-time point, specifically the numbers of infiltrating macrophages were clearly increased, leading to accentuated perivascular granuloma formation (fig 5c, arrowhead) . here, marked lymphocytic cuffing of most blood vessels as well as highly activated endothelial cells (fig 5d, arrowhead) were observed. at both time points, neither pleuritis nor steatitis were present. bacteria were invisible in the he stained sections. after intraperitoneal infection, the hematogeneous spread of e. coli to the lungs had resulted in diffuse, interstitial suppurative pneumonia, diffusely affecting the entire lungs, modelling sepsis-induced ali (fig 6a) . the interalveolar interstitium was heavily infiltrated with neutrophils ( fig 6b) with most prominent aggregation around blood vessels (fig 6c) , consistent with bacterial entry via the circulation. numerous hyaline thrombi were present within small-sized blood vessels (fig 6d, arrowhead) , suggestive of disseminated intravascular coagulopathy (dic) due to bacterial septicemia. large, rod-shaped bacteria were easily detectable only outside the lungs, mostly present in the adipose tissue surrounding the esophagus, possibly due to local spread of e. coli via the abdominal cavity. transnasal infection with mers-cov following adenoviral transduction of human dpp4 yielded an expansive, (fig 7a) interstitial pneumonia with severe alveolar epithelial cell necrosis and infiltration of mainly macrophages, lymphocytes, and fewer neutrophils (fig 7b) . only comparative histopathology of mouse models of acute pneumonia moderate peribronchial ( fig 7c) and perivascular (fig 7d) lymphocytic infiltrations were present while most venous blood vessels had marked fibrinoid degeneration and necrosis of comparative histopathology of mouse models of acute pneumonia vascular walls (fig 7d, asterisk) . additional hallmarks of mers-cov infection were large areas of protein-rich alveolar edema (fig 7e, arrowhead) , pronounced hemorrhage within perivascular and alveolar spaces, and interstitium (fig 7f, arrowhead) , and the formation of hyaline thrombi within small-sized blood vessels. after transnasal infection with iav, mouse lungs displayed a diffusely distributed bronchointerstitial pneumonia restricted to single lung lobes only (fig 8a) . alveolar necrosis was prominent and alveolar septae were diffusely distended by infiltrating inflammatory cells (fig 8b) . bronchial epithelial cells were markedly necrotic (fig 8c, arrowhead) and extensively scaled off into the bronchial lumen. alveoli and interstitium were filled with macrophages and lymphocytes as major effector cells ( fig 8d) and prominent perivascular lymphocytic cuffing ( fig 8e) was a characteristic change. furthermore, large areas of alveolar edema (fig 8f, asterisk) and, albeit to a much lesser extent, areas of hemorrhage within alveoli and interstitium were present, suggesting vascular damage and increased permeability. when mice had been infected with iav prior to infection with s. pneumoniae, a combination and exponentiated phenotype of both models was observed 24 hours later. lesions were widely expansive to the lung periphery but restricted to single lung lobes pre-damaged by iav ( fig 9a) . the character of pneumonia included massive infiltration of neutrophils into alveoli ( fig 9b) and bronchi, typical features of severe, suppurative bronchopneumonia. bronchial epithelium was almost entirely necrotic and bronchi were filled up with pus ( fig 9c) . perivascular spaces were edematous and infiltrated by neutrophils and lymphocytes (fig 9d) whereas only mild lymphocytic perivascular cuffing (fig 9e) was present. a severe proteinrich alveolar edema was seen, similar to that seen in the s. pneumoniae mono-infection (fig comparative histopathology of mouse models of acute pneumonia 9f). pneumococci were difficult to visualize by he stain, possibly due to the lower infectious dose used here when compared to the mono-infection. prior to processing for histopathology, small tissue samples from experimentally infected mouse lungs are commonly removed for molecular analyses of gene and/ or protein expression or other readout systems to receive additional information. to obtain representative data from such samples that can be correlated with the histological changes, it is crucial to know about the homogeneity of the distribution of lesions. also, some experimental protocols recommend to use the left and right halves of the lungs, respectively, for different analytical procedures, again anticipating lesion homogeneity and symmetry. however, when we analyzed the distributions and bilateral symmetry of lung lesions for each of the infection models, we found a wide spectrum of distinct distributions and asymmetries (fig 10) . in principle, lesion distributions followed the route of pathogen entry into the lungs. however, the tendencies to spread towards the periphery of the lobes after aerogenous infection varied between different pathogens despite similar aerogenous infection routes. mostly centrally focused lesions induced by s. aureus and l. pneumophila remained close to the hilus with no trend towards peripheral expansion. infection with s. pneumoniae, a. baumannii and mers-cov resulted in lesions closely surrounding major airway segments with centrifugal expansion towards the periphery. in contrast, lesions induced by k. pneumoniae were mostly located in the periphery of the lobes and airways and much weaker adjacent to the hilus despite aerogenous infection. hematogenously-induced sepsis with e. coli was associated with an entirely diffuse distribution of lesions affecting the whole lung with myriads of inflammatory hot spots, commonly surrounding blood vessels. iav-induced lesions were restricted to individual lung lobes only with a rather homogeneous distribution within affected lobes. superinfection of s. pneumoniae into an iav-pneumonia resulted in a pattern virtually identical to that seen after iav infection alone. except for blood borne e. coli pneumonia which was consistently and evenly distributed throughout the entire lungs, affected areas in all other models tested here were randomly distributed more or less asymmetrically between the right and left halves of the lungs and also between adjacent lobes (fig 10) . for more than 100 years, a wide range of special stains have been used for the histological visualization of pathogens and other relevant structures, based on their more or less specific affinities to certain dyes. here, gram stain modified by brown and brenn was used for the visualization of gram-positive bacteria, including s. pneumoniae (fig 11a, arrowhead) , as easily recognizable, dark blue cocci. in contrast, giemsa stain was conducted predominantly for the detection of gram-negative bacteria such as k. pneumoniae (fig 11b, arrowhead) which then turned into light blue to greenish rods. for more specific pathogen detection on slides, particularly for viruses, immunohistochemistry is the method of choice. here, s. pneumoniae and iav were detected by immunohistochemistry using specific anti-s. pneumoniae or anti-iav antibodies, respectively. s. pneumoniae-positive signals were obtained as myriads of red cocci predominantly in the perivascular interstitium (fig 11c) , within neutrophils in alveoli and interstitium, and on pleural surfaces as well as in mediastinal adipose tissue. in addition, pneumococci were also visualized in the marginal sinuses of tracheal lymph nodes, both in macrophages and extracellularly. iav antigen was localized to the apical surface and cytosol of intact and necrotic bronchial epithelial cells (fig 11d) and within alveolar macrophages. the diversity of lesions and in particular the presence or absence of specific patterns in several of the models used ( table 1 ) strongly suggested that a uniform scoring scheme for the pneumoniae were mostly located in the periphery of the lobes and airways and much weaker adjacent to the hilus. hematogenous infection with e. coli was associated with entirely diffuse distribution of lesions affecting the whole lungs with myriads of inflammatory hot spots, commonly surrounding blood vessels. iav-induced lesions were restricted to individual lung lobes with a rather homogeneous distribution within affected lobes. which lobes were affected followed a rather random and inconsistent pattern. superinfection of s. pneumoniae into an iav-pneumonia resulted in a pattern virtually identical to that seen after iav infection alone. except for e. coli induced pneumonia, virtually all lung lesions were distributed asymmetrically between the left and right lung halves with no tendency of either half to be more often or more strongly affected. https://doi.org/10.1371/journal.pone.0188251.g010 comparative histopathology of mouse models of acute pneumonia semiquantification of mouse pneumonia is inconceivable. instead, scoring systems should take into account the more or less pathogen-specific lesion patterns that can be distilled from the comparative characterizations given above. for this purpose, we carved out the most characteristic lesion patterns that appear suitable for the development of specific scoring schemes for each model (table 2 ). different mouse models of acute pneumonia differ widely, with an obvious strong dependence on pathogen-specific features of virulence and spread, route of infection, infectious dose and other factors. here, we provide a detailed descriptive overview of histopathological features and distributions of lesions within infected lungs and compare them between nine relevant and commonly used infection models at their peaks of injury and inflammation. the models employed here all represent well established protocols that have been optimized and successfully used in previous studies, with model-specific variations in infection doses, routes of pathogen administration and analyzed time points [37-42, 51, 52, 54-56] . our model-specific description parameters (table 2 ) provide a rational for the selection of histopathological quantification criteria, in order to best reflect the model-specific lesion and distribution characteristics, which appear to be most relevant. clearly, the severity of lesions in terms of outcome of quantification systems will depend on several other factors that will have to be addressed separately in each model, such as the infection dose, time point of examination or therapeutic interventions. comparative histopathology of mouse models of acute pneumonia the model-associated characteristics of tissue lesions and immune cell infiltrates are widely consistent with well-established properties of the different pathogens used. for example, the destructive tissue damage with mostly neutrophilic infiltrations as seen in s. pneumoniae, s. aureus, and a. baumannii, are typically seen with extracellular bacteria that express cytotoxic virulence factors, such as pneumolysin and hydrogen peroxide [61] from s. pneumoniae or immunogenic cell wall components such as lipoteichoic acid (lta) from s. aureus [62, 63] . on the other hand, the intracellular pathogen l. pneumophila which primarily infects macrophages [64] resulted in a histiocytic infiltrate at 48 h that developed into granulomatous inflammation at 6 days after infection, typical of a t h 1-response [65, 66] . however, several of the pathogens used were associated with additional distinctive features. for example, histology revealed marked pleuritis and steatitis due to pathogen invasion into adjacent extrapulmonary tissues after infections with s. pneumoniae and k. pneumoniae. this massive bacterial spreading throughout the thoracic cavity was exclusively present in these two models and most likely associated with sepsis, responsible for the rapid clinical progression and unfavourable outcome [41, 67] . however, only k. pneumoniae had the tendency of abscess formation which was not seen in pneumococcal pneumonia. infection with s. aureus and a. baumannii also resulted in similar lesion patterns, except for acinetobacter-induced lesions widely expanding to the lung periphery while staphyloccocus-induced pneumonia was restricted to the lung hilus. a second difference between the two was the presence of prominent vascular thrombosis in a. baumannii-induced pneumonia which was absent from staphylococcus pneumonia. the clinical outcome of mice infected with a. baumannii and s. aureus was more favourable when compared to infection with s. pneumoniae or k. pneumoniae [38] which may be explained by the lack of bacterial spreading throughout the thoracic cavity and adjacent tissues, and possibly sepsis. e. coli infection was included here as a model for sepsis-associated ali [29, 30, 68] and consequently induced wide spread vascular thrombosis and vasculitis, most likely due to its blood borne entry into the lungs and concurrent septicemia with disseminated intravascular coagulopathy and associated vascular lesions. vascular thrombosis with or without vasculitis was also observed in other models, including s. pneumoniae, a. baumannii and mers-cov, however, to a much lesser extent and only within the most strongly affected areas. mers-cov and iav-associated lesions clearly reflected the known cellular tropisms of these viruses with necrosis of alveolar walls or bronchial epithelial cells, respectively, being the most characteristic histopathologic features [69] [70] [71] [72] . typical of virally induced lesions, the inflammatory cell infiltrates in mers-cov and iav infections were dominated by lymphocytes with no or only few neutrophils. nevertheless, the two viral models could be clearly distinguished from each other by additional histological characteristics. only the mers-cov infection led to a marked vascular phenotype with necrosis and degeneration of blood vessels, vasculitis, and consecutive vascular thrombosis as well as pronounced hemorrhages [69, 73] . in contrast, iav-induced pneumonia did not display any of these features, but was dominated by marked perivascular lymphocytic cuffing and alveolar edema [42, 74] . subsequent superinfection with low-dose s. pneumoniae potentiated the severity of the iav-induced lesions and aggravated the course of pneumonia. however, it did not alter the principal histological characteristics of iav-pneumonia. the patterns seen after single infection with s. pneumonia were not repeated in this superinfection model, likely owing to the much lower inoculation dose which is usually rapidly cleared from virus-naive lungs. when the distributions of lesions were compared among the 9 models tested, four distinct patterns could be clearly distinguished. the most common pattern, where lesions were focused around central airways and blood vessels close to the lung hilus with the periphery less or not affected can likely be explained by the aerogenous route of infection and pathogen entry. the opposite pattern characteristic of k. pneumoniae infection where the periphery of the lobes was more strongly affected than their hilus areas despite a similar aerogenous route of infection may be due to the aerosolic intratracheal application [75] of these bacteria which is typical of and necessary in this model [41, [57] [58] [59] . these differences are therefore more likely attributable to the model-specific route of infection rather than pathogen-specific properties. similarly, the very homogenous distribution of e. coli induced pneumonia likely followed the diffuse blood borne entry of the pathogen into the lungs after intraperitoneal infection. again unique among the pathogens tested here, the iav-associated pattern affected entire but only select lung lobes with almost complete sparing of others. this distribution probably followed a random spread of the virus along major airways but why some lobes remained virtually unaffected after transnasal infection remains hard to explain. apart from helping to understand differences in pathogen spread, the uneven and often quite asymmetrical distributions have a tremendous impact in practical terms when acute mouse pneumonia is sampled for molecular studies. when quantitative data on mrna or protein expression levels or other biochemical information are to be compared with one another or with tissue lesions, it is imperative that only identically affected areas are compared. since this is impossible to predict or recognize on the macroscopical level for most models, the practice of sampling different regions of such lungs for different readout systems appears highly problematic. another implication of the distinct lesion characteristics, immune cell reactions and distributions among the different models appears highly relevant for histological scoring systems that aim at first quantitative comparisons [45] . to narrow down the list of parameters appropriate for each pathogen and exclude features that are likely irrelevant for some of the models, we selected 23 single histopathologic criteria for the design of semiquantitative scoring systems suitable for each model. these criteria are partly composed of standard parameters such as the determination of the affected lung area, the distribution of lesions or the type of pneumonia induced. however, numerous other and more model-specific parameters were identified which precisely describe particular aspects and allow for a differentiation between the models, such as the presence of perivascular edema, vascular thrombosis, pleuritis or steatitis. appropriate scoring systems may thus encompass more general parameters when different pathogens are compared to one another or more pathogen-specific parameters in case specific pathogen features are in the focus of an experiment. for example, some of the parameters selected here have proven helpful in the discovery and semiquantification of different phenotypes of mouse pneumonia following genetic engineering of pathogens or mice [38, 51, 76] . however, scoring systems that claim universality for all mouse models of acute pneumonia seem neither generally applicable nor meaningful for all specific experimental goals. even the list of 23 parameters selected here may become inappropriate or insufficient when genetic changes on the pathogen or host side may result in different types of lesions, immune cell responses, time courses or other relevant features. in those cases, the list selected here may have to be adjusted or extended to better meet the specific challenges of each new study. as standard hematoxylin and eosin (he) staining of tissue sections failed to visualize most pathogens, traditional special stains as well as immunohistochemical techniques were employed, depending on specific staining properties of the pathogens and the availability of appropriate antibodies. while s. pneumoniae, k. pneumoniae and e. coli were easily visible in he stained tissue sections in areas with low density of inflammatory cells, e.g., in perivascular spaces, they were very difficult to identify in heavily infiltrated and consolidated lung parenchyma. in contrast, s. aureus, a. baumannii, l. pneumophila and both viruses were entirely invisible by he staining and thus had to be visualized by appropriate histotechnical stains or immunohistochemistry. both approaches will likely also allow for a rough quantification of pathogen numbers in tissues when appropriate image analysis tools are used. in this first comparative study of its kind, we examined previously established models with their optimized routes of infection, time points, and infection doses and volumes specific for each model to reach peaks of lung injury and inflammation. variations of such factors can be expected to result in different lesion severities, composition of the cellular infiltrates, and for some models in different expansions of lesions within the lung. still, the conditions used here are all based on observations that have evolved during extensive previous establishment studies of these models [37-39, 41, 42, 51, 52, 54, 56-58, 60, 77-79] . among the most important reasons, most human pathogens are not pathogenic for mice under non-experimental conditions and the decisive factor for obtaining a useful pneumonia model appears to be the determination of the appropriate infection dose and route of infection. in addition, the exact time points of tissue analysis after infection had to be determined for virtually all models with care to obtain a useful model, including a precise definition of the strain or variant of the pathogen used [51, 53, 80, 81] . another variable to consider is the mouse strain used. except for balb/c mice which were used in the mers-cov infection model here for model-specific reasons [60] , all models were conducted with c57bl/6 mice which is among the most commonly used mouse strain in infection research and therefore allows for comparisons with similar studies. however, variations of the strain or genetic background may have a dramatic impact on the type, severity and outcome of inflammation, particularly in innate immune responses [82] [83] [84] . again, the criteria suggested here for scoring procedures should allow to recognize and quantify such differences related to changes in infection dose and volume, time point of examination, strain and age of mice used, pathogen variant and other variables. histopathology of the lungs may be complex and requires fundamental knowledge in species-specific anatomy, physiology, organ-specific immunology, pathology, and histotechnical procedures. furthermore, various background lesions in mice, including strain specific spontaneous degenerative or inflammatory conditions and the possibility of accidental infections unrelated to the experiment should not be confused with experimental outcome. thus, despite our efforts to specify and simplify the criteria relevant for model-specific assessment and quantification of lesions, it appears crucial that trained histopathology experts be involved in the microscopical examination of mouse lungs [46, 85] . clearly, in addition to descriptive or semiquantitative histology, a number of other parameters may be useful for quantitative comparisons between experimental groups to determine the role of specific cell types, molecules, and therapeutic interventions, depending on the strategy and goal of the study [44] . such parameters could include flow cytometric immune cell identifications and quantifications, elisa or quantitative rt-pcr for the probing of cytokines, chemokines or matrix proteins involved in lung pathology and remodeling, and plaque/colony forming assays for the identification or quantification of pathogens, as previously published for most of the models used here [38, 41, 42, 51-53, 86, 87] . all of these methods, however, lack the spatial resolution that only histological assessments offer. only the combination of these techniques will lead to a better understanding of the disease in the complex context of the entire lung pathology. in conclusion, we have identified a spectrum of pathogen-and model-specific lesion characteristics in mouse models of acute pneumonia. our findings underscore the necessity of model-specific criteria for the accurate histopathological characterization and quantitative assessments of experimental pneumonia. 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subunit expression and regulation in pneumococcal pneumoniacomparison to chlamydial lung infection influenza h3n2 infection of the collaborative cross founder strains reveals highly divergent host responses and identifies a unique phenotype in cast/eij mice strain differences in a murine model of air pollutant-induced nonatopic asthma and rhinitis. toxicologic pathology murine strain differences in inflammatory angiogenesis of internal wound in diabetes the ecvp/esvp summer school in veterinary pathology: high-standard, structured training for young veterinary pathologists miniaturized bronchoscopy enables unilateral investigation, application, and sampling in mice il-37 causes excessive inflammation and tissue damage in murine pneumococcal pneumonia the authors thank charlene lamprecht and angela linke for excellent technical assistance and nancy a. erickson for helpful discussions. key: cord-002823-n55xvwkf authors: halstead, e. scott; umstead, todd m.; davies, michael l.; kawasawa, yuka imamura; silveyra, patricia; howyrlak, judie; yang, linlin; guo, weichao; hu, sanmei; hewage, eranda kurundu; chroneos, zissis c. title: gm-csf overexpression after influenza a virus infection prevents mortality and moderates m1-like airway monocyte/macrophage polarization date: 2018-01-05 journal: respir res doi: 10.1186/s12931-017-0708-5 sha: doc_id: 2823 cord_uid: n55xvwkf background: influenza a viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. application of high gm-csf levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. methods: mice were infected intranasally with influenza a virus (pr8 strain). supra-physiologic levels of gm-csf were induced in the airways using the double transgenic gm-csf (dtgm) or littermate control mice starting on 3 days post-infection (dpi). assessment of respiratory mechanical parameters was performed using the flexivent rodent ventilator. rna sequence analysis was performed on facs-sorted airway macrophage subsets at 8 dpi. results: supra-physiologic levels of gm-csf conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (bal) fluid to near baseline levels. transcriptome analysis, and subsequent validation elisa assays, revealed that excess gm-csf re-directs macrophages from an “m1-like” to a more “m2-like” activation state as revealed by alterations in the ratios of cxcl9 and ccl17 in bal fluid, respectively. ingenuity pathway analysis predicted that gm-csf surplus during iav infection elicits expression of anti-inflammatory mediators and moderates m1 macrophage pro-inflammatory signaling by type ii interferon (ifn-γ). conclusions: our data indicate that application of high levels of gm-csf in the lung after influenza a virus infection alters pathogenic “m1-like” macrophage inflammation. these results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury. electronic supplementary material: the online version of this article (10.1186/s12931-017-0708-5) contains supplementary material, which is available to authorized users. each year, influenza a virus (iav) affects a significant proportion of the population [1] and causes pathologic changes both through direct cellular toxicity causing desquamation, de-ciliation, and cell death, and through indirect effects by stimulating an anti-viral immune response leading to collateral injury [2] . this combination can lead to an ards-like syndrome characterized by increased capillary leak, oxygen diffusion difficulty and ventilation/perfusion mismatch [1] . immune strategies that protect the host's lung function while still allowing for an adequate immune response to clear the viral load and resolve virus-induced pneumonia are needed. a number of pre-clinical studies have tested prophylactic gm-csf both as vaccine adjuvant and local supplementation against iav infection with encouraging results [3] [4] [5] [6] . the effect of local elevation of gm-csf on iav infection in the lung has been investigated in transgenic models with expression of gm-csf under the control of constitutive or doxycycline-inducible promoters in lungs of alveolar or small airway epithelial cells of gm-csf knockout (csf2 −/− ) mice [3, 4] . differential effects on morbidity and mortality from iav infection in these studies was associated with increased alveolar macrophage (am) numbers in the constitutive gm-csf expression models [3, 5] and am differentiation in the gm-csf-inducible model [4] . differential results on morbidity and survival were also obtained after prolonged or brief administration of supra-physiological levels of gm-csf before or at the onset of iav infection [6] . the question of whether therapeutic administration of gm-csf to the airways after establishment of the infection would confer protection has never been addressed. in this study we use a more clinically relevant model to examine whether supra-physiologic levels of gm-csf in the airways, induced after iav infection at the peak of virus replication, provided therapeutic benefit. using gm-csf-inducible mice on the wt c57bl/6 genetic background we show that airway gm-csf over-expression starting at 3 days post infection (dpi) provides protection from mortality and prevents the degeneration of multiple lung mechanical properties. to examine the mechanism of protection conferred by therapeutic gm-csf levels, we measured respiratory and biochemical parameters of lower airway disease, and analyzed the transcriptome of facs-sorted ams and exudative macrophages (em) from iav-infected mice. our findings demonstrate that gm-csf restores proteostasis of exudate proteins and redirects responsiveness of ams and ems from an m1-like to an m2-like activation state, and prevents mortality from influenza-induced ards. the double transgenic gm-csf (dtgm) mice were bred as previously described [4] , but this time on the wild-type c57bl/6 j background. littermate control (lm) mice were defined as being single transgenic littermates of dtgm mice that were only positive for the scgb1a1-rta and thereby did not have the cmv-gm-csf gene, which may potentially be virally induced in the absence of tetracycline (doxycycline) [7] . dtgm mice and lm controls were exposed to 1 mg/ml doxycycline in drinking water, and doxycycline-containing drinking water was replenished every 2-3 days. both male and female mice were used for all experiments; all mice were sex-and age-matched to control mice. the influenza strain a/puerto rico/8/34 (pr8) virus was a kind gift of dr. kevan hartshorn, and was grown in the chorio-allantoic fluid of ten (10) day old specific pathogen free avian supplies (spafas) chicken eggs purchased from charles river laboratories (wilmington, ma) and purified on a discontinuous sucrose gradient as previously described [8] . mice were anesthetized with ketamine/xylazine and intranasally (i.n.) infected with iav virus in 40 μl of pbs. mice were infected in a bsl2 biosafety cabinet and housed within filter-top microisolator cages in the pulmonary immunology and physiology (pip) core, a bsl2 facility in the department of comparative medicine's animal facility at penn state university college of medicine. mice were observed at least twice daily during infections to assess morbidity and mortality. based on our experience at our facility in the last 2 years and the variable clinical presentation of the influenza infection, we used other metrics to monitor morbidity in addition to mouse body weight curves [9] . mice that exhibited immobility, ruffled hair, and labored breathing that had no chance of recovery, coinciding with approximately 30% of body weight loss, were euthanized by ketamine/xylazine overdose and cervical dislocation, and counted as dead. alternatively, mice that were sleeping but had normal breathing and body appearance, i.e., no ruffled hair or labored breathing, reached up to 35-40% body weight loss and then began to recover normally. mice with favorable prognosis but with 30% body weight loss or greater were provided supportive care with food and hydrated gel packs at the bottom of the cage. we have not found a pattern of clinical disease specific to mouse genotype or gender in untreated mice. mice were anesthetized with ketamine/xylazine (130 mg/kg and 10 mg/kg, i.p., respectively). the trachea was cannulated via tracheostomy with a 19g blunt needle and the cannula was secured in place with a suture. mice were kept sedated using isoflurane inhalation (maintenance dosing, 1-5% of inspired air) through the flexivent and were paralyzed with 1 mg/kg vecuronium i.p. mice were ventilated using baseline settings of positive end expiratory pressure (peep) 3 cm h 2 o, tidal volumes (vt) 10 ml/kg, respiratory rate (rr) 150 breaths per minute (bpm) and an fraction of inspired oxygen (fio2) of 0.21. oxygen saturations were measured using the mouseox plus pulse oximeter (starr life sciences, oakmont, pa, usa) via the thigh sensor. lung mechanic parameters were generated from the flexivent rodent ventilator using the forced oscillation technique as previously described [10] . bronchoalveolar lavage (bal) protein measurements bal samples were collected as previously described, and after centrifugation at 150 g for 10 min, bal supernatants were removed and immediately frozen at −80°c until batch analysis. proteins were measured with kits as detailed in additional file 1: table s1 . elisa plate absorbance was measured at 450 nm with a spectramax m2 uv/vis/fluorescence 96-384 plate reader (molecular devices, sunnyvale, ca). cytokines were measured by elisa as described above or by procartaplex cytokine & chemokine 36-plex mouse panel 1a (thermo fisher scientific) via luminex magpix multiplex array (luminex). after iav infection, the entire lung was removed from each mouse and placed in 2 ml of trizol (thermo fisher scientific, waltham ma), weighed, homogenized on ice using a polytron homogenizer for 15-30s intervals, and frozen in aliquots at -80°c until rna extraction. dna was then extracted using chloroform and rna was precipitated using isopropanol. quantitative rt-pcr for iav m1 copies per lung was performed as previously described [11] using the following primers: influenza a/8/puerto rico/34 m1 gene sense: 5'-aagaccaatcctgtcacctctga-3′ and antisense: 5' caaagcgtct-acgctgcagtc -3′ primers, and the mediator probe sequence: 5′-/56 fam/ tttgtgttcacgctc-accgt/36-tamsp/ -3′. data are expressed as m1 viral copies per lung. single cell suspensions were prepared from bal and lung as described in supplemental methods. single cell suspensions from lung digests were placed at 4°c and then surface stained in hank's buffered saline solution (hbss) with 3% fbs with fluorochrome-conjugated monoclonal antibodies (additional file 2: table s2 ), and then stained with a fixable viability dye. for facs-sorting, bal cells were recovered and placed at 4°c and then surface stained in hank's buffered saline solution (hbss) with 3% fbs with fluorochrome-conjugated monoclonal antibodies (additional file 2: table s2 ) and 7-aminoactinomycin d (7-aad) was used to assess viability just prior to acquisition. all flow cytometric data were collected in the penn state hershey flow cytometry core facility using an lsr ii (becton dickinson, bd) instrument, and all facssorting was performed using a facsaria (bd) high speed cell sorter. cells were sorted directly into rna-bee to isolate rna for further rna-sequencing (rna-seq). all facs data analysis was performed using flowjo version 9.9 (treestar, mountain view, ca). rna was phase separated using chloroform and the aqueous phase containing rna was removed following centrifugation and precipitated overnight at −20°c using ice cold isopropanol. rna was washed with 75% ethanol then solubilized in rnase-free water. optical density values of extracted rna were measured using nanodrop (thermo fisher scientific) to confirm an a260:a280 ratio above 1.9. rna integrity number (rin) was measured using bioanalyzer (agilent) rna 6000 pico kit to confirm rin above 7. the cdna libraries were prepared using the smarter® ultra® low input rna kit for sequencing -v3 (clontech) followed by nextera xt dna library prep kit (illumina) as per the manufacturer's instructions. the unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. the final product was assessed for its size distribution and concentration using bioanalyzer high sensitivity dna kit (agilent technologies) and kapa library quantification kit (kapa biosystems). the libraries were diluted to 2 nm in eb buffer (qiagen) and then denatured using the illumina protocol. the denatured libraries were diluted to 10 pm by pre-chilled hybridization buffer and loaded onto truseq sr v3 flow cells on an illumina hiseq 2500 (illumina) and run for 50 cycles using a single-read recipe (truseq sbs kit v3, illumina) according to the manufacturer's instructions. illumina casava pipeline (released version 1.8, illumina) was used to obtain de-multiplexed sequencing reads (fastq files) passed the default purify filter. additional quality filtering used fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit) to keep only reads that have at least 80% of bases with a quality score of 20 or more (conducted by fastq_quality_filter function) and reads left with 10 bases or longer after being endtrimmed with reads with a base quality score of b20 (conducted by fastq_quality_trimmer function). a bowtie2 index was built for the mouse reference genome (grcm38) using bowtie version 2.1.0. the rna-seq reads of each of the 38 samples were mapped using tophat version 2.0.9 [12] supplied by ensembl annotation file; grcm38.78.gtf. gene expression values were computed using fragments per kilobase per million mapped reads (fpkm). differential gene expression was determined using cuffdiff tool which is available in cufflinks version 2.2.1 [13] supplied by grcm38.78.gtf. normalization was performed via the median of the geometric means of fragment counts across all libraries, as described in anders and huber [14] . statistical significance was assessed using a false discovery rate threshold of 0.05. we arbitrarily chose to further analyze the 5% most highly expressed gene transcripts in am or em cell populations from iav-infected dtgm or lm mice using ingenuity pathway analysis (ipa, www.qiagen.com/ingenuity) to identify upstream signaling pathways. significance was measured by fisher's exact test with a q < 0.2 cut-off. all statistical analysis was performed using jmp 12.0.1 software (sas, cary, nc). normally distributed data were analyzed using student's t-test, and non-normally distributed data using wilcoxon signed-rank test. survival analysis was calculated by using the log-rank test. all data points are means ± standard error of the mean (sem) unless otherwise stated. graphs were created using prism 6 for mac os x (graphpad, la jolla, ca). to characterize the pathogenicity of our h1n1 pr8 iav preparation virus, wild-type c57bl/6 j mice (the jackson laboratory, ma) were purchased and we determined the lethal dose 50% (ld 50 ) of our pr8 iav preparation. female wild-type mice were much more susceptible than males with an ld 50 approximately 5fold lower than males (728 vs. 3728 ffu, female and males respectively, additional file 3: figure s1a -d). airway gm-csf levels were conditionally increased following iav infection using a doxycycline inducible promoter in the dtgm mouse model, formerly named the tet-gm +/+ , as previously described [4] . in this conditional transgenic mouse model gm-csf is expressed and secreted by airway club cells via the club cell 10 (cc10, scgb1a1) promoter after oral administration of doxycycline (1 mg/ml in water ad libitum) (fig. 1a) . importantly, in the absence of infection, bal fluid levels of gm-csf in dtgm mice are near the limit of detection, similar to littermate controls (additional file 4: figure s2a ), and their alveolar macrophages appear identical by multiparameter flow cytometry. once doxycycline is administered, bal levels of gm-csf peak after approximately 48 h reaching levels of approximately 500 pg/ml in 2.5 ml of recovered bal fluid and in preliminary experiments the dtgm mice were either administered or not administered doxycycline to create a condition of elevated vs. wild-type levels of airway gm-csf, respectively [4] . however, these preliminary experiments demonstrated that low levels of gm-csf from the scgb1a1 promoter in dtgm mice were endogenously induced by interferons during iav infection (additional file 4: figure s2a ), a finding that has previously been reported [15] . therefore, all subsequent experiments compared the dtgm to lm groups, both exposed to doxycycline, to examine the effect of elevated (dtgm) as opposed to wild-type (lm) levels of airway gm-csf, while also controlling for any off-targets effects of doxycycline. to address our research question of whether "treatment" with gm-csf during severe iav infection would improve survival, dtgm and lm mice were infected i.n. with approximately 1 ld 50 (differential dosing based on sex) of pr8 iav and were administered doxycycline in drinking water. gm-csf overexpression (dtgm) conferred a significant survival advantage as compared to wild-type levels (lm, fig. 1b , **p < 0.005). weight loss and recovery were similar in the two groups, however, because of survivor bias likely artificially elevating the average weights of surviving lm mice (fig. 1c) . of note, doxycycline treatment of lm mice had no effect on survival whereas doxycycline-untreated dtgm mice demonstrated a survival advantage over untreated lm mice, suggesting that even low levels of gm-csf can confer some survival benefit (additional file 4: figure s2b ). lower respiratory tract iav infection can lead to impaired oxygenation due to v:q mismatch and decrease lung compliance due to the infiltration of inflammatory cells and an increase in lung water weight [16] . given the ability of gm-csf to confer survival, we expected elevated gm-csf levels to improve arterial oxygen saturations (% spo 2 ). however, gm-csf did not significantly increase median oxygen saturations (% spo 2 ) levels as compared to lm mice at either 7 or 10 dpi (data not shown). to gain insight into whether gm-csf conferred any lung mechanical benefits, lung mechanics scans were performed by the forced oscillation technique and pv loop curves were generated (fig. 2a, b) . as expected, the pv curve flattens with iav infection due to decreased static compliance (cst), but we were surprised that compliance continued to fall from days 7 to 10 ( fig. 2a-c) . while gm-csf did not affect cst (fig. 2c) or total system resistance (rrs, fig. 2d ), dtgm mice demonstrated less tissue damping or peripheral airway resistance (g, cmh 2 o/ml, fig. 2e) , and significant preservation of newtonian or central airway resistance (rn, cmh 2 o*s/ml, fig. 2f ) and curvature of the deflation limb of the pv curve, a measure of maintenance of alveoli and small airway recruitment (k, 1/cmh 2 o, fig. 2g ) at 10 dpi. given that two of the lung mechanical parameters that are maintained by gm-csf, k and g, are correlated with dynamic processes at the small airway or alveolar level, namely alveolar size [17] and changes in tissue physical properties of small airways [18] respectively, and which can change with lung interstitial edema [19] , we hypothesized that gm-csf may improve lung capillary barrier function and/or enhance alveolar fluid clearance. as surrogate of alveolar fluid content, we measured the concentration of total protein in bal fluid and found that gm-csf overexpression decreased bal fluid total protein levels at 10 (peak inflammation) and 14 dpi (early resolution phase) (fig. 3a) . to further investigate this difference in bal fluid protein content, we examined the concentration of various serum and lung-specific proteins including mouse serum albumin (69 kda), as well as two larger proteins, alpha-2-macroglobulin (180 kda monomer, 720 kda tetramer) and immunoglobulin m (igm, 194 kda monomer, 970 kda pentamer) as markers of capillary leak [20] . gm-csf significantly decreased alpha-2macroglobulin levels at 14 dpi (fig. 3b) , but did not significantly decrease other markers of capillary leak including albumin or igm (additional file 5: figure s3a-b) . we also directly assayed the lung epithelial barrier function with fitc-labeled dextran (mw 10,000), but surprisingly no differences in epithelial barrier function could be detected at 10 dpi (data not shown). additionally, we also investigated whether gm-csf overexpression during iav increased bal levels of the epidermal growth factor family member, amphiregulin. gm-csf overexpression in uninfected mice elevated levels of amphiregulin, though iav infection also induced amphiregulin and gm-csf did not further increase these levels ( fig. 3c) . lastly, we assessed whether elevated gm-csf levels during active infection affected viral clearance. at 7 dpi, the peak of virus levels in our model, we recovered 2-3 × 10 8 m1-copies total lung copies of iav pr8 matrix 1 (m1) via rt-pcr and the viral copies decreased to 0.8-2 × 10 7 by 10 dpi, though there was no statistically significant difference with gm-csf overexpression (fig. 3d) . alveolar macrophages have been shown to be necessary for protection from iav [21] [22] [23] [24] [25] [26] [27] . gm-csf is known to mediate the proliferation and differentiation of monocytes and macrophages; studies using constitutive expression or gm-csf administration before iav infection models both documented an increase in am numbers [5, 6, 28] . therefore we hypothesized that gm-csf would protect siglecf + ams from viral-induced depletion and would increase numbers of total airway (bal-recovered) macrophages. to investigate this immune cells were characterized and enumerated in single cell suspensions of bal and lung enzymatic digests by multi-parameter flow cytometry using a 12-color panel of macrophage and granulocyte surface markers (fig. 4a) . we specifically focused on the two predominant airway macrophage populations present during active iav infection: f4/80 + , cd11b neg/dim , siglecf + cells to discriminate alveolar macrophages (ams) and f4/80 + , cd11b + , siglecf neg/dim cells that have been termed exudative macrophages (ems) [29] . our typical yield of ams recovered from bal fluid of an uninfected mouse is approximately 600,000 cells. at 10dpi, at the height of the inflammatory response to iav, the number of ams to simulate a therapeutic model of gm-csf administration doxycycline was administered to both dtgm and lm control mice starting 3 days after i.n. infection with pr8 iav. doxycycline-containing water was protected from light and changed every three days (a). dtgm (n = 23, red circles/lines) and lm control (n = 15, black squares/lines) mice were administered approximately 2 ld 50 of iav pr8 virus i.n. and administered doxycycline in water starting on +3 dpi, and the effects on survival and body weight are shown. mice were euthanized if they lost >30% body weight and were moribund. gm-csf over-expression (dtgm mice) conferred a significant survival benefit (b) but not a significant effect on weight loss/recovery (c) as compared to wild-type levels (lm mice). results shown represent three independent experiments (**p < 0.005) recovered was much lower, and gm-csf overexpression did not serve to increase this number (fig. 4b) . in contrast, ems become the predominant airway macrophage during iav infection at this time point, but again, gm-csf overexpression did not affect em cell numbers (fig. 4b) . while gm-csf overexpression did not change the number of macrophages, we hypothesized that it changed their phenotype. this is not a new concept as the primary function of gm-csf on ams is to induce differentiation and activation [4, 30, 31] . despite attempting to discriminate the macrophage populations by multiple cell surface markers, we could not distinguish the iav-responding macrophages further than alveolar and exudative macrophages as described in fig. 4a . therefore, we sought to determine whether gm-csf affected the transcriptomes of the am and em populations independently by first facs-sorting the airway macrophages. facs-sorted airway macrophages from bal fluid were obtained at 8 dpi, the time point where the survival curves of the dtgm and lm mice begin to diverge, and next generation rnasequencing was performed on the sorted am and em populations. using an unbiased approach, we identified the transcripts that were significantly affected by gm-csf over-expression during iav infection by comparing the mean value of each transcript from the dtgm and lm groups. for direct comparisons, transcripts that had a mean value of zero (0) fkpm in one of the groups were not analyzed. of the 43,628 genes available in the reference genome, in the am population 23 transcripts were significantly different between the groups with gm-csf over-expression leading to up-regulation of the chemokines ccl17, cxcl3, and ccl6, and the down-regulation of cxcl9, and arg1, the prototypic marker of m2 macrophage polarization (fig. 5a) [32] . in comparison to ams, in ems gm-csf induced more transcripts than it inhibited. only fig. 4 flow cytometric discrimination of alveolar and exudative macrophages by surface marker expression. representative facs plots from an iav-infected lm mouse at 10 dpi, which detail our 12-color flow cytometry gating strategy of single cell suspensions from bal and enzyme-digested lung (a). alveolar macrophages (am) were designated as f4/80 + siglecf + cd11b neg/dim , whereas exudative macrophages (em) were designated as f4/ 80 + siglecf neg/dim cd11b+. supra-physiologic gm-csf levels during iav infection had no effect on the absolute number of either airway (bal-recovered) am or em cell numbers at 10 dpi (b) six transcripts were down regulated by gm-csf including lipg, cxcl10 and ccl12, while gm-csf overexpression induced multiple transcripts in ems including dcstamp, retnla, irgc1, mmp12, and ccl6 (fig. 5b) . our unbiased analysis demonstrated that gm-csf overexpression during iav led to the up-regulation of some transcripts associated with m2 macrophages including matrix metalloprotease 12, mmp12, and ccl17, and the down-regulation of some m1 macrophage-associated transcripts such as cxcl9 and cxcl10. therefore we examined the effect of gm-csf on multiple canonical and novel macrophage polarization markers [33] . interestingly, while gm-csf tended to down-regulate m1 transcripts and up-regulate m2 transcripts, this effect was not absolute in either ams or ems (fig. 5c-f ). to validate these macrophage transcript differences we measured the chemokines ccl17 and cxcl9, and the m2-associated metalloprotease, mmp12, in bal fluid by elisa. ccl17 was significantly induced by gm-csf (not only during iav infection, but also when gm-csf was induced in the absence of iav (fig. 6a) . in comparison, negligible amounts of cxcl9 were present in uninfected mice regardless of gm-csf induction, whereas with iav fig. 5 characterization of the changes in transcriptome patterns of airway macrophages during iav infection. bal airway macrophages were sorted using the gating strategy described in fig. 4a and next generation rna-sequencing was used to profile the complete transcriptome data of ams (a, orange bars) and ems (b, blue bars) at 8 dpi, the time point at which the survival curves diverge (n = 5 mice per group). the effect of supraphysiologic gm-csf levels on each of the 43,628 sequenced macrophage genes was examined: differential gene expression was determined using with transcripts having a q-value <0.2 being included. the relative expression of each transcript was calculated using the equation, log2 expression ratio (dtgm:lm) = log 2 ðx transcript dtgm ) -log 2 (x transcript lm ), and the differential expression of transcripts is shown. to investigate the impact of gm-csf on m1/m2 macrophage polarization, the log2 expression ratios were plotted against known m1 and m2 macrophage-associated transcripts from ams (c, d) and ems (e, f) infection gm-csf overexpression there was a trend toward decreased expression (fig. 6b, p = 0 .09). the concentration of mmp12 was approximately 6-fold higher in gm-csf overexpressing mice at 10 dpi as compared to lms (fig. 6c, p < 0.01) . we also examined the ratio of the two chemokines (cxcl9: ccl17) as an intrinsic property of the balf to probe macrophage polarization by chemokine expression and supra-physiologic gm-csf levels significantly decreased this ratio more than ten-fold in iav-infected mice (fig. 6d , p < 0.005). lastly, we attempted to determine which signaling pathways were affected by gm-csf overexpression during iav infection by analyzing our transcriptomes with ingenuity pathway analysis (ipa) software (qiagen). we analyzed the effect of gm-csf overexpression on the mean log 2 expression ratios for the 5% most expressed genes in each of the macrophage type groups (am vs. em). the ipa software allows the construction of an upstream analysis that calculates the likelihood that an upstream regulator is involved given the gene set provided (p-value of overlap), as well as a composite score of activation depending on the state of downstream genes being increased or decreased in quantity (activation z-score). for both the upstream regulator analysis, and the subsequent canonical pathway analysis, we used a stringent p-value of overlap cutoff of 1e-10. ipa predicted that gm-csf activates (table 1a ) several upstream regulators of signaling pathways in both ams and ems including il-10 receptor alpha (il10ra), transcription factor tripartite motif-containing 24 (trim24), and the atypical chemokine receptor 2 (ackr2). conversely, ipa predicted that gm-csf over-expression inhibited multiple inflammatory signaling pathways in both ams and ems including interferon regulatory factor 3 (irf3), irf7, interferon gamma (ifng), interferon alpha/beta receptor (ifnar), tir domain-containing adapter molecule 1 (ticam1, or trif), signal transducer and activator of transcription 1 (stat1), rapamycin-insensitive companion of mammalian target of rapamycin (rictor), toll-like receptor 4 (tlr4), dexd/hbox helicase 58 (ddx58, or retinoic acid-inducible gene 1 [rig-1]), and inhibitor of nuclear factor kappa-b kinase subunit beta (ikbkb). in terms of canonical pathway analysis one pathway, "eukaryotic initiation factor 2 (eif2) signaling", was activated in both ams and ems, whereas "fc-γ receptormediated phagocytosis in macrophages and monocytes" was inhibited in both populations (table 1c, d) . "interferon signaling" was inhibited in ems (table 1d) , and trended towards significance in the am population [−log(p-value) 7.66, z-score − 1.5], even though the levels of type i, ii and iii interferons were unchanged in fig. 6 effect of gm-csf overexpression on airway levels of ccl17, cxcl9 and mmp12. mouse ccl17 (a), cxcl9 # (b), and mmp12 # (c) were measured by elisa in bal fluid from doxycycline-treated lm (black) and dtgm (red) uninfected and iav-infected (10 dpi) mice. furthermore, the ratio of cxcl9:ccl17 # in each bal sample was determined to examine the relative effect of supraphysiologic gm-csf levels on macrophage chemokine polarization (d). results from three independent experiments. ( # please note the log 10 scale, *p < 0.05, **p < 0.005) bal fluid from dtgm as compared to wt mice (additional file 6: figures s4 a-c ). in this study we examined the effect of elevated gm-csf levels during iav infection on clinical, lung physiologic and biochemical markers in a mouse model, and then used rna-sequencing to ascertain the differential effects of elevated gm-csf levels on the transcriptomes of the two predominant airway macrophages present during the peak of iav infection. our finding that elevation of airway gm-csf during active iav infection confers protection from mortality from iav is novel. multiple preclinical mouse studies have described the observations that the absence of gm-csf increases susceptibility to iav [3, 4, 25] , while supra-physiologic levels of gm-csf achieved by constitutive overexpression or exogenous administration are beneficial [5, 6, 28] . importantly, however, the publications that have demonstrated positive effects of supra-physiologic levels of gm-csf against iav infection have used either constitutive expression models [3] [4] [5] or have administered gm-csf either before [6, 28] or on the day of infection [5] . to our knowledge this is the first description of the use of a therapeutic model of gm-csf wherein it is "administered" to the airways well after establishment of the infection (+3 dpi) and still confers protection. table 1 ingenuity pathway analysis predictions of the effects of supra-physiologic levels of gm-csf on airway macrophages during iav. bal airway macrophages were sorted and rna-sequencing was performed to compare the gene expression between iav-infected lm (n = 5 mice) and dtgm (n = 5 mice) treated with doxycycline at 8 dpi. using the means of each group, the 5% (2181 genes) most expressed transcripts from each of the genotypes, dtgm and lm, were analyzed using qiagen's ingenuity pathway analysis (ipa) software. ipa was used to identify differential upstream regulators between ams (a) and ems (b) of dtgm and lm mice, and upstream regulators were included in the table if their p-value of overlap was <1e-10 and the activation z-score was < −2 or > +2. ipa was also used to identify differential effects of gm-csf on canonical pathways of ams (c) and ems (d). ingenuity canonical pathways were included in the table if their -log(p-value) was >10 and the z-score of pathway activation was < −2 or > +2 gm-csf over-expression led to an increase in macrophage expression and bal fluid levels of ccl17 and mmp12, whereas a decrease in cxcl9 or monokine induced by gamma interferon (mig). these protein data, in addition to our macrophage transcriptome data, suggest that high levels of gm-csf push the typically classically activated m1-like monocytes/macrophages in the lung during iav towards an m2-like phenotype. interestingly, a recent investigation showed that the presence of m1-like monocytes are a major determinant of iav pathogenicity in patients and strengthened this notion with a mouse model demonstrating that adoptive transfer of m2 as opposed to m1 macrophages results in better outcomes [34] . the observation that gm-csf is pushing macrophages towards an m2-phenotype is in stark contrast to a large body of in vitro literature that defines m1 monocytes/macrophages as being induced by gm-csf, whereas m2 monocytes/macrophages are differentiated by macrophage colony-stimulating factor (m-csf) [35] [36] [37] . on the other hand, alveolar macrophages from gm-csf-deficient csf2−/− mice exhibit a mixed m1/m2 phenotype, not a strictly m2 phenotype as in vitro data would suggest [38] . and our data also suggests that the polarization was not at all absolute: e.g., in ams, gm-csf led to lower transcript levels of the prototypic m2 macrophage marker, arg1 (fig. 5a) . thus, while the m1/m2 macrophage polarization schema has been helpful [39, 40] , perhaps a more nuanced view of macrophage polarization [41] , where their intrinsic differentiation plasticity allows them to attend to specific needs of their local immune environment [42] , could explain these results. ipa also predicted the activation of the il-10 receptor alpha-chain in both ams and ems. given that il-10 levels in bal fluid were not elevated in dtgm as compared wt mice (additional file 6: figure s4d ), it is possible that gm-csf overexpressing during iav somehow potentiates il-10 signaling in the lung microenvironment. the role of interferons during iav infection is also nuanced. while it has been shown using ifnar −/− and ifngr1 −/− mice that interferon signaling is necessary for protection from iav [43] , it is possible that this requirement only extends to epithelial cells. interferon-γ may not be necessary during iav infection and may in fact be detrimental, e.g., nitrogen oxide synthase 2 deficient (nos2−/−) mice are more protected from iav [44] , and sun and metzger demonstrated that treatment with an anti-ifnγ mab clone xmg1.2 had little effect on the course of the viral infection, but inactivation of ifn-γ protected against secondary bacterial pneumonia [45] . recently, califano et al. showed that ifnγ −/− mice on both the balb/c and c57bl/6 backgrounds demonstrated improved survival to lethal iav infection [34] . in their model, ifnγ serves to restrict protective innate lymphoid cell group 2 (ilc2) function, whose production of il-5 and amphiregulin may improve lung barrier function. another group has also demonstrated that gm-csf can induce amphiregulin in a smoke model of copd followed by iav infection [46] , however our data (fig. 3c) suggest that pretreatment with gm-csf is necessary for this effect on amphiregulin levels. furthermore, our gm-csf over-expression is started after iav infection, amphiregulin levels at 10 dpi were not different in gm-csf over-expressing mice, and therefore amphiregulin is likely not an active player in our model. it is possible that our inducible gm-csf model may be replenishing gm-csf that otherwise would be produced by ilc2s whose functions have been restricted by ifnγ [34] . our data suggest that high levels of gm-csf inhibit interferon signaling in airway macrophages, though the mechanism is not clear. canonically, gm-csf signaling acts through jak2/stat5 [47] , though the beta-chain itself can activate nf-kb, and this activation is dependent on tnfr-associated factor 6 (traf6) [48, 49] , an e3 ubiquitin ligase with multiple immune functions [50] . interestingly, our upstream analysis predicts that gm-csf activates trim24, (aka tif1α), a negative regulator of interferon signaling that acts by binding the retinoic acidresponsive element of the stat1 promoter [51] , thus inactivating multiple interferon pathways. trim24 is also an e3-ubiquitin ligase, and the tumor suppressor protein, p53, serves as a ligand for both ligases: trim24 targets p53 for degradation [52] while traf6 restricts p53 mitochondrial translocation [53] . furthermore, a recent microarray study examining the relative pathogenicity of a mouse adapted strain of iav (ma-ca/04) described negative inhibition of trim24 and early sustained interferon responses as important factors [54] . however, we detected only very low levels of trim24 transcripts in our sorted airway macrophages, but gm-csf over-expression did lead to increased expression of another trim family member, trim16, that also acts as a e3 ubiquitin ligase that can heterodimerize with other trims [55] . future studies are needed to determine the exact cellular signaling pathways linking gm-csf and interferon. gm-csf enhanced exudative macrophage expression, and 8 dpi bal fluid levels of mmp12, or macrophage elastase, which is best known for its requirement for the development of smoke-induced emphysema in mice [56] . however, it may also regulate acute inflammatory responses by proteolysis of chemokines [57] , and through its divergent effects on ifn-α signaling depending on its intracellular (activating) vs. extracellular (inactivating) localization [58] . a recent report demonstrated in two separate mouse models inflammation (peritonitis and arthritis) that macrophages resolve inflammation through multiple mechanisms via mmp12 including dampening neutrophil infiltration, clearing actin and fibrin from nets, terminating complement activation, and by activating prothrombin thus exhibiting procoagulant activity [59] . cd4+ t cells and stat4/6, at least in a mouse model of pneumocystis pneumonia, are necessary for m2 macrophage mmp12 expression and relm-α and ccl17 production [60] . while our data suggest that gm-csf may block m1-like polarization in the lung during iav infection, it is not yet clear what in the lung microenvironment could promote m2-like macrophage responses. recently it was shown that macrophage polarization may be pushed towards a il-4 dependent pathway in the lung and liver by the presence of surfactant protein a (sp-a) and complement component c1q, respectively [61] . the relationship between supraphysiologic gm-csf levels and sp-a during iav infection remains to be investigated and will be the subject of future studies. our current model of gm-csf induction on the wildtype background differs from our previous work using the inducible model generated on the gm-csf knockout (csf2 −/− ) genetic background [4] . the present study is not confounded by prior immaturity of ams and defective surfactant catabolism, nor potential defects in migratory dendritic cell subsets, nk cells, and other myeloid cells outside the alveolar compartment in the lung and in other tissues of csf2 −/− mice [62] [63] [64] [65] , or disruption of gm-csf secretion by immune and non-immune cells that may elaborate gm-csf in response to infection. studies in csf2 −/− /spc-gm mice, in which t2aecs express high levels of gm-csf constitutively, came to disparate conclusions as to the role of ams, dendritic cells and epithelial cells [3, 5] in host resistance to iav infection. however, the life-long overexpression of gm-csf in the spc-gm +/+ model results in non-physiological proliferation of both t2aec cells and ams [66] that obscures assessment of temporal responses to iav. the spc-gm +/+ model also illustrates that prolonged lung exposure to supraphysiologic levels of gm-csf leads to desquamative interstitial pneumonia (dip) [4] . we did not observe any similar findings of dip in our model, but this is not surprising given our model creates only a temporary doxycyclineinduced overxpression, and the overriding inflammatory effects of iav infection likely masks any differences. in our model, the ability of supra-physiologic levels of gm-csf to beneficially alter disease progression after iav infection delineates a time frame for possible future therapeutic intervention to arrest development of acute lung injury. in this regard, administration of gm-csf in humans has shown promise in the treatment of ards [67] . concentration-dependent signaling via the gm-csf receptor affecting differentiation, proliferation, 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article macrophage matrix metalloproteinase-12 dampens inflammation and neutrophil influx in arthritis experimental pneumocystis lung infection promotes m2a alveolar macrophage-derived mmp12 production local amplifiers of il-4r α -mediated macrophage activation promote repair in lung and liver origin of the lamina propria dendritic cell network distinct cd11b+−monocyte subsets accelerate endothelial cell recovery after acute and chronic endothelial cell damage intestinal lamina propria dendritic cell subsets have different origin and functions the concerted action of gm-csf and flt3-ligand on in vivo dendritic cell homeostasis the concerted action of gm-csf and flt3-ligand on in vivo dendritic cell homeostasis gm-csf enhances lung growth and causes alveolar type ii epithelial cell hyperplasia in transgenic mice inhaled granulocyte/macrophage colony-stimulating factor as treatment of pneumonia-associated acute respiratory distress syndrome functions of granulocyte-macrophage colony-stimulating factor the granulocyte-macrophage colony-stimulating factor receptor: linking its structure to cell signaling and its role in disease regulation of dendritic cell development by gm-csf : molecular control and implications for immune homeostasis and therapy specific contributions of csf-1 and gm-csf to the dynamics of the mononuclear phagocyte system guide for the care and use of laboratory animals. national research council (us) committee for the update of the guide for the care and use of laboratory animals we would like to thank nate sheaffer and joseph bednarzyk from the penn state hershey flow cytometry core facility, as well as the institute for personalized medicine (ipm) at penn state hershey college of medicine, for assistance. we would also especially like to thank kevan hartshorn and mitchell white for providing the iav pr8 virus preparation used in all experiments. availability of data and materials all rna-seq data is available from the gene expression omnibus (geo) database, and the other datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. our data demonstrate that in vivo high airway levels of gm-csf profoundly rescue mice from lethal influenza pneumonia. while in vitro gm-csf is canonically described as an m1-polarizing cytokine, our data demonstrates that in vivo, during iav infection, gm-csf instead temporizes the type ii interferon-induced m1 polarization of airway macrophages. the exact mechanism through which high levels of gm-csf block m1macrophage polarization is still not known, and is the focus of our ongoing research. additional file 1: table s1 . all protein concentration measurements were made as described in the manuscript text using the reagents and kits listed. (tiff 3075 kb) additional file 2: table s2 . multi-parameter flow cytometry was utilized to characterize the alveolar and exudative macrophages as shown in fig the mouse influenza a virus infections and tissue harvesting were carried out by wg, md, tu, ly, sh and ekh. the rna sequence analyses were performed by yik, ps and jh. overall experimental design, analysis and interpretation were performed by esh with the mentorship of zcc. all authors read and approved the final manuscript.ethics approval and consent to participate all animal procedures were approved by the institutional animal care and use committee (iacuc) at pennsylvania state university college of medicine under protocols #43629 and 47,450, and were cared for as previously described [11] . the regulation of the use of mice in research falls under the public health service policy on humane care and use of laboratory animals (phs policy), and is enforced by the office of laboratory animal welfare (olaw) under assurance number a3045-01. in order to comply with the phs policy, our institution adheres to the us government principles for the utilization and care of vertebrate animals used in testing, research and training and the guide for the care and use of laboratory animals 8th edition [72] . not applicable, the authors agree to pay the journal processing fee should the manuscript be accepted for publication. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-325192-italbsed authors: desai, tanay m.; marin, mariana; chin, christopher r.; savidis, george; brass, abraham l.; melikyan, gregory b. title: ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion date: 2014-04-03 journal: plos pathog doi: 10.1371/journal.ppat.1004048 sha: doc_id: 325192 cord_uid: italbsed interferon-induced transmembrane proteins (ifitms) inhibit infection of diverse enveloped viruses, including the influenza a virus (iav) which is thought to enter from late endosomes. recent evidence suggests that ifitms block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. consistent with this mechanism, excess cholesterol in late endosomes of ifitm-expressing cells has been reported to inhibit iav entry. here, we examined iav restriction by ifitm3 protein using direct virus-cell fusion assay and single virus imaging in live cells. ifitm3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. although late endosomes of ifitm3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. these results imply that excess cholesterol in late endosomes is not the mechanism by which ifitm3 inhibits the transition from hemifusion to full fusion. the ifitm3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. we propose that ifitm3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. alternatively, ifitm3 may redirect iav fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes. the recently identified interferon-induced transmembrane proteins (ifitms) inhibit infection of diverse enveloped viruses [1] [2] [3] . ectopic expression of ifitm1, -2 and -3 restricts a growing number of unrelated viruses, including iav [1, 2, [4] [5] [6] [7] . ifitm3 has been shown to potently restrict infection by iav and the respiratory syncytial virus in vivo [8] [9] [10] . in contrast, arenaviruses and some retroviruses, such as murine leukemia virus (mlv), are resistant to ifitm restriction [2, 6] . the ifitms have been reported to inhibit hiv-1 entry, albeit less potently than iav and apparently in a cell type-dependent manner [11] [12] [13] . the mechanism by which ifitms inhibit infection of diverse viruses is not fully understood. ifitm2 and -3 are predominantly found in late endosomes (le) and lysosomes [13, 14] , whereas ifitm1 is also found at the cell periphery [4, 15] . different membrane topologies of ifitms have been proposed [16] , but recent data suggests that ifitm3 is a type ii transmembrane protein [17] . accumulating evidence implies that ifitms may interfere with virus-endosome fusion [1, 2, 5, 13, 14] . the fact that ifitms seem to expand acidic intracellular compartments [13] indicates that the fusion block is downstream of the low ph trigger. effective restriction of viruses that enter from the le, such as iav, ebola virus (ebov) and sars coronavirus seems consistent with the cellular localization of ifitm2 and -3 proteins. however, these proteins also restrict vesicular stomatitis virus (vsv) that appears to fuse with early endosomes [18] . ifitms have been reported to curtail viral infection by modifying properties of cellular membranes, such as fluidity and spontaneous curvature [3, 5, 14] . these effects could be related, in part, to the accumulation of cholesterol in le as a result of ifitm-mediated disruption of the interaction between the vesiclemembrane-protein-associated protein a (vapa) and oxysterolbinding protein (osbp) [14] . since lipids play an important role in membrane fusion, these findings offer an attractive paradigm for a broad antiviral defense mechanism that involves altering the lipid composition of cellular membranes. the recent finding that amphotericin b, which forms complexes with sterols [19] , rescues iav infection in ifitm2-and ifitm3-expressing cells [20] is in line with the notion that cholesterol may be directly or indirectly involved in iav restriction. however, lipid composition-based models do not readily explain the lack of restriction of amphotropic mlv and arenaviruses, which enter cells via distinct endocytic routes [21, 22] . these findings indicate that ifitms may restrict virus entry from a subset of intracellular compartments. in order to define the mechanism of ifitm restriction, it is important to identify the viral entry step(s) targeted by these proteins, define compartments in which restriction occurs, and elucidate potential changes in intracellular membranes that may be responsible for this phenotype. here, we examined the mechanism of ifitm3 restriction of iav using single particle imaging and a direct virus-cell fusion assay. our results show that ifitm3 does not inhibit the lipid mixing stage of iav fusion but blocks the release of viral contents into the cytosol, and that this phenotype does not correlate with cholesterol accumulation in intracellular compartments. specifically, ifitm3 inhibits the conversion of hemifusion to fusion through a mechanism that does not rely on cholesterol accumulation. together these findings reveal a previously unappreciated view of ifitm-mediated restriction and suggest new avenues of investigation to delineate the mechanism by which these proteins block infection. we chose to focus on ifitm3 to study the mechanism of iav restriction because this protein potently inhibits infection in vitro and in vivo [8] [9] [10] . since published data suggest that ifitm3 likely inhibits the viral fusion step, a direct virus-cell fusion assay was employed to evaluate the extent of restriction in different cell lines [23] . hiv-1 particles carrying the b-lactamase-vpr (blam-vpr) chimera and pseudotyped with the influenza ha and na proteins from the h1n1 a/wsn/33 strain (referred to as iavpp) were allowed to fuse with cells transduced with an empty vector or with an ifitm3-expressing vector. the resulting cytosolic blam activity was measured as previously described [24] . out of several cell lines tested, a549 and mdck cells over-expressing ifitm3 were least permissive to iavpp fusion (fig. 1a) . in agreement with the previous reports [2, 13] , we found that ifitm3 overexpression partially inhibited vsv g glycoprotein-mediated fusion of pseudoviruses (vsvpp) carrying the blam-vpr chimera (fig. 1a) . similar to inhibition of iavpp fusion, the ifitm3mediated restriction of vsvpp was most potent in a549 and mdck cells. as expected, fusion of particles pseudotyped with the lassa fever virus glycoprotein (lasvpp), which directs virus entry through an ifitm3-resistant pathway [2, 6] , was not considerably affected by ifitm3 over-expression. we next checked if the strong suppression of virus fusion in a549 and mdck cells was related to the level of ifitm3 expression. immunostaining for ifitm3 in these and cho cells which exhibited modest restriction of viral fusion (fig. 1a ) did not reveal a clear correlation between ifitm3 expression and inhibition of iavpp or vsvpp fusion (fig. 1b) . of note, potent iav restriction in a549 and mdck cells was not related to the usage of hiv-1 corebased pseudoviruses. influenza virus-like particles containing the iav blam-m1 chimera [25] also failed to efficiently fuse with a549-ifitm3 and mdck-ifitm3 cells while fusing well with vectortransduced cells (fig. 1c) . we also found that both vectortransduced a549 and mdck cells were highly susceptible to iav infection, as determined by virus titration (see materials and methods). these two cell lines were therefore chosen for studies of ifitm3-mediated restriction described below. ifitm-based restriction has been studied using a cell-cell fusion model, as well as by forcing viral fusion with the plasma membrane by lowering the ph [5, 20] . since fusion with the plasma membrane is more amenable to mechanistic studies than endocytic entry, we asked whether ifitm3 can restrict forced iav fusion. exposure to acidic buffer induced iavpp fusion with a549-vector cells pretreated with bafilomycin a1 (bafa1), which blocked low ph-dependent entry from endosomes (fig. 1d ). the extent of forced fusion was lower compared to the conventional entry route. by contrast, forced iavpp fusion with a549-ifitm3 cells was ,3-fold more efficient than endocytic fusion with cells not treated with low ph or bafa1, showing that ifitm3 does not restrict iavpp fusion at the cell surface. interestingly, ifitm1 suppressed iavpp-plasma membrane fusion at low ph (fig. 1d ), in agreement with the jaagsiekte sheep retrovirus (jsrv) and iav fusion data [5, 20] . the inability of ifitm3 to block iav fusion with the plasma membrane is consistent with its lower abundance at the cell surface [13, 14, 20] and shows that the mechanism of restriction must be studied in intracellular compartments. preponderance of evidence implies that hemifusion is a universal intermediate (reviewed in [26, 27] ) that precedes the formation of a fusion pore. having shown that ifitm3 overexpression inhibits viral fusion (fig. 1a, c) , we asked whether this protein also blocks the upstream hemifusion step. this was accomplished by labeling the a/pr/8/34 virus membrane with a self-quenching concentration of vybrant did (vdid), using a modification of the previously published protocol [28] . incorporation of self-quenching quantities of a lipophilic dye enables the visualization of single lipid mixing events based on the marked increase in fluorescence upon dye redistribution to an endosomal membrane (see for example [28, 29] ). significantly, to control for fluctuations in the vdid fluorescence caused by deviation from a focal plane, the viral surface proteins were labeled with the amine-reactive alexafluor-488 (af488) dye. the relatively steady af488 signal before and after hemifusion is allowed correcting for the vdid intensity fluctuations due to moving in and out of focus. the vdid/af488 co-labeling interferon-induced transmembrane proteins (ifitms) block infection of many enveloped viruses, including the influenza a virus (iav) that enters from late endosomes. ifitms are thought to prevent virus hemifusion (merger of contacting leaflets without formation of a fusion pore) by altering the properties of cell membranes. here we performed single iav imaging and found that ifitm3 did not interfere with hemifusion, but prevented complete fusion. also, contrary to a current view that excess cholesterol in late endosomes of ifitm3-expressing cells inhibits iav entry, we show that cholesterol-laden endosomes are permissive for virus fusion. the ability of ifitm3 to block the formation of fusion pores implies that this protein stabilizes the cytoplasmic leaflet of endosomal membranes, either directly or indirectly, through altering its physical properties. ifitm3 may also redirect iav to a non-productive pathway by promoting fusion with intralumenal vesicles of late endosomes instead of their limiting membrane. protocol only modestly (,2-fold) reduced iav infectivity compared to the mock-labeled viruses (fig. s1a ). immunofluorescence staining of af488-labeled virions with anti-ha antibodies revealed an excellent co-localization of the two signals (fig. s1b , c), thus supporting the notion that af488/vdid-labeled particles are bona fide virions. . cells were fixed, permeabilized and immunostained for ifitm3 (red), as described in materials and methods. the nuclear stain, hoechst-3342, is shown in blue. (c) ifitm3 restricts fusion of influenza virus-like particles containing b-lactamase reporter protein fused to the influenza matrix protein-1 (blam1). experiments were carried out as described above. data are means and sem from 2 independent triplicate experiments. (d) exposure to low ph overcomes the ifitm3-mediated block of iavpp fusion. to force pseudovirus fusion at the plasma membrane, a549 cells transduced with ifitm1, ifitm3 or an empty vector were pretreated with 50 nm bafa1 for 30 min at 37uc or left untreated. iavpp/blam-vpr pseudoviruses (moi = 1) were bound to cells of in the cold and exposed to either a prewarmed ph 5.0 mes-citrate buffer or neutral buffer for 10 min at 37uc and further incubated in growth medium (with or without bafa1) for 90 min at 37uc. data are means and sem from 2 independent triplicate experiments. ***, p,0.001 by two-tailed t-test. doi:10.1371/journal.ppat.1004048.g001 labeled viruses were allowed to enter a549-vector cells, and the resulting lipid mixing activity was examined by single particle tracking. a fraction of virions exhibited a marked increase in the vdid signal ( fig. 2a, b) . redistribution of vdid was mediated by low ph-dependent conformational changes in the iav ha glycoprotein, as evidenced by potent inhibition of lipid mixing by anti-ha antibodies (fig. 2c ) and by nh 4 cl (fig. 3a) . without simultaneous monitoring of the viral content release into the cytoplasm, vdid dequenching does not discriminate between hemifusion (operationally defined as lipid mixing without content transfer [30] ) and full fusion. to avoid over-interpreting dequenching events, we will refer to these events as lipid mixing or hemifusion. a similar vdid dequenching pattern was observed in mdck cells transduced with an empty vector (data not shown). analysis of lipid mixing showed that 2.260.4% and 5.660.6% of cell-bound particles released vdid in a549 and mdck cells, respectively (fig. 3a) . by comparison, a much greater fraction of virions (38.360 .6%) hemifused with cho cells (data not shown), in agreement with the previously reported data [28] . importantly, iav lipid mixing was readily detected in ifitm3 + a549 and mdck cells (figs. 2d-g and 3a). not only was lipid mixing not inhibited in a549-ifitm3 cells, but a .3-fold greater fraction of particles released vdid in these cells compared to control cells ( fig. 3a , p,0.001). by comparison, ifitm3 overexpression in mdck cells did not significantly promote vdid dequenching (fig. 3a) . thus, contrary to the cell-cell fusion results [5] , ifitm3 does not inhibit and can even promote iav lipid mixing, consistent with the block of virus entry at a posthemifusion stage. accordingly, the addition of oleic acid, which augments hemifusion by altering spontaneous membrane curvature, did not rescue iavpp or vsvpp fusion with a549-ifitm3 cells (fig. s2 ). this is in agreement with the recent infectivity results [20] , but in contrast with the rescue of fusion between jsrv env-and ifitm-expressing cells by this fatty acid [5] . the higher frequency of vdid dequenching in a549-ifitm3 cells could be caused by the increased endosome acidity compared to control cells [13] . however, the distribution of waiting times to the onset of lipid mixing was independent of ifitm3 expression or the type of target cells (a549 vs. mdck, fig. 3b , p = 0.37). the fact that the kinetic curves do not reach plateau indicates that iav entry into a549 and mdck cells is not completed within the first hour. our results thus demonstrate that ifitm3 restricts the iav fusion at a post-hemifusion step, most likely at the point of fusion pore opening, as evidenced by the dramatic decrease of the blam signal in a549 and mdck cells expressing this protein (fig. 1a ). under our conditions, vdid dequenching was typically completed within a few minutes for both control and ifitm3 + cells (fig. 2) . this dequenching rate is much slower than sudden increases in fluorescence of the iav membrane markers described previously [28, 31] . while a portion of vdid dequenching could be completed within seconds (fig. s3 ), these fast events were not common. slow dequenching was also typical with the vdid/ af488-labeled x31 virus, as well as with the x31 virus labeled with a 15-fold excess of did, using the published protocol for single virus imaging [28] (data not shown). slow vdid dequenching during the first hour of virus-cell coincubation did not appear to result from iav degradation in le/ lysosomes, since the surface-exposed af488 label persisted long after vdid dequenching was completed and because anti-ha antibodies blocked vdid dequenching (fig. 2 ). in addition, we did not detect any correlation between the lag before the onset of lipid mixing and the vdid dequenching slope (fig. s4a ). this result reinforces the notion that late lipid mixing events are mediated by ha and not by virus degradation. control experiments, in which samples were not exposed to laser light during the first 30 min at 37uc, did not reveal fast dequenching events reaching completion in less than 1 min (data not shown). this control argues against phototoxicity-related attenuation of virus fusogenicity as the cause for sluggish lipid redistribution. since free vdid diffusion between a virus and a small endosome should be completed in less than a second [32, 33] , an initial membrane connection between iav and an endosome must severely impair lipid movement. to assess whether early fusion intermediates in control and ifitm3 + cells restrict vdid diffusion to the same extent, we examined the rate of vdid dequenching. single particle analysis revealed that, in a549 cells, the average vdid dequenching profile (fig. 3c ) was independent of ifitm3 expression, as were the initial slopes of vdid dequenching ( fig. s4b , p.0.5). these results indicate that ifitm3 over-expression does not affect the properties of fusion intermediates responsible for vdid redistribution, such as the size and/or architecture of a hemifusion site (e.g., [34, 35] ). we then asked whether the rate of vdid dequenching varied depending on the cell type. the average rate of vdid fluorescence increase in mdck cells was ,2-fold greater than in a549 cells (figs. 3c and s4b, p,0.02). this demonstrates our ability to detect changes in the rate of vdid transfer and shows that lipid transfer lasts several minutes irrespective of the cell type. we also examined the final extent of vdid dequenching, which is proportional to the surface area of a target membrane over which it redistributes. this parameter was not significantly affected by ifitm3 expression in a549 cells or by the cell type (mdck vs. a549 cells, fig. 3d ). together, similar kinetics and extents of viral lipid dilution in control and ifitm3 + cells suggest that neither the size/architecture of early fusion intermediates nor the surface area of target endosomes is considerably affected by ifitm3 expression. to investigate the relationship between lipid mixing and productive iav infection, we compared the fraction of cells ''receiving'' at least one vdid dequenching event in live cell imaging experiments to the fraction of cells that got infected under the same conditions. the only difference was that virus imaging was not continued beyond 1 h after initiation of fusion, whereas infection proceeded overnight. we found that one or more vdid dequenching events occurred in 15% of a549 cells while 44% of cells got infected (fig. s5 ). under the same conditions, 20% of mdck cells ''hosted'' one or more dequenching events and 36% were infected. the greater fraction of infected cells compared to those permissive to hemifusion is likely due to the shorter time widow for single virus imaging, which is likely to miss late vdid dequencing events (fig. 3b ). the lower apparent fraction of cells supporting vdid dequenching could also be caused by the presence of viruses that did not incorporate self-quenching amounts of vdid. importantly, the comparable efficiencies of lipid mixing and infection, indicate that the former events likely culminate in productive infection. to determine whether ifitm3 impairs the iav's ability to form small fusion pores, we attempted to load the virus with a content marker by soaking in a concentrated solution of sulforhodamine b, as described in [36] . however, only a small fraction of af488-labeled particles stained with sulforhodamine, and the retained dye was lost in live cell experiments under conditions that blocked iav fusion (data not shown). we therefore ifitm3 blocks influenza a virus fusion pore plos pathogens | www.plospathogens.org resorted to using hiv pseudoviruses bearing a/wsn/33 ha and na glycoproteins and co-labeled with the capsid marker, yfp-vpr, and the content marker, gag-icherry [24, 37] . upon virus maturation, the ''internal'' mcherry is proteolytically cleaved off the hiv-1 gag-icherry and released through a fusion pore, as manifested by the loss of the red signal ( fig. 4 and [37] ). the yfp-vpr signal, which remained associated with the viral core after fusion, provided a reference signal for single particle tracking. under our conditions ,1% of double-labeled pseudoviruses entering a549-vector cells lost their content marker, while approximately 2% fused with mdck-vector cells. in sharp contrast, the mcherry release in ifitm3 + a549 and mdck cells or in vector-transduced cells in the presence of nh 4 cl could not be detected (fig. 4e , p,0.001). thus, ifitm3 does not adversely affect iav hemifusion but severely inhibits viral content release into the cytoplasm. together these findings suggest that the mechanism of ifitm3-mediated restriction arises from the entrapment of viruses at a hemifusion intermediate prior to fusion pore formation. a recent study has shown that, through disrupting the interaction between vapa and osbp, ifitm3 causes cholesterol accumulation in le [14] . based on this finding, the authors proposed that high levels of endosomal cholesterol may inhibit iav fusion and/or the release of nucleocapsid. staining with filipin revealed that ifitm3 + a549 cells exhibited increased levels images of vdid dequenching (extended projections) and particle fluorescence intensities obtained by tracking virions in a549 cells. a schematic illustration of iav hemifusion with an endosome (gray), which leads to vdid dequenching, is overlaid on the graph. i 1 and i 2 are fluorescence intensities immediately before dequenching and at the peak of dequenching, respectively. (c) iav lipid mixing activity in a549-vector cells is blocked in the presence of anti-ha antibody. af488and vdid-labeled iav were pre-incubated with 20 mg/ml of polyclonal anti-iav antibody (millipore, billerica, ma) for 1 h at room temperature. viruses were then bound to a549-vector cells in the cold by spinoculation, and entry was initiated with warm imaging buffer supplemented with 20 mg/ml of the antibody. images were collected from 12 fields and the average fraction of af488 particles with the vdid signal above the threshold level was determined and normalized to control conditions without the antibody. ***, p,0.001. (d, e) representative images and analysis of lipid mixing in a549-ifitm3 cells. (f, g) representative images and analysis of lipid mixing in mdck-ifitm3 cells. the ratio of vdid and af488 signals (blue line) shows robust increase in the red signal in spite of variations in the green channel caused by axial displacement of the virus. thick lines were obtained by smoothing raw fluorescence intensity data (thin lines). cell contours are shown by dashed lines in a and d. see also corresponding movies s1, s2 and s3. doi:10.1371/journal.ppat.1004048.g002 of intracellular cholesterol (fig. 5a) . however, the filipin signal was still primarily associated with the plasma membrane and the total cellular cholesterol was not elevated in ifitm3 + cells (fig. s6 ). in addition, the overall intensity of intracellular cholesterol poorly correlated with the level of ifitm3 expression (fig. 5c ). by comparison, pretreatment of a549-vector cells with u18666a, which inhibits transport of ldl-derived cholesterol from le/lysosomes (reviewed in [38] ), resulted in a dramatic shift in the filipin staining pattern from the plasma membrane to endosomes (fig. 5b ). aberrant accumulation of cholesterol in le is also known to occur in cells lacking the functional npc1 cholesterol transporter [39] . we therefore knocked down npc1 expression in a549 cells using shrna (shnpc1, fig. 5d ) and examined the resulting cholesterol distribution (fig. 5b) . reduced npc1 expression correlated with excess cholesterol in intracellular compartments, which was also much more pronounced than endosomal filipin staining in a549-ifitm3 cells. we next asked whether the cholesterol accumulation induced by u18666a pretreatment or by down regulation of npc1 can phenocopy the ifitm3-mediated restriction of viral fusion. neither iav lipid mixing (vdid dequenching) nor fusion (blam signal) was inhibited by silencing npc1 in a549 cells (fig. 5e, f) . vsvpp also fused with shnpc1-transduced cells as efficiently as with control cells (fig. 5e) . these results show that excess cholesterol does not inhibit viral fusion or hemifusion. in control experiments, silencing the npc1 expression potently suppressed fusion of ebola gp-pseudotyped particles (ebovpp, fig. 5e ), which use npc1 as a receptor [40, 41] . similar to the npc1 knockdown phenotype, pretreatment of a549 cells with 10 mm u18666a, which caused cholesterol buildup in endosomes (fig. 5b) , did not inhibit fusion of iavpp or vsvpp (fig. 5g) . as will be shown below for mdck cells, higher doses of u18666a can inhibit viral fusion (fig. 5g) , but this effect is due to elevation of endosomal ph as opposed to cholesterol accumulation in endosomes. to generalize the effects of excess cholesterol in a549 cells, we tested whether endosomal cholesterol can inhibit viral fusion in mdck cells. as in a549 cells, ifitm3 over-expression in mdck cells caused moderate accumulation of cholesterol in endosomes (fig. 6a) , while pre-treatment with u18666a caused a much more dramatic buildup of intracellular cholesterol (fig. 6b) . however, unlike a549 cells, iavpp and vsvpp fusion was significantly inhibited in u18666a-treated mdck cells (fig. 6c ). since prolonged exposure to u18666a has been reported to raise endosomal ph [42] , we sought to determine if insufficiently acidic ph could prevent iav hemifusion/fusion with pretreated mdck cells. the ph in iav-carrying endosomes was measured using virions co-labeled with the ph-insensitive af488 (green) and cypher5e (red), which fluoresces brighter at acidic ph [28] (fig. s7a) . cells were incubated with viruses for 45 min, and the red/green signal ratio from individual particles was measured (fig. s7b) . the average ph in virus-containing endosomes of mdck-ifitm3 cells was slightly less acidic than in control cells: 5.3860.03 (n = 498) vs. 4.9860.04 (n = 242), respectively ( fig. 6d and f, p, 0.001). interestingly, as shown in figure 6e , endosomal ph in u18666a-treated mdck cells was markedly shifted to neutral values (6.4460.05, n = 160, p,0.001). since the ph threshold for triggering a/pr/8/34 fusion is reported to be around 5.6 [43] , elevation of endosomal ph in u18666a-treated mdck cells is the likely cause of inhibition of viral fusion. together our results we also took advantage of the available cho cell line that does not express npc1 [44] to further ascertain the role of endosomal cholesterol in iav fusion. these cells (designated cho-npc1 2 ) exhibited exaggerated endosomal cholesterol staining, in sharp contrast to a peripheral staining pattern in parental cho cells (fig. 7a) . in spite of the high endosomal cholesterol content in cho-npc1 2 cells and of the elevated level of total cholesterol (fig. s6 ), iavpp fused with these cells as efficiently as with parental cells (fig. 7c) . the npc1-null cells also supported iav lipid mixing, albeit at somewhat reduced level compared to control (figs. 7d and s8) . pretreatment of cho cells with u18666a also trapped cholesterol in endosomes and raised the total cholesterol content (figs. 7b and s6 ), but only modestly diminished the extent of iavpp or vsvpp fusion (fig. 7e) . interestingly, in contrast to the decreased endosome acidity in mdck cells, endosomes in u18666a-treated cho cells were more acidic than in control cells (fig. s9) . in control experiments, both the lack of npc1 expression and u18666a pretreatment blocked ebovpp fusion (fig. 7c, e) , consistent with its reliance on npc1 receptor and high sensitivity to disruptions of cholesterol transport [45] . together, our results show that the cholesterol accumulation achieved through two different interventions -u18666a pretreatment and npc1 silencing -does not phenocopy ifitm3mediated restriction of viral fusion. this implies that (i) elevated levels of endosomal cholesterol do not generally confer resistance to viral fusion, and (ii) the mechanism by which ifitm3 blocks transition from hemifusion to full fusion is not through the mislocalization of cholesterol. the ifitms restrict the cellular entry of multiple pathogenic enveloped viruses. recent studies lead to a model that ifitms inhibit virus-host hemifusion [5] and that the membranerigidifying properties of cholesterol may contribute to antiviral actions [14] . in contrast to these studies, our results now demonstrate that ifitm3 prevents the release of viral genomes into the cytosol by inhibiting viral entry after hemifusion but prior to fusion pore formation (fig. 8) . moreover, we found that ifitm3 can promote hemifusion in some cells, perhaps secondary to its acidifying the endosomal pathway. ifitm3 therefore does not negatively regulate the properties of contacting leaflets involved in hemifusion, but stabilizes the cytoplasmic leaflet of the endosomal membrane, thereby disfavoring the formation of fusion pores [35] . in one potential scenario ifitm3 is located directly at the site of arrested hemifusion, perhaps ''toughening'' the endosomal membrane to create a barrier to viral entry (pathway 1). a considerable colocalization of ifitm3 with internalized iav ( [3] and fig. s10 ) is consistent with pathway 1's direct mechanism of inhibition. alternatively, ifitm3 might arrest hemifusion through an indirect mechanism, perhaps involving modulation of lipid and/or protein composition of the cytoplasmic leaflet (pathway 2). recent findings that changes in global membrane properties interfere with productive entry would appear to support an indirect mechanism [5, 14] . lipids, such as unsaturated fatty acids and cholesterol that confer negative spontaneous curvature to membranes can promote hemifusion (a net negative curvature structure) and disfavor a fusion pore (a net positive curvature intermediate), as has been previously shown for oleic acid [35] . although this prediction is consistent with efficient lipid mixing in endosomes of ifitm3 + cells observed in our imaging experiments, several studies [20, [46] [47] [48] and our own results do not support cholesterol accumulation as playing a role in fusion inhibition. we found that cholesterolladen endosomes in cells pretreated with u18666a or expressing undetectable/low levels of npc1 supported efficient viral fusion. it is thus possible that ifitm3 interferes with cellular functions of vapa other than the interaction with osbp, such as regulation of snares and modulation of lateral mobility of membrane proteins (reviewed in [49] ). ifitm3 appears to induce the formation multivesicular bodies and increase the number of ilvs [13, 14] . one can therefore envision that ifitm3 may inhibit infection by redirecting viruses to a non-productive pathway, perhaps involving fusion with ilvs instead of the limiting membrane of le (fig. 8, pathway 3) . if, as suggested in [14] , ifitm3 disallows back fusion of ilvs with the limiting membrane, then virus-ilv fusion products will likely be degraded. indeed, back fusion has been implicated in the vsv core release into the cytosol following the virus-ilv fusion [50] . it should be stressed that this ''fusion decoy'' model does not explain the ability of ifitm1 to interfere with fusion at the cell surface ( [5] and fig. 1d ). it is also not clear why the old world arenaviruses, which have been reported to enter from mvbs [51] , are not restricted by ifitms. the indistinguishable extents of vdid dequenching in control and ifitm3 + cells (fig. 3d) indicate that target endosomes have similar sizes. while this appears to argue against redirection of iav fusion to small ilvs, the lack of a post-hemifusion decay of vdid fluorescence in a549 and mdck cells (figs. 2 and s3) is consistent with iav fusion with abundant ilvs in endosomes of ifitm3 + cells. this is because a lipophilic dye in the limiting membrane of an endosome should be quickly removed through membrane trafficking [24, 31, 52] . because post-dequenching decay was not observed irrespective of the level of ifitm3 expression, it is possible that iav may infect several cell lines by fusing with small intralumenal vesicles followed by the nucleocapsid release through back fusion (fig. 8, dashed black arrows) . this pathway could explain the similar extents and rates of vdid dequenching in control and ifitm3-expressing cells, which are indicative of similar lipid intermediates and of the size of a target membrane, respectively. as discussed above, slow vdid dequenching observed by single iav imaging can be rationalized in the context of fusion with the limiting membrane of endosomes (pathways 1 and 2), as well as in the context of fusion with ilvs (pathway 3). slow dilution of this dye in pathway 3 could occur through multiple rounds of iav fusion with small ilvs, whereas pathways 1 and 2 would predict restricted lipid diffusion through early fusion intermediates formed at the limiting membrane. although the latter notion is in agreement with the reported restriction of lipid movement through hemifusion sites and small fusion pores [34, 35, 53, 54] , these intermediates are usually short-lived under physiological conditions and tend to resolve into larger structures that do not impair lipid movement [28, 32, 35] . clearly, more detailed studies of virusendosome hemifusion and fusion are needed to understand the nature of slow lipid redistribution between iav and endosomes. the ifitms may now arguably be one of the most broadly acting and clinically relevant restriction factor families [1, 3] . while both ifitm3's membrane-associated topology and its localization to the site of viral attenuation suggest it acts to restrict viral entry via a direct mechanism, additional work remains to be done to fully elucidate its actions. nonetheless, as the primary effector of ifn's anti-iav actions, ifitm3 represents a previously unappreciated class of restriction factor that prevents viral entry by stabilizing a hemifusion intermediate, likely comprised of an invading virus fatally tethered to the interior of the endosome's limiting membrane. future single virus experiments combining the detection of both viral lipid and content release events (see for example [52] ) should provide further insights into iav entry pathways and the mechanism of ifitm3-mediated restriction. indeed, such efforts may also bring to light unknown viral countermeasures, which are perhaps employed by the ifitmresistant new and old world arenaviruses. cell lines, plasmids and reagents hek 293t/17 cells and human lung epithelial a549 cells were obtained from atcc (manassas, va) and grown as previously described [55] . wild-type cho cells and cho-npc1 2 cells, a gift from dr. l. liscum (tufts university) [44] , were grown in alpha-mem (quality biological inc, gaithersburg, md) supplemented with 10% fbs and penicillin-streptomycin. the a549, mdck, helah1 and cho cells stably expressing ifitm3 or ifitm1 were obtained by transducing with vsv-g-pseudotyped viruses encoding wild-type ifitm3 and ifitm1 or with the vector pqcxip (clontech) and selecting with puromycin, as described previously [2] . the pr8denv, blam-vpr, pcrev, hiv-1 gag-icherrydenv and pmdg vsv g expression vectors were described previously [37, 55] . the yfp-vpr was a gift from dr. t. hope (northwestern university). the pcaggs vectors encoding influenza h1n1 wsn ha and na were provided by donna tscerne and peter palese, and the pcaggs blam1 (wsn) plasmid was a gift from dr. a. garcia-sastre (mount sinai). vectors expressing phcmv-gpc lassa and pcdna3.1-ebola gp (zaire) were gifts from dr. f.-l. cosset (universitã© de lyon, france) [56] and dr. l. rong (university of illinois) [57] , respectively. u18666a was from tocris bioscience (bristol, uk). poly-llysine, filipin, sulphorhodamine b bafilomycin a1 and the cholesterol kit were from sigma-aldrich. alexafluor-488 amine-reactive carboxylic acid, vybrant-did (vdid, 1,19-dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt), hoechst-33342 and live cell imaging buffer were purchased from life technologies (grand island, ny). cypher5e mono nhs ester was from ge healthcare (pittsburgh, pa). antibodies used were rabbit anti-ifitm3 (to n-terminus) from abgent (san diego, ca), mouse anti-iav-np and goat anti-iavpolyclonal antibodies from millipore (billerica, ma), rat antimouse-igg-fitc from ebioscience (san diego, ca), and goat anti-rabbit-cy5 from jackson immunoresearch (west grove, pa). pseudovirus production and titration were described previously [58] . pseudoviruses were produced by transfecting hek293t/17 cells using jetprime transfection reagent (polyplus-transfection sa, ny). for lasv and ebov pseudoviruses, 5 mg of the phcmv-gpc lassa or 5 mg of the pcdna3.1-ebola gp was included in the transfection mixture. fluorescently labeled influenza pseudoviruses were produced using 1 mg of pr8denv, 2 mg of hiv-1 gag-icherrydenv [37] , 2 mg of yfp-vpr, 1 mg of pcrev, and 2 mg of each wsn ha-and na-expressing vectors. ebola gp pseudoviruses were concentrated 106, using lenti-x tm concentrator (clontech, mountain view, ca). to generate influenza blam1 vlps, hek293t cells were transfected with pcaggs-blam1 (5 mg) and 2.5 mg of each pcaggs-wsn ha and pcaggs-wsn na. after 12 h, the transfection reagent was removed, and cells were further cultivated in phenol red-free growth medium. the influenza virus surface proteins and the lipid membrane were labeled with af488 and vdid, respectively. a hundred mg of influenza virus from the purified h1n1 a/pr/8/34 stock (2 mg/ ml, charles river, ct) was diluted in 95 ml of sodium bicarbonate buffer (ph 9.0) supplemented with 50 mm af488. the mixture was incubated for 30 min at room temperature, after which time, 5 ml of vdid (from 1 mm stock in dmso) was added followed by an additional incubation for 90 min in the dark at room temperature with mild agitation. the labeled viruses were purified through a nap-5 gel filtration column (ge healthcare) in 145 mm nacl solution buffered with 50 mm hepes, ph 7.4. approximately 50% of af488-labeled particles incorporated detectable amounts of vdid with minimal contamination by free dye aggregates. the infectious iav titer was determined in mdck or a549 cells after incubation with serially diluted inoculum for 15 h at 37uc. cells were fixed, permeabilized, blocked and incubated with rabbit r2376 anti-wsn ha antibody (a gift from dr. d. steinhauer, emory university) for 2 h at room temperature. cells were then washed and incubated with secondary cy5-conjugated goat anti-rabbit antibodies (jackson immunoresearch, pa) in 10% fbs-containing buffer supplemented with 10 mg/ml hoechst-33342 for 1 h. the number of infected cells per image field was determined by fluorescence microscopy and normalized to the total number of cells (stained nuclei). the infectious titer (iu/ml) was calculated by taking into account the ratio of the area of well and the image area and correcting for dilution and volume of viral inoculum. the b-lactamase (blam) assay for virus-cell fusion was carried out as described previously ( [24] and methods s1). briefly, pseudoviruses bearing b-lactamase-vpr chimera (blam-vpr) were bound to target cells by centrifugation at 4uc for 30 min at 15506g. unbound viruses were removed by washing, and fusion was initiated by shifting to 37uc for 90 min, after which time cells were placed on ice and loaded with the ccf4-am substrate (life technologies). the cytoplasmic blam activity (ratio of blue to green fluorescence) was measured after an overnight incubation at 12uc, using the synergy ht fluorescence microplate reader (bio-tek, germany). iav was pre-bound to a549-ifitm3 cells in the cold, followed by incubation at 37uc for 90 min and immunostaining with mouse anti-iav-np (millipore, billerica, ma) (when applicable) and rabbit anti-ifitm3 antibody (n-terminus, abgent, san diego, ca), as described in [13] . rat anti-mouse-igg-fitc (ebioscience, san diego, ca) and goat anti-rabbit-cy5 antibodies were used for secondary staining. cellular distribution of cholesterol was examined by incubation with 0.25 mg/ml filipin added during the incubation with secondary antibodies. images were collected on a lsm 780 laser scanning microscope (carl zeiss, germany) using a 636 oil immersion objective. all staining methods involved fixation with 2% paraformaldehyde, permeabilization with 0.25% triton-x100, blocking in with 10% fbs and dilution in phosphate buffered saline (with calcium and magnesium), and sequential incubation with primary and secondary antibodies for 2 h and 1 h, respectively. to silence the npc1 gene, a549 cells were transduced with five shrnas encoded by plk0.1 lentiviral vector (sigma) and selected with puromycin. the samples for western blotting were processed as described in [24] . the npc1 protein band was detected with rabbit anti-npc1 (abcam, cambridge, ma) and horseradish peroxidase-conjugated protein g (bio-rad, hercules, ca), using a chemiluminescence reagent from ge healthcare. stage of an lsm 780 confocal microscope. virus entry was initiated by adding 2.5 ml of pre-warmed imaging buffer and imaged at 37uc using a c-apo 406/1.2na water-immersion objective. three z-stacks separated by ,2 mm were acquired every 7-8 s through the multitime macro (carl zeiss). to block iav hemifusion and fusion, experiments where performed in hbss supplemented with 50 mm hepes/70 mm nh 4 cl (ph 7.6) or containing 200 nm of bafa1. the time lapse images were first visually inspected to identify vdid dequenching or loss of mcherry events. the number of relevant events in each experiment was independently determined by two trained individuals. particle trajectories and their mean/total fluorescence intensities were obtained using volocity (perkinelmer, ma). the onset of lipid mixing and the initial slope of vdid dequenching were determined by fitting to a pair of straight lines (fig. s11 ). iav particles were co-labeled with the af488 dye (phinsensitive) and cypher5e, which fluoresces brighter at acidic ph. the ratios of the cypher5e and af488 signals were converted to ph values using a calibration curve obtained by exposing coverslip-immobilized viruses to citrate-phosphate buffers of different acidity (fig. s7 ). images were collected from 3 different fields, and sum of single-particle fluorescence was calculated. the mean ratios of cypher5e to af488 signals as a function of ph were used for the calibration curve. cells were inoculated with labeled viruses for 45 min at 37uc, as described above. images were collected from at least 10 different fields, and single particle-based ratio of fluorescence signals was calculated. outliers with a near-background cypher5e signal were rejected to reduce the uncertainty in ph measurements. statistical significance was assessed using the pairwise t-test or rank sum test. single-particle fusion events in control and ifitm3 expressing cells were compared by the z-test. figure s5 relationship between iav lipid mixing activity and infection. the fraction of a549 cells where at least one lipid mixing event was observed within 1 h at 37uc, and the fraction of cells that became infected within 15 h at 37uc were estimated as described in methods s1. infectivity data were collected from 5 image fields each, with .30 cells per field. particle-to infectivity ratio was calculated from the fraction of infected cells and the average number of virions bound to cells. figure s7 calibration of labeled iav as a ph-sensor. af488-and cypher5e-labeled iav particles were attached to poly-l-lysine coated coverslips, and the ratio of two fluorescence signals was measured in citrate-phosphate buffers of different acidity. (a) top and bottom panels are images of labeled iav at neutral ph and low ph, respectively. (b) the total signal for each dye was determined after thresholding and the cypher5e/af488 ratio at different ph are plotted. error bars are standard deviations for 3 different imaged fields for each ph value. the line indicates a first order polynomial fit to the data, which served as a ph calibration curve. (pdf) figure s8 an example of single iav lipid mixing event in cho cells. (a) image panels show entry of an af488 (green) and vdid (red) labeled virus into a cho cell that culminates in vdid dequenching (arrow). (b) fluorescence intensity profiles of af488 and vdid obtained by tracking the virion shown in panel a. (pdf) figure s9 ph distribution in iav carrying endosomes of cho cells. shown are the distributions of endosomal ph in cho cells pretreated with 40 mm of u18666a for 12 h or left untreated. cells were incubated with af488/cypher5e-labeled iav, and endosomal ph was measured as described in materials and methods. u18666a increased endosomal acidity (p,0.001). (pdf) figure s10 incoming iav tends to colocalize with ifitm3-positive endosomes. a549-ifitm3 cells were allowed to internalize iav for 90 min at 37uc and immunostained for the iav-np using mouse antibody (millipore, billerica, ma) and for ifitm3. the enlarged boxed area is shown on the right. iav and ifitm3 puncta were identified by thresholding and object identification. the extent of colocalization was estimated by counting iav puncta, which exhibited a volumetric overlap of at least 50% with ifitm3 puncta, and normalizing over all iav puncta. the number in the right corner is the mean % colocalization and standard deviation for 7 image fields. (pdf) figure s11 a line-fitting approach to determining the onset and the initial rate of vdid dequenching in single iav fusion experiments. fitting the vdid dequenching traces with two straight lines yields the time of hemifusion (t h ) and the initial slope of dequenching. (pdf) methods s1 description of additional methods employed in this study. movie s1 lipid mixing between single vdid-labeled iav and an endosome in a549 cells. iav co-labeled with af488 (green) and vdid (red) was incubated with a549 cells at 37uc. the lipid mixing event (hemifusion) is manifested in marked increase of vdid fluorescence. the numbers in the upper right corner show time after raising the temperature (min:sec:msec). scale bar is 10 mm. for details, see fig. 2a movie s3 lipid mixing upon entry of single vdidlabeled iav into an mdck-ifitm3 cell. iav co-labeled with af488 (green) and vdid (red) was incubated with cells at 37uc. lipid mixing (hemifusion) is seen as marked increase in the vdid signal. the numbers in the upper right corner show time after raising the temperature (min:sec:msec). for details, see the broad-spectrum antiviral functions of ifit and ifitm proteins the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitms restrict the replication of multiple pathogenic viruses ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever virus ifitm proteins restrict viral membrane hemifusion distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections ifitm3 limits the severity of acute influenza in mice ifitm3 restricts the morbidity and mortality associated with influenza defining the range of pathogens susceptible to ifitm3 restriction using a knockout mouse model characteristics of ifitm, the newly identified ifninducible anti-hiv-1 family proteins the ifitm proteins inhibit hiv-1 infection ifitm3 inhibits influenza a virus infection by preventing cytosolic entry the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 interferon-induced transmembrane protein 3 is a type ii transmembrane protein differential requirements of rab5 and rab7 for endocytosis of influenza and other enveloped viruses interaction of polyene antibiotics with membrane lipids: physicochemical studies of the molecular basis of selectivity amphotericin b increases influenza a virus infection by preventing ifitm3-mediated restriction different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses caveola-dependent endocytic entry of amphotropic murine leukemia virus a sensitive and specific enzymebased assay detecting hiv-1 virion fusion in primary t lymphocytes hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes an enzymatic virus-like particle assay for sensitive detection of virus entry membrane hemifusion: crossing a chasm in two leaps the energetics of membrane fusion from binding, through hemifusion, pore formation, and pore enlargement visualizing infection of individual influenza viruses characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking gpi-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy diffusion and redistribution of lipid-like molecules between membranes in virus-cell and cell-cell fusion systems membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion the pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation singleparticle kinetics of influenza virus membrane fusion fusion of mature hiv-1 particles leads to complete release of a gag-gfp-based content marker and raises the intraviral ph cellular mechanism of u18666a-mediated apoptosis in cultured murine cortical neurons: bridging niemann-pick disease type c and alzheimer's disease lipid and cholesterol trafficking in npc ebola virus entry requires the cholesterol transporter niemann-pick c1 small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection regulation of the v-atpase along the endocytic pathway occurs through reversible subunit association and membrane localization ph-dependent hemolysis by influenza, semliki, forest virus, and sendai virus the transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in npc1 cells multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin sterols and sphingolipids strongly affect the growth of fusion pores induced by the hemagglutinin of influenza virus multiphasic effects of cholesterol on influenza fusion kinetics reflect multiple mechanistic roles non-vesicular lipid transport by lipid-transfer proteins and beyond endosome-tocytosol transport of viral nucleocapsids old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion the lipid-anchored ectodomain of influenza virus hemagglutinin (gpi-ha) is capable of inducing nonenlarging fusion pores restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion inhibition of hiv-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion characterization of lassa virus cell entry and neutralization with lassa virus pseudoparticles comprehensive analysis of ebola virus gp1 in viral entry multifaceted mechanisms of hiv-1 entry inhibition by human alphadefensin we wish to thank dr. david steinhauer (emory university) for the gift of r2376 antibody, dr. laura liscum (tufts university) for cho-npc1 2 cells, dr. f.-l. cosset (universitã© de lyon, france) and dr. l. rong (university of illinois) for lassa virus and ebola virus gp-expressing vectors, respectively. we are also grateful to dr. n. gahlaut for help with image analysis and lauren byrd-leotis for the initial efforts to label and image iav, as well as to dr. leonid chernomordik (nichd) and the members of melikyan laboratory for stimulating discussions. key: cord-321673-v5o49ees authors: nieto-torres, jose l.; verdiá-báguena, carmina; castaño-rodriguez, carlos; aguilella, vicente m.; enjuanes, luis title: relevance of viroporin ion channel activity on viral replication and pathogenesis date: 2015-07-03 journal: viruses doi: 10.3390/v7072786 sha: doc_id: 321673 cord_uid: v5o49ees modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. in general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. besides boosting virus propagation, viroporins have also been associated with pathogenesis. linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. this article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology. cells maintain optimum subcellular compartment ionic conditions, different to those of the extracellular media by controlling ion transport through lipid membranes. different cell organelles present particular ion compositions. these asymmetric distributions of ions among biological membranes generate electrochemical gradients, essential for the proper cell functioning [1] . crucial aspects of the cell are governed by the membrane potential, ca 2`s tores in the endoplasmic reticulum (er) and the golgi apparatus, and different ph conditions found in the organelles of the secretory pathway, which benefit from those ion gradients. the coordinate action of a multitude of ion channels and transporters generates and tightly controls these ionic milieus found within cells. it is well known that viruses exploit and modify host-cell ion homeostasis in favor of viral infection. to that purpose, a wide range of viruses encode viroporins [2] . viroporins constitute a large family of multifunctional proteins broadly distributed in different viral families, and are mainly concentrated in rna viruses [2] . highly pathogenic human viruses, such as influenza a virus (iav), human immunodeficiency virus 1 (hiv-1), hepatitis c virus (hcv), several picornaviruses, respiratory syncytial virus (rsv), and coronaviruses (covs), such as the one responsible for the severe acute respiratory syndrome (sars-cov), and the etiologic agent of middle east respiratory syndrome (mers-cov), encode at least one viroporin [3] [4] [5] [6] [7] [8] [9] . these are transmembrane proteins that stimulate crucial aspects of the viral life cycle through a variety of mechanisms. noticeably, these proteins oligomerize in cell membranes to form ion conductive pores, which generally display mild ion selectivity, indicating that viroporins do not show preference for particular ionic species. the measurements of channel conductance are in accordance with the formation of relatively wide pores, supporting the non-specificity of viroporins. the influence of the lipid charge in channel function is a distinctive feature of some viroporins, as reported in the case of sars-cov e protein [10, 11] . ion channel (ic) activity is relevant for virus propagation and may have a great impact on host-cell ionic milieus and physiology [2, 12, 13] . once inserted on cell membranes, viroporins tune ion permeability at different organelles to stimulate a variety of viral cycle stages that will be described below. ic activity ranges from almost essential, to highly or moderately necessary for viruses to yield properly. besides modifying cellular processes to favor virus propagation, the loss of ion homeostasis triggered by viral ic activity may have deleterious consequences for the cell, from stress responses to apoptosis [2, 14, 15] . that is why cells have evolved mechanisms to sense the ion imbalances caused by infections and elaborate immune responses to counteract viruses. interestingly, the ic activity of several viroporins triggers the activation of a macromolecular complex called the inflammasome, key in the stimulation of innate immunity [16] [17] [18] [19] [20] [21] . inflammasomes control pathways essential in the resolution of viral infections. however, its disproportionate stimulation can lead to disease. in fact, disease worsening in several respiratory viruses infections is associated with inflammasome-driven immunopathology [22, 23] . taking into consideration the relevance of ic activity in viral production, and its direct effect in pathology and disease, ion conductivity and its pathological stimulated pathways can represent targets for combined therapeutic interventions. in general, viroporins are small proteins (less than 100 amino acids) with at least one amphipathic helix that constitutes its transmembrane domain, spanning lipid membranes [2] . larger viroporins have also been described in covs. this is the case of sars-cov 3a protein, porcine epidemic diarrhea virus (pedv) 3 protein, or human coronavirus 229e (hcov-229e) 4a protein [24] [25] [26] . to form the ion conductive pore, viroporins self-assemble and oligomerize, which is a key feature of this family of proteins. structural studies for either the transmembrane domain or for full-length viroporins have revealed the molecular architecture of these viral ion channels, which can present different oligomerization statuses. iav m2, picornavirus 2b and chlorella virus kcv form tetrameric structures [27] [28] [29] [30] , whereas pentamers have been described for hiv-1 vpu, sars-cov and mers-cov e proteins, and rsv sh protein [9, [31] [32] [33] [34] . hcv p7 and human papillomavirus e5 proteins form hexameric channels [35, 36] . in addition, most measurements of channel conductance are in accordance with the formation of relatively wide pores, again in agreement with the non-specificity of viroporins [6, 10, 37, 38] . ion selectivity of ion channels indicates the preference of the pore for a specific ion and defines the functional roles that the channel may display. it is known that iav m2 protein channels are highly selective for protons and ion conductance is activated at low ph [39] [40] [41] . likewise the iav m2 channel, the kcv protein of chlorella virus is another highly selective channel, which contains a conserved k`selectivity filter [29] . nevertheless, most viroporins usually show mild ion selectivity, which means that, in general terms, these channels do not display preference for a particular ion. hiv-1 vpu protein displays a mild cationic selectivity in nacl and kcl electrolyte solutions [4] . similarly, hcv p7 channels are selective for monovalent cations (na`and k`) over monovalent anions (cl´) [42] . moreover, functionally relevant h`transport has also been identified for this protein in cell culture [12] . orf4a protein of hcov-229e forms a channel that prefers cations over anions but does not show a clear specificity for a particular type of cation [25] . still, a lot of electrophysiology experiments remain to be done for a proper characterization of viroporin selectivity. setting aside a few highly proton selective viroporins, the vast majority display a weak selectivity, either cationic or anionic, which is strongly dependent on the lipid charge of their host membrane, as discussed below in detail. interestingly, under some circumstances the selectivity of these channels can be modulated. sh protein exhibits a poor cationic selectivity at neutral ph, which turns into anionic at acidic ph [43] . this is consistent with the titration of histidines, the only titratable residues of the sh protein. the presence of ca 2`i n selectivity experiments performed in kcl solutions reduced the cationic preference of p7 channels, which may indicate that ca 2`a ffect the selectivity filter [42] . probably, the most striking mechanism influencing ic selectivity and conductance has been recently reported for sars-cov e protein [10, 11] . it was observed that the lipids are an integral component of the pore structure because electrophysiological measurements proved that the lipid charge modulates the ion transport properties of the sars-cov e protein. these findings suggested that viroporins can assemble into alternative complex structures, forming protein-lipid pores ( figure 1 ). viruses 2015, 7 4 figure 1 . ion channels formed by viroporins. left depiction represents a channel exclusively formed by protein monomers (blue cylinders) inserted on a lipid membrane. schematic on the right shows a protein-lipid pore. in this latter case, the lipid head groups (cyan circles) are oriented towards the channel pore, modulating ion conductance and selectivity. the ic activity of a number of transmembrane proteins, as well as of small peptides and antimicrobial peptides, is strongly dependent on the lipid environment. the case of sars-cov e protein is a clear example of how lipid membrane charge influences the main ion transport properties of an ic. e protein behaves quite differently when reconstituted in neutral or negatively-charged membranes ( figure 2 ). depictions represent e protein channels inserted in non-charged membranes (left) or negatively-charged membranes (right), under low solute concentrations and neutral ph. in these circumstances, each e protein monomer presents two negative charges provided by glutamic acid residues, and a positive charge conferred by an arginine. when reconstituted in fully or partially negatively-charged membranes, lipid head groups provide additional negative charges to the pore, which makes e protein channel more selective for cations and more conductive. conductance experiments showed that in non-charged phosphatidylcholine membranes, the e protein channel acts as a non-selective neutral pore, since the channel conductance changes linearly with bulk solution conductivity [11] . in contrast, in negatively charged phosphatidylserine membranes the variation of channel conductance with solution concentration follows different regimes depending on the salt concentration; a characteristic trait of charged pores [10, 11, 44] . these experiments, together with ion selectivity measurements [11] , suggest that e protein behaves as a charged pore in negativelycharged membranes. parallel observations are made when reconstituting sars-cov e protein in membranes containing a small percentage of charged lipids and, in particular, in membranes containing figure 1 . ion channels formed by viroporins. left depiction represents a channel exclusively formed by protein monomers (blue cylinders) inserted on a lipid membrane. schematic on the right shows a protein-lipid pore. in this latter case, the lipid head groups (cyan circles) are oriented towards the channel pore, modulating ion conductance and selectivity. the ic activity of a number of transmembrane proteins, as well as of small peptides and antimicrobial peptides, is strongly dependent on the lipid environment. the case of sars-cov e protein is a clear example of how lipid membrane charge influences the main ion transport properties of an ic. e protein behaves quite differently when reconstituted in neutral or negatively-charged membranes ( figure 2 ). left depiction represents a channel exclusively formed by protein monomers (blue cylinders) inserted on a lipid membrane. schematic on the right shows a protein-lipid pore. in this latter case, the lipid head groups (cyan circles) are oriented towards the channel pore, modulating ion conductance and selectivity. the ic activity of a number of transmembrane proteins, as well as of small peptides and antimicrobial peptides, is strongly dependent on the lipid environment. the case of sars-cov e protein is a clear example of how lipid membrane charge influences the main ion transport properties of an ic. e protein behaves quite differently when reconstituted in neutral or negatively-charged membranes ( figure 2 ). depictions represent e protein channels inserted in non-charged membranes (left) or negatively-charged membranes (right), under low solute concentrations and neutral ph. in these circumstances, each e protein monomer presents two negative charges provided by glutamic acid residues, and a positive charge conferred by an arginine. when reconstituted in fully or partially negatively-charged membranes, lipid head groups provide additional negative charges to the pore, which makes e protein channel more selective for cations and more conductive. conductance experiments showed that in non-charged phosphatidylcholine membranes, the e protein channel acts as a non-selective neutral pore, since the channel conductance changes linearly with bulk solution conductivity [11] . in contrast, in negatively charged phosphatidylserine membranes the variation of channel conductance with solution concentration follows different regimes depending on the salt concentration; a characteristic trait of charged pores [10, 11, 44] . these experiments, together with ion selectivity measurements [11] , suggest that e protein behaves as a charged pore in negativelycharged membranes. parallel observations are made when reconstituting sars-cov e protein in membranes containing a small percentage of charged lipids and, in particular, in membranes containing depictions represent e protein channels inserted in non-charged membranes (left) or negatively-charged membranes (right), under low solute concentrations and neutral ph. in these circumstances, each e protein monomer presents two negative charges provided by glutamic acid residues, and a positive charge conferred by an arginine. when reconstituted in fully or partially negatively-charged membranes, lipid head groups provide additional negative charges to the pore, which makes e protein channel more selective for cations and more conductive. conductance experiments showed that in non-charged phosphatidylcholine membranes, the e protein channel acts as a non-selective neutral pore, since the channel conductance changes linearly with bulk solution conductivity [11] . in contrast, in negatively charged phosphatidylserine membranes the variation of channel conductance with solution concentration follows different regimes depending on the salt concentration; a characteristic trait of charged pores [10, 11, 44] . these experiments, together with ion selectivity measurements [11] , suggest that e protein behaves as a charged pore in negatively-charged membranes. parallel observations are made when reconstituting sars-cov e protein in membranes containing a small percentage of charged lipids and, in particular, in membranes containing the same amount of charged lipid ("20%) as the ergic-golgi membranes, where e protein channel is presumably localized [10] . these observations support the view that some polar lipid heads line the pore lumen together with the protein transmembrane helices and that both, protein and lipids, contribute to channel selectivity. an additional proof reinforcing this hypothesis is provided by electrophysiology experiments using lipids with negative intrinsic curvature. it is known that the protein-lipid pore formation is favored in membranes with positive curvature [45] . in fact, when e protein is reconstituted in dioleoyl phosphatidylethanolamine membranes, a lipid with intrinsic negative curvature, it is much more difficult to achieve channel insertion because the membrane negative curvature becomes energetically unfavorable for the assembly of a proteolipidic structure [10] . the e protein-lipid channel may not constitute a unique case, as poliovirus 2b permeabilization displayed a strict requirement for anionic phospholipids in the membrane composition [28] , which may indicate that lipid molecules are involved in the pore formation. regulatory mechanisms, such as gating, operate in some viroporins. histidine residues present in the iav m2 transmembrane domain become protonated when encountering low ph conditions, and experience a conformational change that opens the channel pore allowing h`flux [46] . the influence of the histidine residues in the ic is also observed in the sh protein. in this protein the conductance is ph-dependent and consistent with the titration of these residues [43] . viroporins could be regulating the transport capacity of cellular ion transporters, besides showing their intrinsic ic activity. a well-defined case is that of human papillomavirus oncoprotein e5, which interacts with vacuolar h`atpase (v-atpase) modulating its function and leading to cell transformation [47, 48] . interestingly, other viroporins interact with cellular ion pumps. sars-cov e protein binds the alpha subunit of na`/k`atpase in infected cells [49] . iav m2 protein binds na`/k`atpase beta1 subunit [50] . it is well known that the ion transport capacity of cellular pumps such as na`/k`atpase and sarcoendoplasmic reticulum ca 2`a tpase (serca) may be influenced by the interaction with other proteins. fxyd proteins and phospholamban are well-characterized examples [51, 52] . these regulatory proteins are small transmembrane proteins that oligomerize and eventually form ics when reconstituted in artificial lipid membranes [53] , as happens for viroporins. some examples of viroporins modifying the activity of cellular ion channels have also been reported. hiv-1 vpu interacts with the k`channel task-1 promoting viral release [54] . in addition, indirect inhibition of epithelial sodium channels has been reported for iav m2 and sars-cov e proteins [55, 56] . the possibility of a dual role of ion modulation by viroporins, depending both on their intrinsic transport properties and on their possible regulatory activity on key cellular ion transporters, increases the potential relevance of this family of proteins. viroporins are involved in processes relevant for virus production. in general, these proteins do not affect viral genome replication, but stimulate other key aspects of the viral cycle such as entry, assembly, trafficking and release of viral particles [2, 13] . as a consequence, partial or total deletion of viroporins usually leads to significant decreases in viral yields. hcv p7 viroporin is indispensable for virus propagation, as no infectious viruses are recovered when p7 protein is eliminated [57] . other viruses are more tolerant to viroporin deletion. thus, iav or hiv-1 lacking m2 and vpu, respectively, can be efficiently rescued. nevertheless, in general, viroporin defective viruses show significantly lower virus yields, ranging from 10-to 100-fold [58, 59] . the relevance of e viroporin in coronavirus production seems to be species-specific. deletion of e gene in transmissible gastroenteritis virus (tgev) and mers-cov generates replication-competent propagation-deficient viruses [60, 61] . e gene is not essential for virus production in sars-cov and mouse hepatitis virus (mhv), although in its absence viral titters are reduced from 100-to 1000-fold, respectively [62, 63] . silencing of sars-cov 3a protein, pedv 3 protein, and hcov-229e 4a protein expression in virus infection cause a reduction in virus titers [24, 25, 64] , supporting that these viroporins are involved in virus production. in the case of sars-cov 3a protein, this has also been shown in mice infected with a sars-cov variant missing 3a protein (c. castaño-rodriguez, j.l. nieto-torres and l. enjuanes, unpublished results). in some cases, viroporin deletion induces a growth restriction that can be cell or tissue specific. thus, rsv lacking the sh gene, shows an efficient growth in cell culture, but a limited production in the nasal turbinates of infected mice or chimpanzees, as compared with the wild type virus [65, 66] . collectively, previous data support that viroporins stimulate virus propagation. a key issue is to understand the relevance of their ic conductance on this activity. both viroporin ic activity and other functions not related to ion conductivity apparently affect virus propagation. some non-conductive viroporin domains are important players in viral morphogenesis. the cytoplasmic tail of several viroporins actively participates in virus production. a few examples are now briefly described. cov e protein c-terminal domain interacts with the viral membrane (m) protein during the morphogenesis process [67] . in fact, the last nine amino acids of sars-cov e protein c-terminus stimulate virus growth [68] . this protein sequence includes a pdz-binding motif involved in interactions with cellular proteins and virulence. iav m2 cytoplasmic domain interacts with the m1 protein to favor virus assembly at the site of budding [69] . an amphipathic helix located in the cytoplasmic tail of the iav m2 protein induces bending of cellular membranes, necessary for budding, and excision of the nascent particles [70] . this functional domain works independently to the m2 ion conductive properties. hiv-1 vpu also facilitates budding termination of newly formed viruses in an ic independent manner. vpu deleted viruses are less efficiently released and remain attached to the plasma membrane of infected cells [59] . in this case, the transmembrane domain of vpu accounts for this phenotype by establishing critical interactions with cellular proteins such as one with the interferon-stimulated protein tetherin. this binding prevents tetherin from inhibiting the release of viral particles at the plasma membrane of infected cells [71, 72] . both ic activity and tetherin inhibitory property are concentrated in the vpu transmembrane domain, nevertheless these functions work independently [73] . accumulating reports support that viroporin ion conductivity favors virus yields. inhibition of viroporin ic properties, through subtle mutations that may not interfere with other functions of the protein, affects viral growth to different extents. point mutations that blocked hcv p7 ic activity either completely abrogated virus production or resulted in a 100-fold restriction in virus yields, depending on the virus strain [12, 57] . influenza viruses lacking m2 ion conductivity, presented either a 15-fold reduction of viral titer in tissue culture [74] , or showed a standard production in cell culture but a restricted growth in the nasal turbinates of infected mice [75] . sars-cov e protein ion conductivity also supports viral production and fitness. e protein ic activity was knocked down by introducing point mutations in key residues of its transmembrane domain [10] . although not essential for virus production, e protein ic activity stimulated viral propagation. viruses lacking e protein ion conductivity were outcompeted by others displaying this function [22] . in fact, ic defective viruses tended to restore ion conductance by introducing compensatory mutations in the transmembrane domain of the protein both in cell cultures and in mice [22] . in agreement with these data, iav lacking m2 ic activity showed fitness defects, as it was outgrowth by the parental virus, in an even faster manner than that observed in sars-cov [74] . the relevance of ion conductivity in boosting virus production remains unknown for the moment in other viral systems. initial findings argued that scrambling of the hiv-1 vpu transmembrane domain inactivated ion conductivity and partially inhibited viral production [76] . however, this alteration of the transmembrane domain affected the vpu-tetherin interaction and ic activity. recent reports showed that mutations that specifically inhibited ion conductivity, but not vpu-tetherin interaction, did not affect vlp production; therefore, this interaction with tetherin seems to be responsible for the observed phenotype [73] . pharmacological inhibition of viroporin ion conductance further supports the role of ic in virus propagation. several compounds inhibit viroporin ion conductivity in artificial lipid membranes, and some of them efficiently reduce viral growth when administered to infected cells. inhibition of m2 protein ic activity mediated by amantadine prevents or decreases viral growth in a strain specific manner [39, 77] . amantadine also inhibits hcv growth [13] . hcov-229-e and mhv growth is restricted by hexamethylene amiloride, which blocks e protein ion conductivity [78] . the viral growth inhibitory properties of these compounds encourage its use as potential antivirals. this aspect will be further addressed below. widely conserved pathways influenced by viroporin ion conductivity, and others that seem to be species-specific are now described and graphically summarized to facilitate their overview ( figure 3 ). early key aspects of virus cycle, such as viral entry, can be boosted through ion conductivity. non-enveloped viral particles lack viroporins as structural components, and these proteins are poorly represented in the membrane of enveloped viruses [63, [79] [80] [81] . nevertheless, the viroporin molecules embedded in the viral envelope can actively participate in viral entry; iav m2 protein is a well characterized example. iav is internalized within the cell through an endocytic process. endosomes containing viral particles fuse with acidic organelles to form endolysosomes. the h`atpase of these organelles pumps h`inside their lumen lowering the ph. the few m2 copies present in the iav envelope are activated by the low ph conditions and allow the flux of h`inside the viral particle. the acidification of the endocytosed virion drives a series of conformational changes in the viral hemagglutinin (ha) protein, leading to the exposure of a fusion peptide [82, 83] . in parallel, the m1 protein layer, which underlies the viral envelope, and protects the viral ribonucleoproteins, is disassembled under these ph conditions [84] . the fusion peptide finally triggers the fusion of the viral and endosomal membranes, resulting in the release of viral ribonucleoproteins in the cell cytoplasm. inhibition of m2 protein ic activity by amantadine results in incomplete uncoating of the virion in some iav strains [39, 77] . this mechanism does not seem relevant for hcv virus, as its entry is independent of p7 ic activity [12] . whether or not this pathway, or related ones, participate in the entrance of other viral species remains to be established. viruses 2015, 7 8 figure 3 . pathways stimulated by viroporin ion channel activity leading to virus production. viral-membrane embedded viroporins (red ellipses) transport h + inside the endocytosed virion. this causes structural changes in fusion and matrix proteins facilitating the uncoating of viral ribonucleoproteins (1); viroporin-mediated ions leak from intracellular organelles such as the endoplasmic reticulum (er) or the golgi apparatus towards the cytoplasm causes a blockade of vesicle transport and/or hijacking of autophagic membranes. these processes finally result in the accumulation of membranous structures that will serve as platforms for viral replication and morphogenesis. blue, red and green structures show the viral replicase (2); in addition, equilibration of golgi and secretory pathway organelles' ph protect both viral proteins involved in entry (blue structures and green ellipses) and newly formed virions that can be sensitive to acidic environments (3); viroporins (red and blue ellipses), locate in the budding neck of some enveloped viruses. these proteins may interact and oligomerize, rearranging the formation of channels, which additionally could facilitate virion scission (4). early key aspects of virus cycle, such as viral entry, can be boosted through ion conductivity. non-enveloped viral particles lack viroporins as structural components, and these proteins are poorly represented in the membrane of enveloped viruses [63, [79] [80] [81] . nevertheless, the viroporin molecules embedded in the viral envelope can actively participate in viral entry; iav m2 protein is a well characterized example. iav is internalized within the cell through an endocytic process. endosomes containing viral particles fuse with acidic organelles to form endolysosomes. the h + atpase of these organelles pumps h + inside their lumen lowering the ph. the few m2 copies present in the iav envelope + figure 3 . pathways stimulated by viroporin ion channel activity leading to virus production. viral-membrane embedded viroporins (red ellipses) transport h`inside the endocytosed virion. this causes structural changes in fusion and matrix proteins facilitating the uncoating of viral ribonucleoproteins (1); viroporin-mediated ions leak from intracellular organelles such as the endoplasmic reticulum (er) or the golgi apparatus towards the cytoplasm causes a blockade of vesicle transport and/or hijacking of autophagic membranes. these processes finally result in the accumulation of membranous structures that will serve as platforms for viral replication and morphogenesis. blue, red and green structures show the viral replicase (2); in addition, equilibration of golgi and secretory pathway organelles' ph protect both viral proteins involved in entry (blue structures and green ellipses) and newly formed virions that can be sensitive to acidic environments (3); viroporins (red and blue ellipses), locate in the budding neck of some enveloped viruses. these proteins may interact and oligomerize, rearranging the formation of channels, which additionally could facilitate virion scission (4). modification of the ionic content of intracellular compartments can ultimately result in functional and morphological transformations. the ion conductivity of several viroporins affects protein transport. alteration of the ionic content of the golgi apparatus and the endosomes interfere with cellular protein processing and sorting [85] . iav m2 causes a lag in protein transport through the golgi apparatus [86] . this delay on transport can be inhibited by the addition of amantadine, an ic activity inhibitor of m2 protein. furthermore, monensin, an h`/na`antiporter, causes similar defects. both m2 and monensin induce dilation of golgi cisternae, although to different extents. infectious bronchitis virus (ibv) e protein also affects protein transport through the secretory pathway, whereas the mutant predicted to inhibit ic activity does not [87] . coxsackievirus 2b protein disturbs ph and calcium homeostasis in the golgi and the er, thereby inhibiting protein transport. mutations inhibiting 2b ion conductivity restored proper protein trafficking [88] . ca 2`i s involved in membrane fusion events, and its leakage to the cell cytoplasm causes anterograde vesicle trafficking blockage. sars-cov 3a protein is both necessary and sufficient for sars-cov golgi fragmentation and promotes the accumulation of intracellular vesicles that may be used for virus formation or for non-lytic release of virus particles [89] . the exact relevance of protein transport delay on virus production remains to be established. it has been speculated that alteration of ionic homeostasis in transport vesicles affects anterograde trafficking leading to conglomeration of cell membranes that will serve as the platforms for viral replication and budding [88] . in addition, ionic efflux from intracellular organelles can lead to pathways triggering the accumulation of autophagic vacuoles as platforms for virus replication. in fact, rotavirus nsp4 viroporin allows ca 2`e fflux form er thereby triggering autophagy in favor of viral infection [90] . in many cases, viral proteins, and even virions, have to progress through the secretory pathway, encountering the low ph found within the golgi apparatus lumen [91] . these acidic conditions may inactivate forming virions, which eventually are acid-sensitive [12] . viroporin ion conductivity is critical in viral progeny protection and, occasionally, it can be trans-complemented by heterologous viroporins. iav m2 ic activity is thought to keep the ph of the golgi apparatus and its associated vesicles above a threshold, in order to avoid conformational changes in ha which may lead to its premature activation rendering non-infectious viruses. in fact, blocking of m2 ion conductivity by amantadine induces irreversible activation of ha [92] . this effect can be overcome by the treatment of infected cells with monensin that alkalizes the golgi in an analogous manner to m2 protein. similarly to what has been shown for m2 protein, hcv p7 protein mediates a h`leak from intracellular organelles, and this alkalinization is required for the production of infectious viruses [12] . mutant viruses lacking p7 ion conductivity were not rescued, and treatment with amantadine greatly diminished the production of wild type infectious virions. furthermore, in the absence of p7 ic activity, alternative approaches to balance the ph of intracellular organelles result in partial recovery of infectious viruses. either bafilomycin a1 treatment, an inhibitor of a h`vacuolar atpase (v-atpase), or expression of iav m2, but not the m2 ic mutant, rescued virus production [12] . the increase of cellular permeability by viroporins, ic activity may ultimately result in cell lysis, a requisite for the egress of non-enveloped viruses [2] . it has also been speculated that disruption of ion gradients may trigger the membrane fusion events necessary for budding termination and release of enveloped viruses [2] . nevertheless, in the latter case other non-ion conductive viroporin functions, as those previously described for m2 and vpu, may also be relevant in the release process. in addition, we propose that viroporins, located in the budding neck of forming viruses [70] , could oligomerize to form an ic facilitating membrane fusion and budding termination. besides their key role in virus propagation, viroporins are also virulence factors in different viral systems. total or partial deletion of non-essential viroporins usually leads to attenuated phenotypes. interestingly, these viroporin-deleted viruses have been successfully used as potential vaccine candidates. mice infected with an iav lacking m2 protein showed no weight-loss, nor pathology, and were protected against the wild type virus [93] . an rsv defective for sh protein showed growth attenuation in the upper respiratory tracts of mice and chimpanzees and caused mild disease [65, 66] . a classical swine fever virus (csfv) missing full-length or partial-length p7 protein, similar to hcv p7 viroporin, lacked virulence [94] . a sars-cov lacking the full-length e protein (sars-cov-∆e) was attenuated in hamsters, transgenic mice expressing the human sars-cov receptor (hace2), and in both young and elderly mice. sars-cov-∆e induced increased stress and limited nf-κb driven inflammation [62, 95, 96] . in addition, elimination of defined e protein regions of 6-12 amino acids in its carboxy-terminus leads to viral attenuation [68, 97] . several of these mutants are promising potential vaccine candidates for sars-cov. sars-cov 3a protein regulates host-cellular responses involved in the activation of pro-inflammatory genes such as c-jun and nf-κb [98] [99] [100] , and in the production of pro-inflammatory cytokines and chemokines such as il-8 or ccl5 [99] . furthermore, 3a protein ic activity has been linked to its pro-apoptotic function [101] . pedv 3 protein has also been involved in pathogenesis as a pedv with a 49-nucleotide deletion in 3 gene lacks ic activity and is attenuated [24] . viroporins are frequently associated with virus propagation, as their deletion from the viral genome usually causes viral titer drops that, by themselves, could contribute to the attenuated viral phenotype. however, viroporin removal can modulate cellular signaling pathways leading to virulence. it has been proposed that the protein transport blockage exerted by the ic activity of viroporins, such as that from coxsackievirus 2b and coronavirus e proteins causes a delay in the transport of major histocompatibility complex class i (mhc-i) molecules towards the plasma membrane [87, 88, 102] . this mhc-i trafficking defect allows the evasion of adaptive immune responses, leading to more productive infections (figure 4) . over stimulation of immune responses can also lead to pathogenesis. indeed, ic activity is an important trigger of immunopathology, as demonstrated for sars-cov e protein. mutant viruses lacking ion conductivity, which showed proficient replication in mice lungs, presented an attenuated phenotype. animals infected with the sars-covs lacking e protein ic activity showed a reduced mortality in comparison with those inoculated with the parental virus [22] . interestingly, mice infected with the ic defective viruses presented much less edema accumulation in the lung airways, the ultimate cause of acute respiratory distress syndrome (ards) [103] . flooded bronchioles and alveoli fail to interchange gases, leading to hypoxemia and eventually to death. noticeably, there was a good correlation between edema accumulation and the disassembly of the airway epithelia which, when intact, drive edema resolution through an ion mediated water reabsorption. edema accumulation and airway epithelia damage is accompanied by an exacerbated proinflammatory response in the lung parenchyma [103] . animals infected with the ic deficient viruses presented decreased levels of il-1β, tnf and il-6 in the lung airways, key mediators of the ards progression. il-1β is a key orchestrator of the inflammatory response and plays a critical role in counteracting invading viruses, however overstimulation of this pathway can lead to unwanted deleterious effects for the organism. in fact, over production of il-1β is related with important pathologies such as gout, atherosclerosis, diabetes, ards and asthma [104] [105] [106] [107] . as a consequence, il-1β production is tightly regulated in the organism, through the inflammasome multiprotein complex. figure 4 . pathways stimulated by viroporin ion channel activity leading to pathology. molecular patterns associated with viral infections are recognized by cellular sensors (signal 1), which activate the transcription and translation of the nlrp3 inflammasome components (nlrp3, asc and procaspase-1) and the inactive pro-il-1β. viroporins inserted in the intracellular organelles, such as the endoplasmic reticulum (er) or the golgi apparatus, favor the leak of ca 2`a nd h`ions that move following their electrochemical gradient into cell cytoplasm. this ionic imbalance (signal 2) induces the assembly of the inflammasome complex, which triggers the maturation of pro-il-1β into il-1β through the action of caspase-1. secreted il-1β mediates a potent pro-inflammatory response that can be deleterious for the cell and the organism, when overstimulated. in addition, alteration of ionic milieus in intracellular compartments comes along with a protein transport delay or blockage. this results in a decrease of the levels of mhc-i molecules (blue rectangles) at the plasma membrane, preventing the infected cell to be recognized by the immune system. protein transport blockage also diminishes the levels and activity in the cell surface of ion channels and transporters, crucial in the resolution of edema accumulation. epithelial sodium channels (green structure) and na`/k`atpase (purple rectangles) impairment have been related to the worsening of viral respiratory diseases such as those caused by sars-cov, iav or rsv. how can viral ic activity stimulate this exacerbated inflammatory response leading to pathology and disease? inflammasomes participate in pathogen recognition by sensing disturbances in cellular milieus, including intracellular ionic concentrations [108, 109] . the nlrp3 inflammasome is one of the most extensively studied [110] . two signals are generally required to activate this complex: the first one is usually triggered by molecular patterns associated to the viral infection, such as double-stranded rna. this leads to the transcription and translation of the different components of the inflammasome and the immature pro-il-1β [16] . only when a second signal is simultaneously present, the components of the inflammasome are assembled, leading to the cleavage and activation of caspase-1. this protein processes pro-il-1β into its active form il-1β that is released to the extracellular media to promote proinflammation [16] . interestingly, viroporin ic activity represents the second signal required for inflammasome activation, inducing the release of active il-1β (figure 4 ). in general, viroporins induce an efflux of ions, such as h`and ca 2`t hat move from their intracellular stores into the cytoplasm following strong electrochemical gradients, triggering the nlrp3 inflammasome. iav m2 was the first noticed ion conducting viral protein activating this pathway [16] . nowadays, several other viroporins stimulating the inflammasome have been identified. rsv sh, human rhinovirus 2b, encephalomyocarditis virus 2b, hcv p7, and iav pb1-f2, among others, are additional examples [17] [18] [19] [20] 23, 111] . this mechanism of overstimulation of il-1β production seems to have key consequences in pathogenesis. the severity of human rhinovirus infection is linked to overinflammation and release of cytokines, including il-1β. rhinovirus 2b protein causes a ca 2`l eakage from the er and golgi apparatus essential for inflammasome activation and il-1β production [19] . similarly, rsv sh activates this pathway resulting in immunopathology [18] . given the wide variety of effects that viroporin ic activity has on viral propagation and its influence on pathogenesis, inhibition of ic conductivity has been a promising target for therapeutic interventions. a growing list of compounds interfering with viroporin ic activity of several viruses such as iav, hcv, hiv-1, coronaviruses, and rsv, has been described [43, 78, [112] [113] [114] . despite being active in artificial lipid bilayers, and sometimes in cell culture, the pharmacological use of antagonists of ic activity in humans is still limited to a reduced number of cases. amantadine was the first inhibitor of viroporin ic activity approved for use in humans, and it has been utilized in clinics for around 20 years in the treatment of iav [113] . amantadine binds m2 protein at the n-terminal lumen of the channel and at the c-terminal surface of the protein with high and low affinities, respectively, blocking ion conductance and interfering with viral replication [115] . however, iav variants containing mutations in the m2 transmembrane domain that conferred resistance to amantadine emerged [115, 116] . amantadine and hexamethylene amiloride are effective inhibitors of hcv p7 ic activity as shown using in vitro systems [42, 112] . hcv shows a genotype-dependent sensitivity to amantadine, when the drug is applied at high doses, which may explain its inefficacy in infected patients, limiting the application of amantadine in hcv disease treatment [117] . similarly, high concentrations of hexamethylene amiloride are required to inhibit p7 conductivity in cell culture, which increased its toxicity, making this drug unsuitable for clinical administration [117] . however, drug screenings have identified other promising compounds interfering p7 ic activity, such as long alkyl iminosugars derivatives and bit225, with the latter showing modest but successful restriction of hcv load in infected patients [6, 118] . these compounds may represent the basis for a therapeutic treatment of hepatitis c. taking into consideration the fast selection of drug-resistant viruses under drug pressure, the simultaneous inhibition of ic activity, and other signaling pathways stimulated by ion conductivity leading to pathology, may represent a better 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small-molecule inhibitor of the nlrp3 inflammasome for the treatment of inflammatory diseases the work done by the authors was supported by grants from the government of spain the authors declare no conflict of interest. key: cord-298458-p7rvupjo authors: schmidt, megan e.; varga, steven m. title: the cd8 t cell response to respiratory virus infections date: 2018-04-09 journal: front immunol doi: 10.3389/fimmu.2018.00678 sha: doc_id: 298458 cord_uid: p7rvupjo humans are highly susceptible to infection with respiratory viruses including respiratory syncytial virus (rsv), influenza virus, human metapneumovirus, rhinovirus, coronavirus, and parainfluenza virus. while some viruses simply cause symptoms of the common cold, many respiratory viruses induce severe bronchiolitis, pneumonia, and even death following infection. despite the immense clinical burden, the majority of the most common pulmonary viruses lack long-lasting efficacious vaccines. nearly all current vaccination strategies are designed to elicit broadly neutralizing antibodies, which prevent severe disease following a subsequent infection. however, the mucosal antibody response to many respiratory viruses is not long-lasting and declines with age. cd8 t cells are critical for mediating clearance following many acute viral infections in the lung. in addition, memory cd8 t cells are capable of providing protection against secondary infections. therefore, the combined induction of virus-specific cd8 t cells and antibodies may provide optimal protective immunity. herein, we review the current literature on cd8 t cell responses induced by respiratory virus infections. additionally, we explore how this knowledge could be utilized in the development of future vaccines against respiratory viruses, with a special emphasis on rsv vaccination. introduction given its continuous exposure to the outside environment, the respiratory mucosa is highly susceptible to viral infection. the human respiratory tract can be infected with a variety of pulmonary viruses, including respiratory syncytial virus (rsv), influenza virus, human metapneumovirus (hmpv), rhinovirus (rv), coronavirus (cov), and parainfluenza virus (piv) (1) . the severity of disease associated with respiratory viral infection varies widely depending on the virus strain as well as the age and immune status of the infected individual. symptoms can range from mild sinusitis or cold-like symptoms to more severe symptoms including bronchitis, pneumonia, and even death. rsv is the leading cause of severe lower respiratory tract infection in children under 5 years of age (2) . rsv is commonly associated with severe lower respiratory tract symptoms including bronchiolitis, pneumonia, and bronchitis and is a significant cause of hospitalization and mortality in children, the elderly, and immunocompromised individuals (2) (3) (4) (5) (6) . similarly, piv commonly infects children and is a major cause of croup, pneumonia, and bronchiolitis (7, 8) . seasonal influenza infections, most often of the influenza a virus (iav) subtype, are responsible for 3-5 million cases of severe infection annually (9) . seasonal iav infections also result in approximately 290,000-650,000 deaths per year, most commonly in either young children or elderly populations (9) (10) (11) . however, infection with emerging pandemic iav strains, such as the 2009 h1n1 pandemic strain, primarily induces severe disease and mortality in otherwise healthy adults younger than 65 years of age (12) . in contrast, respiratory infection with hmpv, rv, and cov are most commonly associated with symptoms of the common cold (13) (14) (15) . two notable exceptions are severe acute respiratory syndrome (sars) cov and middle east respiratory syndrome (mers) cov, which cause acute respi ratory distress and mortality in infected individuals (16) (17) (18) . despite their profound impact on human health, most common respiratory viruses lack an approved vaccine. the strategy employed most often in vaccine development is the induction of robust neutralizing antibody responses. however, the hallmark of many respiratory viral infections, including rsv, hmpv, and rv, is the ability for reinfections to occur frequently throughout life (19) (20) (21) . this suggests that the antibody response to these respiratory viruses may wane over time. indeed, despite a correlation between pre-existing nasal iga and protection from reinfection, the development of long-lasting rsv-and rv-specific mucosal iga responses was poor in infected adults (22, 23) . although iav-specific neutralizing antibodies are elicited efficiently through either infection or vaccination, iav vaccine formulations must be redeveloped annually to account for the rapid mutations of ha and na genes in seasonal strains (24) . therefore, vaccinations that solely promote the induction of neutralizing antibodies may not be optimal in providing protection against many respiratory virus infections. the induction of cellular immune responses has thus far received little attention in respiratory virus vaccine development. cd8 t cells play a critical role in mediating viral clearance following many respiratory virus infections including rsv, iav, and hmpv (25) (26) (27) . in addition, recent murine studies utilizing cd8 t cell epitope-specific immunization strategies observed significantly reduced lung viral titers following iav, rsv, or sars challenges (28) (29) (30) . therefore, the induction of virus-specific cd8 t cell responses has the potential to improve upon the efficacy of current vaccination strategies. here, we review the current literature on cd8 t cell responses following respiratory virus infections and discuss how this knowledge may best be utilized in the development of future vaccines. following an acute respiratory infection, dendritic cells (dcs) that have taken up viral antigen stimulate the activation of naive cd8 t cells in the lung draining lymph node to induce robust virus-specific cd8 t cell responses [reviewed in ref. (31) (32) (33) ]. respiratory virus infection in mouse models results in an increase in the frequency and number of total and antigenspecific cd8 t cells in the lungs and airways. rsv-specific cd8 t cell responses typically reach peak numbers in the lung at approximately day 8 following an acute infection (34) (35) (36) (37) . the kinetics of virus-specific cd8 t cells are slightly more delayed following other respiratory virus infections with peaks occurring at approximately day 10 for iav, days 10-14 for hmpv, and days 12-14 for pneumonia virus of mice (pvm), a model respiratory virus (38) (39) (40) (41) (42) . the peak of the antigen-specific cd8 t cell response generally corresponds to lung viral clearance following rsv, iav, hmpv, and pvm infections (36, (40) (41) (42) (43) . human cd8 t cell kinetics following respiratory virus infections are less well known given the lack of identified cd8 t cell epitopes and the difficulty in obtaining respiratory tract samples from children following initial virus exposure. the frequency of total activated cd8 t cells in tracheal aspirates peaked at approximately 10 days after the onset of symptoms in children with rsv, iav, rv, or cov infections (44). rsv-specific cd8 t cells were detected in the tracheal aspirates of children; however, the evaluated epitopes were present at very low frequencies, comprising up to only 2% of the total cd8 t cell response (44). following peak expansion, cd8 t cell contraction occurs and a memory population of virus-specific cd8 t cells remains within the lung. the majority of virus-specific cd8 t cells are located within the lung parenchyma, rather than the pulmonary vasculature, following localized respiratory infections in mice (37) . similarly, human rsv-and iav-specific cd8 t cells were enriched within the lung compared to the blood (45). rsv-specific cd8 t cells in tracheal aspirates of children remain elevated during convalescence following a severe rsv infection, in contrast to murine studies (46). these studies suggest that cd8 t cell responses in the airways may be more prolonged following viral clearance in humans compared to mice. following respiratory virus infection, cd8 t cells become activated and develop the ability to produce inflammatory cytokines. virus-specific cd8 t cells in the lung and airways of mice upregulate expression of markers associated with activation including cd11a, cd25, nkg2a, and cd44 as well as downregulate expression of the lymphoid homing receptor cd62l (34, 35, 47, 48) . activated cd8 t cells also acquire effector functions following viral infection. virus-specific cd8 t cells rapidly produce cytokines including ifn-γ and tnf as well as degranulate, as measured by cd107a expression, following ex vivo peptide stimulation (35, 38, 41, 48) . human virusspecific cd8 t cells also acquire an activated phenotype and effector functions following a respiratory virus infection. cd8 t cells from the tracheal aspirates of children following rsv, rv, or cov infections expressed elevated levels of the activation markers cd38 and hla-dr and the proliferation marker ki-67 (44). expression of effector molecules such as granzyme b and perforin were also increased. similarly, cd8 t cells from bronchiolar lavage (bal) fluid samples exhibited increased expression of ki-67, granzyme b, cd38, and hla-dr following either experimental rsv infection of adults or severe, natural rsv infection of infants (46, 49). additionally, human virusspecific cd8 t cells produce cytokines following respiratory virus infection, as peripheral blood cd8 t cells secreted ifn-γ, tnf, and il-2 following stimulation with peptides derived from rsv, iav, hmpv, or rv (49-53). following contraction, a subset of virus-specific cd8 t cells remain in the host to form a long-lasting memory population that provides protection against subsequent infection. cd8 t cell contraction to form long-term memory populations in the lung is regulated in part by inflammatory chemokine signaling (54). mice deficient in either cxcr3 or cxcr3 and ccr5 exhibit a significant increase in the number of memory cd8 t cells following iav infection, suggesting that chemokine signaling through cxcr3 and ccr5 plays a critical role in t cell memory generation (54). following respiratory viral infections in mice and humans, virus-specific cd8 t cells can be detected up to several months post-infection (47, 49, 55, 56). however, respiratory virus-specific memory cd8 t cell populations decline in magnitude with age in the peripheral blood (57) . interestingly, adult rsv-specific cd8 t cell responses are significantly reduced compared to iav-specific cd8 t cell responses in the peripheral blood, suggesting that memory cd8 t cell responses to iav in humans may be more stable than rsv (57) . memory cd8 t cells rapidly expand in the lung following a secondary respiratory virus infection in both mice and humans (35, 38, 39, 44, 49) . the observed expansion is primarily due to the migration of circulating cd8 t cells into the lung and airways, rather than proliferation of resident cells (58) . the expansion of virus-specific cd8 t cells in the lung and airways following infection corresponds with an increase in cxcr3-and ccr5-binding chemokines, supporting a role for chemokine-mediated migration of cd8 t cells following secondary infection (59) . indeed, ccr5 expression on memory cd8 t cells is required for their early recruitment into the airways after secondary infection, but not to the lung parenchyma (59) . following secondary expansion, memory cd8 t cells rapidly produce effector cytokines such as ifn-γ and tnf (30, 38, 60) . additionally, virus-specific memory cd8 t cells express high levels of cd11a and produce cytolytic molecules, such as granzyme b, after infection (61, 62) . these effector functions of respiratory virus-specific memory cd8 t cells are critical for mediating viral clearance and protecting against infection, as discussed below. based on the expression of activation marker cd45ra and lymphoid homing receptor ccr7, human memory cd8 t cells have been broadly separated into four major subsets: (1) naive (cd45ra + ccr7 + ), (2) central memory (tcm; cd45ra -ccr7 + ), (3) effector memory (tem; cd45ra − ccr7 − ), and (4) late effector memory (temra; cd45ra + ccr7 − ) (63) . due to their expression of ccr7, tcm home primarily to secondary lymphoid organs, while tem migrate to peripheral tissues and rapidly exert effector functions. temra are a subset of tem cells that have re-expressed cd45ra. they exhibit reduced proliferative and functional capacity, and thus are considered to be terminally differentiated cells. human virus-specific memory cd8 t cell populations are typically composed of a combination of tem and temra within the peripheral blood (44, 46, 50, 52, 55). alternatively, rsv-specific memory cd8 t cells located in the airways in both adults and infants are primarily of tem phenotype and also express high levels of cd27, cd28, and ccr5 and low levels of cd62l (46, 49). together, these studies indicate that tem cd8 t cells are dominant following respiratory virus infection in humans. given the frequent exposure to viruses in the respiratory tract, tem cells may be critical for the rapid employment of cd8 t cell effector mechanisms following reinfection. recently, an additional population of memory cd8 t cells that persist within peripheral tissues has been identified, termed tissue-resident memory cd8 t cells (trm) (64) . trm have been observed within several peripheral organs including the intestine, skin, female reproductive tract, and lung. trm generated following a respiratory virus infection represent a non-circulating population of memory cd8 t cells that are maintained within the lung parenchyma (65) . virus-specific trm are located along the wall of large airways and within pulmonary tissue surrounding bronchioles and alveoli (66, 67) . respiratory virus infection also induces trm within the airway lumen (68, 69) . airway trm downregulate cd11a expression and can be distinguished from recently trafficked cd8 t cells that express high levels of cd11a (70, 71) . the localization of lung and airway trm following respiratory virus infection is distinctly different from that of tcm, which traffic through the pulmonary vasculature and accumulate in the lung-draining lymph node (72, 73) . virus-specific tem are also differentially located from trm residing primarily in the pulmonary vasculature or within the lung tissue near blood vessels, spacially distinct from regions that contain trm (67, 73). following either rsv or iav infection in mice, lung and airway trm are induced and can be identified by their expression of the canonical resident memory markers cd69 and cd103, which promote their migration to and retention within the lung tissue (37, 65, (74) (75) (76) . iav-and rsv-specific trm are also generated in the lungs of mice that have been locally vaccinated via an intranasal route, but not mice that have been immunized systemically (30, (77) (78) (79) . importantly, iav-specific trm expressing cd69 were also detected in human lung tissue sections but were absent from the spleen (65) . similarly, rsv-specific trm expressing both cd69 and cd103 were identified in the human bal but were not present in the peripheral blood (49). following secondary viral infection, trm expand prior to the recruitment of circulating memory cd8 t cell populations from the peripheral blood and rapidly produce ifn-γ (60, 66). thus, trm provide a crucial first-line of defense for protecting the host from re-infection with a respiratory virus. however, in contrast to other memory cd8 t cell subsets that remain stable for long periods of time, iavspecific trm exhibit limited longevity and enhanced apoptosis with time following infection (66, 80) . the loss of iav-specific trm corresponds to an increase in viral titers and weight loss following a heterosubtypic iav infection (66, 80) . interestingly, infant mice generate fewer lung trm following iav infection or vaccination and exhibit reduced heterosubtypic protection compared to mice initially infected as adults (79) . given the role of lung trm in providing protection against respiratory virus infections, identifying strategies to promote the generation of long-lived trm will be critical for future vaccines, particularly for infant populations. it has been well established that cd8 t cells are critical for viral clearance following an acute respiratory virus infection in mice. adoptive transfer of cd8 t cell clones resulted in significantly reduced viral titers in the lung following rsv, iav, or hmpv infections (25, 27, (81) (82) (83) (84) . similarly, the transfer of either rsv-or iav-immune splenic cd8 t cells accelerated viral clearance in the lung following infection (85) (86) (87) (88) (89) . accordingly, rsv infection of mice depleted of cd8 t cells resulted in significantly increased lung viral titers at day 7 post-infection, although the virus was ultimately cleared by day 11 (26) . in contrast, depletion of cd8 t cells alone did not alter clearance of hmpv (40). instead, depletion of both cd4 and cd8 t cells together elevated lung virus titers at day 7 following infection with hmpv. importantly, cd8 t cells have been shown to be sufficient to mediate viral clearance in the lung following acute respiratory infections (87, 88) . athymic nude mice, which lack t cells, fail to clear either rsv or iav resulting in persistent infections (87, 90) . however, the transfer of either rsv-or iav-immune splenic cd8 t cells into athymic mice resulted in significantly reduced lung viral titers by day 15 and 21, respectively (87, 88) . together, these studies indicate that cd8 t cells play a critical role in mediating viral clearance following acute respiratory infections in mice. although studies are limited, a role for cd8 t cells in the elimination of respiratory viruses has also been established in humans. early studies demonstrated that immunocompromised children with t cell defects experienced prolonged viral shedding following rsv, iav, or piv infections compared to immunologically normal children (3, 91, 92) . following bone marrow transplantation of an rsv-infected child with severe combined immunodeficiency, a marked reduction in nasal viral load was observed that correlated with an elevation of cd8 t cell counts (92) . recently, it has been demonstrated that the number of pre-existing virus-specific cd8 t cells in the airway of adults experimentally infected with rsv correlated with reduced overall viral load in the nasal cavity and bronchial brushings (49). in addition to pre-existing cd8 t cell numbers, cd8 t cell effector functions also correlate with reduced viral load. cd8 t cell target cell lysis activity measured by chromiumrelease assay correlated with a lack of viral shedding in the nasal washes of adults experimentally infected with h1n1 iav (93) . additionally, individuals with the lowest frequencies of ifn-γ + cd8 t cells exhibited the highest viral titers following natural h7n9 iav infection (94) . these studies support the role of cd8 t cells in respiratory virus clearance in humans, consistent with the numerous murine studies. cd8 t cells mediate viral clearance by utilizing a variety of effector mechanisms to induce the apoptosis of virus-infected cells (95) . cd8 t cells can use direct cell-cell contact to eliminate target cells through the interactions of surface molecules such as fas (cd95) and fasl (cd95l). additionally, trail expressed on cd8 t cells can interact with its receptors dr4 and/or dr5 to induce the destruction of infected cells. cd8 t cells can also secrete perforin and granzymes to cause membrane pore formation and induce apoptosis. lastly, cd8 t cells produce inflammatory cytokines, such as ifn-γ and tnf, which may either directly or indirectly promote the cell death of virus-infected cells. while the exact mechanism utilized is unclear, many of these effector functions have been associated with cd8 t cell-mediated clearance of respiratory viruses. fas/fasl interactions and the perforin pathway have been established as the primary mechanisms by which cd8 t cells eliminate infected cells following an iav infection (96, 97) . studies utilizing trail-deficient mice and antibody-mediated trail blockade have also demonstrated a role for trail in cd8 t cell-mediated clearance of iav (98, 99) . similarly, fas/ fasl and perforin pathways have also been associated with virus elimination following rsv infection. perforin-deficient and fasl-deficient gld mice exhibit significantly delayed viral clearance (100, 101) . however, both perforin-deficient and gld mice achieve complete viral clearance by day 10 post-infection, suggesting that cd8 t cells compensate for those deficiencies through alternative mechanisms. one such mechanism is likely tnf production, as neutralization of tnf in perforin-deficient and gld mice significantly increased viral titers compared to igg-treated controls (101) . this is in contrast to studies following pvm and iav infections, where viral clearance occurs independently of tnf (102, 103) . ifn-γ does not appear to play a prominent role in cd8 t cell-mediated viral clearance, as both ifn-γ-deficient mice and mice that received ifn-γdeficient cd8 t cells exhibit equivalent viral titers to wild-type mice following rsv, pvm, or iav infections (101, (103) (104) (105) . together, these studies demonstrate that cd8 t cells use multiple complementary mechanisms to eliminate virally infected cells following a respiratory virus infection. given the ability of cd8 t cells to mediate viral clearance following an acute viral infection, it is no surprise that memory cd8 t cells also play a critical role in protecting against secondary respiratory virus infections. the adoptive transfer of airway iav-specific memory cd8 t cells resulted in significantly reduced lung titers following iav challenge compared to pbs transfer controls (60) . similarly, transfer of airway rsv-specific memory cd8 t cells reduced lung viral load and weight loss following subsequent rsv infection (76) . these studies indicate that transferred memory cd8 t cells are capable of providing protection against secondary respiratory virus challenge. memory cd8 t cell-mediated protection against secondary infection has been shown more convincingly in mouse models through the use of vaccination strategies to generate virus-specific memory cd8 t cells. recombinant baculovirus or murine cytomegalovirus (mcmv) vectors expressing the rsv m2 protein induced m2-specific cd8 t cells that mediated the reduction of lung viral titers following rsv challenge (78, 106) . whole protein vaccination with hmpv virus-like particles containing f and m proteins elicited hmpv-specific cd8 t cells that reduced viral titers in μmt mice, which lack antibodies (107) . cd8 t cell epitope vaccines against either rsv or hmpv have also demonstrated cd8 t cell-mediated protection following challenge by reducing lung viral load and histopathology compared to unimmunized controls (108, 109) . a similar strategy utilizing dc-peptide vaccination to generate pre-existing pvm-specific memory cd8 t cells also resulted in enhanced viral control following pvm infection (42). recently, several studies have utilized dc-prime, recombinant listeria monocytogenes-(dc-lm) or vaccinia virus-boost (dc-vv) vaccination protocols to generate a high frequency of pre-existing antigen-specific memory cd8 t cells in the absence of virus-specific cd4 t cell memory and antibodies (28) (29) (30) . prime-boosted mice exhibited significantly reduced lung viral titers following rsv, iav, or sars infections compared to controls lacking virus-specific memory cd8 t cells. additionally, memory cd8 t cells were able to reduce weight loss and mortality following lethal challenges with either iav or sars. overall, these studies provide clear evidence that memory cd8 t cells provide protection against secondary respiratory virus infection by reducing viral titers. the most well studied example of memory cd8 t cellmediated protection from a secondary respiratory virus infection is heterosubtypic immunity to iav subtypes. iav-specific neutralizing antibody responses recognize the surface glycoproteins hemagglutinin (ha) and neuraminidase (na), which vary between subtypes as a result of genetic reassortment, known as antigenic drift. however, the internal proteins of the virus are often conserved between iav subtypes. therefore, memory cd8 t cells that recognize epitopes within conserved viral proteins may be capable of providing cross-protection between iav viruses of differing subtypes. evidence of heterosubtypic immunity was first demonstrated by the protection of h1n1 iav-immune mice from a lethal h2n2 iav challenge without the induction of a neutralizing antibody response (110) . since then, a memory cd8 t cell-mediated role in accelerating clearance of a heterosubtypic iav strain has been well-established in mouse, chicken and non-human primate models (66, (111) (112) (113) (114) . recently, it has been demonstrated that trm are essential in providing cross-protection against secondary iav infection with a heterosubtypic strain (60, 66, 80) . mice with cd103 + trm in the lung exhibit more efficient viral clearance and reduced weight loss following heterosubtypic challenge than mice lacking a trm response (66) . importantly, the protection was provided solely by lung-resident memory cd8 t cells, as blocking the ability of recently proliferated tcm cells from trafficking to the lung did not impact protection (66) . consistent with the limited lifespan of iav-specific trm, heterosubtypic protection by memory cd8 t cells wanes over time, with a decline observed as early as 60 days following the initial infection (80, 111) . interestingly, systemic immunization with cognate antigen is capable of boosting the trm pool by expanding the circulating tem population that seeds the lungs (80) . therefore, it is possible that trm-mediated heterosubtypic protection could be re-established by vaccination after a waning of the protective trm population in the lung. while protection in mouse models is well established, whether memory cd8 t cells play a critical role in protection following secondary respiratory infection in humans is currently unclear. similar to studies in murine models, evidence for heterosubtypic immunity mediated by memory cd8 t cells has also been demonstrated in humans. individuals lacking h1n1-specific neutralizing antibody titers exhibited an inverse correlation between memory cd8 t cell activity and viral shedding following their first exposure with h1n1 iav (93) . more recently, it was demonstrated that the frequencies of pre-existing crossreactive memory cd8 t cells correlated with reduced symptoms, including fewer patients with fever, sore throat, and cough, following infection with the 2009 pandemic h1n1 iav strain (50). similarly, a correlation between pre-existing h3n2-specific memory cd8 t cells and reduced risk of viral shedding following 2009 pandemic h1n1 iav infection was observed (115) . thus, memory cd8 t cell-mediated heterosubtypic protection is also likely to be critical in humans. following experimental rsv infection in humans, the frequency of pre-existing rsv-specific memory cd8 t cells in the airways correlates with a reduction in both cumulative and lower respiratory tract symptom scores, suggesting a possible role for memory cd8 t cells in protection against rsv in humans (49). however, evidence has also been provided suggesting that memory cd8 t cells may not contribute to protection following respiratory virus infections in humans. natural reinfection of infants with rsv did not result in a boosting of the cd8 t cell response (116) . similarly, the frequency of rsv-specific memory cd8 t cells in the peripheral blood of healthy adults is significantly reduced compared to iavspecific memory cd8 t cells (57) . therefore, the extent to which memory cd8 t cells play a role in providing protection against rsv infection in humans remains unclear. despite their beneficial role in mediating viral clearance and protecting against secondary infection, cd8 t cells have also been associated with the induction of immunopathology following respiratory virus infection. although mice depleted of cd8 t cells exhibited elevated lung viral titers, weight loss and symptom illness scores were significantly reduced in cd8 t cell depleted mice following acute rsv infection (26) . similarly, the adoptive transfer of cd8 t cell lines exacerbated weight loss following an acute rsv infection, despite accelerating viral clearance (82) (83) (84) . similar reduction in disease severity was also demonstrated following either hmpv or pvm infection of cd8 t cell depleted mice or mice genetically deficient in cd8 t cells, respectively (40, 103). in addition to the induction of immunopathology following acute respiratory virus infections, we recently demonstrated that memory cd8 t cells also mediate severe immunopathology following secondary rsv infection (30) . large frequencies of systemic, pre-existing rsv-specific memory cd8 t cells generated through dc-lm immunization induced significant weight loss, pulmonary dysfunction, and mortality following rsv challenge, despite a significant reduction in lung viral titers. this result was in contrast to studies using similar immunization strategies to either iav or sars, in which memory cd8 t cells mediated protection against lethal viral challenge in the absence of immunopathology (28, 29) . interestingly, the immunopathology induced by rsv-specific memory cd8 t cells occurred only in the context of an rsv infection, as mice challenged with a recombinant iav strain expressing an rsv-derived cd8 t cell epitope exhibited significantly reduced morbidity and were protected from mortality (30) . this result is consistent with several studies that demonstrate cd8 t cells enhance viral clearance while preventing mortality following iav infection (25, 81, 85, 87, 117) . together, these studies demonstrate a clear role for cd8 t cells in the development of immunopathology following primary and secondary infections with some respiratory virus infections, particularly rsv. antiviral mechanisms utilized by cd8 t cells to mediate viral clearance following respiratory virus infection also contribute to the development of immunopathology. removal of the fas/ fasl pathway in gld mice resulted in significant amelioration of weight loss and symptom illness scores following rsv infection (101) . similarly, rsv-infected perforin-deficient mice exhibited prolonged weight loss and symptom illness scores compared to wild-type mice (100) . tnf contributes substantially to immunopathology, as antibody-mediated depletion of tnf prior to either rsv or iav infection significantly reduced weight loss (101, 102, 118) . additionally, mice that are ifn-γ-deficient, depleted of ifn-γ, or received an adoptive transfer of ifn-γ-deficient cd8 t cells prior to rsv infection lost significantly less weight than controls (89) . cd8 t cell production of tnf and ifn-γ following pvm infection also induced pulmonary immunopathology by initiating a cytokine storm (119) . in addition to causing disease in acute respiratory infections, ifn-γ produced by memory cd8 t cells mediated the severe and fatal immunopathology following rsv infection of dc-lm prime-boosted mice (30) . the role of cd8 t cells in the development of pathology following respiratory infections in humans remains unclear. the best evidence supporting a pathogenic role for cd8 t cells in humans infected with respiratory viruses comes from a study evaluating an rsv-infected severe-combined immunodeficiency patient after bone marrow transplantation (92) . the patient exhibited increased cd8 t cell counts following bone marrow transplant, which corresponded to a sharp reduction in rsv nasal titers. however, the appearance of cd8 t cells also correlated with a marked increase in respiratory rate indicative of reduced pulmonary function. also supporting a pathogenic role of cd8 t cells is the finding that children requiring mechanical ventilation due to severe rsv infection expressed significantly increased levels of activated granzymes and more cd8 t cells producing granzyme b compared to healthy controls (120) . in contrast, a study of infants following either fatal iav or rsv infections revealed a near absence of cd8 t cells from affected lung regions by immunohistochemical staining (121, 122) . similarly, infants with severe rsv infection exhibited an underexpression of genes related to cd8 t cells in the peripheral blood (123) . in support of a protective, rather than pathogenic, role of cd8 t cells, correlations have been identified between increased cd8 t cell cytolytic activity and cytokine production with reduced symptom score, faster recovery, and fewer fatalities following h1n1 or h7n9 iav infections (93, 94) . therefore, whether cd8 t cells play a primary role in mediating pathology versus protection following human respiratory virus infection remains controversial and is an important topic of future investigation. given the potential of cd8 t cell effector functions to cause immunopathology following respiratory virus infection, the immune system has evolved critical regulatory mechanisms to prevent prolonged cd8 t cell effector activity following viral clearance. cd8 t cell effector functions, including production of ifn-γ and tnf, are suppressed in the lung following the resolution of iav and rsv infections (124) (125) (126) (127) . one of the primary mechanisms utilized to limit the cd8 t cell response is through suppression by regulatory cd4 t cells (tregs). tregs accumulate in the lungs following either rsv or iav infection peaking at approximately day 6 post-infection, prior to the peak of the cd8 t cell response (36, (128) (129) (130) . antibody-mediated depletion of cd25 + tregs prior to rsv infection resulted in exacerbated weight loss, pulmonary dysfunction, and lung inflammation (128) . this enhanced illness corresponded to an increased frequency of rsv-specific cd8 t cells and elevated levels of ifn-γ and tnf protein in the lung (36, 128) . consistent with the treg depletion studies, increasing rsv-specific tregs prior to rsv infection using rsv peptide-immunization resulted in an amelioration of weight loss and a reduction in cd8 t cell numbers in the blood and spleen, but not the lung (131) . tregs also can suppress cd8 t cell effector functions following a secondary infection with iav (130) . antibody-mediated cd25 + treg depletion prior to heterosubtypic iav challenge resulted in enhanced inflammation and pulmonary dysfunction corresponding to an increase in cd8 t cell numbers and ifn-γ production. one mechanism through which tregs may suppress cd8 t cell responses is through the production of the anti-inflammatory cytokine il-10. foxp3 + tregs secrete il-10 following primary infection with rsv or iav (130, (132) (133) (134) . infection of either il-10-deficient mice or mice treated with il-10 receptor blocking antibody resulted in increased numbers of either ifn-γ + or ifn-γ + tnf + cd8 t cells, suggesting that il-10 suppresses cd8 t cell effector functions following respiratory virus infection (132) (133) (134) . interestingly, il-10 production by foxp3 − cd4 t cells and cd8 t cells following either rsv or iav infection has also been reported, indicating that effector t cell responses may self-regulate their effector functions (132) (133) (134) (135) . together, these studies demonstrate that tregs and il-10 production play a critical role in regulating cd8 t cells following primary and secondary respiratory virus infections to prevent immunopathology. interactions between inhibitory receptors on cd8 t cells with their ligands represents another important mechanism mediating the inhibition of cd8 t cell effector functions following infection. regulation of cd8 t cells through the pd-1:pd-l1 pathway is a common inhibitory pathway utilized following respiratory virus infection. expression of pd-1 on pulmonary cd8 t cells is upregulated following rsv, iav, or hmpv infection in mice (41, 136, 137) . blockade of pd-l1 in primary rsv, iav, or hmpv and secondary hmpv infections results in enhanced cd8 t cell effector functions, including ifn-γ, tnf, and granzyme b production (41, [136] [137] [138] . cd8 t cell effector functions are also enhanced following either hmpv or iav infections in pd-1-deficient mice (41). importantly, the pd-1:pd-l1 pathway has also been associated with human cd8 t cell responses. human cd8 t cells in the nasal cavity significantly upregulated pd-1 following rsv infection compared to cd8 t cells from the blood of either healthy or rsv-infected individuals (137) . pd-1 and pd-l1 are also both upregulated in the lung figure 1 | critical factors for an optimal cd8 t cell-mediated respiratory syncytial virus (rsv) vaccine. a future rsv vaccine designed to elicit a cd8 t cell response will require a balance between cd8 t cell-mediated protection and immunopathology, which may be achieved through the consideration of three important aspects: (1) magnitude, (2) localization, and (3) regulation. an optimal magnitude of the cd8 t cell response will be one that achieves efficient viral clearance in the absence of immunopathology. the vaccination route will be critical in determining the localization of the cd8 t cell response. a pulmonary route of vaccination will induce trm in the lung that provides superior protection compared to a systemic immunization that would likely not generate protective trm. lastly, regulation of the cd8 t cell response generated through vaccination will be crucial, as uncontrolled effector functions, particularly ifn-γ production, can result in immunopathology. frontiers in immunology | www.frontiersin.org april 2018 | volume 9 | article 678 tissue following severe infections with either rsv or the 2009 h1n1 iav pandemic strain (41). in vitro human studies have demonstrated that pd-l1 is constitutively expressed on human airway and bronchial epithelial cells, but expression is significantly upregulated following either iav or rsv infection (136, 139) . similar to in vivo mouse studies, in vitro pd-l1 blockade resulted in significantly increased cd8 t cell production of ifn-γ, il-2, and granzyme b following rsv infection (139) . together, these studies demonstrate a critical role for pd-1 in the suppression of cd8 t cell-mediated immunopathology and cytokine production in both mice and humans. in the absence of pd-1 signaling following hmpv infection, cd8 t cell ifnγ production remains impaired, suggesting the involvement of compensatory inhibitory pathways (140) . antigen-specific lung cd8 t cells express inhibitory receptors tim-3, lag-3, and 2b4 following hmpv infection and exhibit enhanced cytokine production following in vitro blockade of each receptor individually (140) . in vivo blockade of lag-3 partially restored cd8 t cell ifn-γ production in pd-1-deficient mice following hmpv infection (140) . tim-3 has also been demonstrated to be critical in suppressing cd8 t cell responses in vivo, as tim-3 receptor (galectin-9)-deficient mice exhibited significantly enhanced cd8 t cell responses following both primary and secondary iav infections (141) . together, these studies indicate that multiple inhibitory receptor pathways are utilized following pulmonary virus infection to dampen the pathogenic cd8 t cell response and prevent immunopathology. successful vaccinations against the majority of respiratory viruses remain elusive. the goal of most vaccination strategies is to induce robust virus-specific neutralizing antibody responses. however, the antibody response generated by infection with many respiratory infections, including rsv and rv, wanes over time. therefore, neutralizing antibody responses as the sole mediator of a vaccine against most respiratory viruses may not provide long-term protection without yearly vaccination. vaccination strategies that include the induction of virus-specific cd8 t cell responses, either alone or in combination with humoral immunity, may be advantageous by providing many benefits associated with cellular immune responses. cd8 t cells are critical for the elimination of virusinfected cells, and viral clearance was prolonged in the absence of cd8 t cells following acute respiratory virus infections. additionally, robust memory cd8 t cell responses efficiently reduced lung viral titers in the absence of neutralizing antibodies following rsv, iav, or sars secondary infections. an important property of cd8 t cells is that they often recognize conserved viral proteins, allowing for cross-protection between different virus strains. this is particularly important for heterosubtypic protection of iav strains, as neutralizing antibodies are not capable of recognizing iav strains of differing subtypes. despite their benefits in mediating viral clearance and providing protection against secondary infections, memory cd8 t cell responses have been associated with the induction of immunopathology following respiratory virus infections. the same antiviral mechanisms employed by memory cd8 t cells to accelerate viral clearance also contribute to immunopathology, including the fas/fasl pathway-and perforin-mediated cytolysis and ifn-γ and tnf cytokine secretion. thus, the efficient elimination of respiratory viruses by memory cd8 t cells comes at a cost of disease for the host. cd8 t cell-mediated immunopathology appears to be virus-specific. although high frequency, systemic, antigen-specific memory cd8 t cells induced severe disease and mortality following rsv infection, no pathology was observed using similar systems for iav and sars infections. therefore, induction of memory cd8 t cells as the sole immune mediator may be particularly dangerous for an rsv vaccine, but significantly less so in either an iav or a sars vaccine. to be able to include cd8 t cell responses within a future respiratory virus vaccine, it will be extremely important to determine how to balance cd8 t cell-mediated protection versus immunopathology following respiratory infection. for rsv in particular, three critical aspects to consider in this balance include magnitude, localization, and regulation of the rsv-specific cd8 t cell response (figure 1) . dc-lm immunization generated m282-90-specific cd8 t cells at a frequency of approximately 20% in the peripheral blood, but induced fatal immunopathology following rsv challenge (30) . however, dc-prime only and trivax immunizations generated a much lower frequency of total m282-specific cd8 t cells, and rsv induced significantly reduced disease in these mice (30, 109) . thus, identifying the optimal magnitude of rsv-specific cd8 t cells for protection in the absence of immunopathology is crucial. it is also clear from recent studies in mouse models that localization of rsv-specific cd8 t cells is a significant factor for both their efficacy of mediating viral clearance and their ability to induce immunopathology following infection. intranasal immunization with mcmv-m generated trm within the lung tissue that accelerated viral clearance. in contrast, mice administered mcmv-m systemically did not generate trm and exhibited delayed viral clearance (78) . similar results were observed with local immunization with dc-iav-m282 (30) . m282-specific lung trm generated by pulmonary immunization did not induce immunopathology following rsv infection, in contrast to systemic dc-lm immunization, which resulted in severe pathology in the absence of trm cells. thus, vaccination strategies against rsv will likely be the most effective when administered through a pulmonary route to generate trm that will provide protection within the lung following reinfection without inducing immunopathology. lastly, identifying ways to regulate vaccine-generated cd8 t cell responses will likely reduce immunopathology following subsequent infection. ifn-γ produced by cd8 t cells was the primary mediator of immunopathology following rsv infection of dc-lm vaccinated mice (30) . however, neutralization of ifn-γ had no effect on lung viral titers, suggesting that cd8 t cells utilize other antiviral mechanisms to mediate viral clearance in this system. since cd8 t cells are able to reduce viral titers in the absence of ifn-γ, reducing the amount of ifn-γ produced by cd8 t cells would likely result in ameliorated disease following rsv infection. if vaccination strategies can identify mechanisms by which cd8 t cell cytokine production, particularly ifn-γ, can be attenuated without altering their ability to eliminate virusinfected cells, the pathology induced by cd8 t cells would likely also be decreased. the development of a cd8 t cell-mediated vaccine should be pursued given the limitations of antibody responses to respiratory viruses. it is possible that the ideal vaccine for respiratory virus infections will include the induction of both virus-specific cd8 t cells and neutralizing antibodies. a vaccination approach combining both arms of the adaptive immune response may allow for optimal viral control in the absence of disease symptoms. however, before cd8 t cells can be developed further as a mediator of protective immunity, the balance between protection and pathology must be achieved. future studies evaluating aspects of memory cd8 t cell magnitude, localization, and regulation will greatly assist in reaching this balance. both authors wrote and edited the manuscript. incubation periods of acute respiratory viral infections: a systematic 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infection control of pathogenic effector t-cell activities in situ by pd-l1 expression on respiratory inflammatory dendritic cells during respiratory syncytial virus infection programmed death-1 impairs secondary effector lung cd8+ t cells during respiratory virus reinfection rsv-induced bronchial epithelial cell pd-l1 expression inhibits cd8+ t cell nonspecific antiviral activity multiple inhibitory pathways contribute to lung cd8+ t cell impairment and protect against immunopathology during acute viral respiratory infection t cell immunoglobulin and mucin protein-3 (tim-3)/ galectin-9 interaction regulates influenza a virus-specific humoral and cd8 t-cell responses key: cord-347304-1o4fb3na authors: yang, xiaoyun; zhao, chunling; bamunuarachchi, gayan; wang, yang; liang, yurong; huang, chaoqun; zhu, zhengyu; xu, dao; lin, kong; senavirathna, lakmini kumari; xu, lan; liu, lin title: mir‐193b represses influenza a virus infection by inhibiting wnt/β‐catenin signalling date: 2019-01-25 journal: cell microbiol doi: 10.1111/cmi.13001 sha: doc_id: 347304 cord_uid: 1o4fb3na due to an increasing emergence of new and drug‐resistant strains of the influenza a virus (iav), developing novel measures to combat influenza is necessary. we have previously shown that inhibiting wnt/β‐catenin pathway reduces iav infection. in this study, we aimed to identify antiviral human micrornas (mirnas) that target the wnt/β‐catenin signalling pathway. using a mirna expression library, we identified 85 mirnas that up‐regulated and 20 mirnas that down‐regulated the wnt/β‐catenin signalling pathway. fifteen mirnas were validated to up‐regulate and five mirnas to down‐regulate the pathway. overexpression of four selected mirnas (mir‐193b, mir‐548f‐1, mir‐1‐1, and mir‐509‐1) that down‐regulated the wnt/β‐catenin signalling pathway reduced viral mrna, protein levels in a/pr/8/34‐infected hek293 cells, and progeny virus production. overexpression of mir‐193b in lung epithelial a549 cells also resulted in decreases of a/pr/8/34 infection. furthermore, mir‐193b inhibited the replication of various strains, including h1n1 (a/pr/8/34, a/wsn/33, a/oklahoma/3052/09) and h3n2 (a/oklahoma/309/2006), as determined by a viral reporter luciferase assay. further studies revealed that β‐catenin was a target of mir‐193b, and β‐catenin rescued mir‐193b‐mediated suppression of iav infection. mir‐193b induced g0/g1 cell cycle arrest and delayed vrnp nuclear import. finally, adenovirus‐mediated gene transfer of mir‐193b to the lung reduced viral load in mice challenged by a sublethal dose of a/pr/8/34. collectively, our findings suggest that mir‐193b represses iav infection by inhibiting wnt/β‐catenin signalling. influenza a virus (iav) infection causes annual epidemics and recurring pandemics, leading to substantial morbidity and mortality, as well as significant economic losses. iav belongs to the family orthomyxoviridae and has a segmented, negative-sense, and singlestranded rna genome. although vaccines remain a major means of prevention, a significant amount of time is required to develop and produce an effective vaccine against a new virus strain (soema, kompier, amorij, & kersten, 2015) . furthermore, vaccines need to be reformulated annually due to the frequent emergence of new viruses (houser & subbarao, 2015) . antiviral drugs, on the other hand, are essential for treatment and prophylaxis. however, the error-prone nature of the influenza rna polymerase, due to its lack of proofreading-repair activity, makes the virus highly susceptible to mutation, resulting in its resistance to antivirals (watanabe et al., 2014) . for example, there has been rapid emergence of iav strains that are resistant to amantadine and rimantadine, and these antivirals are thus no longer recommended for anti-influenza treatment (barr et al., 2007; bright et al., 2005) . resistance against neuraminidase inhibitors, such as oseltamivir and zanamivir as well as newly developed peramivir and laninamivir, has also been reported (barrett & mckimm-breschkin, 2014; hurt et al., 2009; kamali & holodniy, 2013; orozovic, orozovic, jarhult, & olsen, 2014) . therefore, it is increasingly urgent to develop drugs that target host factors rather than viral proteins, which is less likely to cause drug resistance. the small coding capacity of iav requires it to utilise the host cell machinery for its life cycle (watanabe, watanabe, & kawaoka, 2010; york, hutchinson, & fodor, 2014) . many host proteins and signalling pathways regulate iav infection at different stages. early in 2003, wurzer et al. (2003) discovered that efficient iav propagation depends on the activation of host caspase-3, a central player in apoptosis, as the presence of a caspase-3 inhibitor in cells strongly impairs viral replication. several studies have shown that iav stabilises the p53 protein, activates p53 signalling and consequently induces apoptosis in host cells (nailwal, sharma, mayank, & lal, 2015; turpin et al., 2005; zhirnov & klenk, 2007) . recently, cyclophilin a was found to interact with the iav m1 protein and thus to impair early viral replication (x. liu et al., 2012) . iav also interacts with many other cellular pathways, including the nf-κb, pi3k/akt, mapk, pkc/pkr, and tlr/rig-i signalling cascades, to overcome host defences against the virus (gaur, munjhal, & lal, 2011; ludwig & planz, 2008; . micrornas (mirnas) are~22-nt small noncoding rnas that posttranscriptionally regulate gene expression by binding the 3′-untranslated region (3′-utr) of a target mrna to inhibit protein translation or degrade mrna (y. wang, stricker, gou, & liu, 2007) . several thousand mirnas have been identified in plants, animals, and viral genomes (akhtar, micolucci, islam, olivieri, & procopio, 2016) . mirnas are key modulators in diverse signalling pathways (zamore & haley, 2005) . increasing evidence indicates that mirnas also participate in host-virus interactions and play a pivotal role in the regulation of viral replication. for example, mir-122, a liverspecific mirna, facilitates viral replication by targeting the 5′-utr of hepatitis c virus rna (thibault et al., 2015) . cellular mir-24 and mir-93 target the viral large protein (l protein) and phosphoprotein (p protein) genes of vesicular stomatitis virus (otsuka et al., 2007) . in addition, mir-323, mir-491, and mir-654 inhibit replication of h1n1 iav by binding the pb1 gene (song, liu, gao, jiang, & huang, 2010) . mirna can also modulate the type i interferon (ifn) system to combat viral infection. wang et al. has demonstrated that ifninduced mir-155 positively regulates the host antiviral innate immune response via type i ifn signalling by targeting suppressor of cytokine signalling 1 (socs1; p. wang et al., 2010) . we have recently demonstrated a beneficial role of wnt/βcatenin signalling in iav infection. the activation of wnt/β-catenin with wnt3a enhances influenza virus replication, whereas the inhibition of the pathway with icrt14 decreases influenza infection (more et al., 2018) . in the present study, we sought to identify mirnas that regulate iav replication by modulating the wnt/β-catenin pathway. here, we report the systematic screening of a mirna expression library and identification of 20 mirnas that modulate the activity of the wnt/β-catenin pathway. four selected mirnas that inhibit the pathway, mir-193b, mir-548f-1, mir-1-1, and mir-509-1, were found to have anti-iav activities. mir-193b suppressed iav replication by targeting β-catenin. the wnt/β-catenin signalling pathway plays an important role in various biological processes. several studies have shown that a number of mirnas modulate the activity of wnt/β-catenin signalling by regulating the expression of the pathway-related components (anton, chatterjee, simundza, cowin, & dasgupta, 2011; t. hu et al., 2016; k. huang et al., 2010) . to systematically identify mirnas that regulate wnt/β-catenin signalling, we constructed a mirna expression library composed of 738 human mirna precursor expression vectors and performed screening using a topflash reporter luciferase assay as a readout for wnt/β-catenin signalling activity ( figure 1a ). the topflash reporter vector contains three copies of t-cell factor (tcf)/lymphoid enhancer-binding factor (lef) binding sites upstream of the luciferase reporter gene. the initial screening was performed in 96-well plates with three replicates. human hek293t cells were transfected with a topflash luciferase reporter plasmid, mirna expression plasmid, and the normalisation vector, prl-tk vector, for 24 hr and were then stimulated with wnt3a for 24 hr, followed by the dual luciferase assay. among 738 mirnas, 85 mirnas were found to up-regulate and 20 to down-regulate luciferase activity with a fold change ≥2, resulting in a hit rate of 14.2% (figure 1a and table s1 ). to further validate the primary screen data, we repeated the reporter assay with a fopflash vector containing mutated tcf binding sites as a negative control for topflash activity. if a mirna changed the fopflash activity similar to the topflash activity, the mirna was considered to be a false positive. we selected approximately equal numbers of up-and down-regulating mirnas (up, 24 mirnas with a greater than or equal to threefold change, and down, 20 mirnas with a ≥2-fold change, figure 1b ). among the 20 down-regulating mirnas, mir-548f-1, 548f-2, and 548f-4 have an identical mature mirna, and thus, mir-548f-2 and 548f-4 were excluded. topflash and foplash firefly luciferase activities, normalised to prl-tk renilla luciferase activity, are shown in figure 1c for the up-regulating mirnas and figure 1d for down-regulating mirnas. a ratio of normalised topflash/normalised fopflash are presented in figure 1e ,f. using a topflash/fopflash ratio of 1.5 as a cut-off value, we identified 15 and five mirnas that up-and down-regulated the wnt/β-catenin signalling pathway, respectively. we then tested whether the above-identified mirnas that downregulated the wnt/β-catenin pathway could inhibit iav infection. among the five mirnas (mir-193a, mir-193b, mir-548f-1, mir-1-1, and mir-509-1) that down-regulated wnt/β-catenin signalling, mir-193a and mir-193b are isoforms of the same family. because mir-193b showed greater inhibition of wnt/β-catenin signalling than mir-193a, we tested the anti-iav activities of mir-193b and the other mirnas. we overexpressed each mirna in hek293 cells for 24 hr, infected the cells with a/pr/8/34 at a multiplicity of infection (moi) of 0.01 for 48 hr, and determined viral mrna and protein expression in infected cells and the titre in the culture medium. the mirna expression vector contains an enhanced green florescent protein (egfp) marker, which can be used to monitor the transfection efficiency. egfp images showed an approximately 75-85% transfection efficiency (figure 2a) , and overexpression of mirnas was confirmed by real-time polymerase chain reaction (pcr; figure 2b ). western blot analysis showed that overexpression of mir-193b, mir-548f-1, mir-1-1, and mir-509-1 in hek293 cells reduced viral np protein expression by 73 ± 14%, 42 ± 9%, 61 ± 25%, and 35 ± 19%, respectively, and ns1 protein expression by 98 ± 1%, 61 ± 21, 63 ± 18, and 30 ± 15%, respectively (figure 2c,d) . these mirnas also reduced the mrna levels of np, ns1, and pb1 in cells by 30-60% ( figure 2e ). furthermore, progeny virus production in the culture media was suppressed by all of the mirnas (figure 2f ). immunostaining of np revealed that these mirnas also led to a decrease in iav-infected cells ( figure 2g ). it is noteworthy that mir-509-1 inhibited the wnt/β-catenin signalling, the second most but merely showed moderate suppression of the iav infection, suggesting a possible offtarget effect of the mirna. taken together, the data showed that the mirnas that down-regulated the wnt/β-catenin signalling pathway reduced iav infection. given that mir-193b suppressed wnt/β-catenin signalling and the viral mrna and protein levels more than other mirnas, we selected mir-193b for further investigation. first, we determined whether the effects of mir-193b on iav infection were cell type dependent. the lung is the primary site of iav infection, and thus, we chose human lung epithelial a549 cells for this purpose. due to the low transfection efficiency of plasmid into a549 cells, we used a lentivirus to overexpress mir-193b. lentivirus infection efficiencies under our conditions were nearly 100% as revealed by egfp images (figure 3a (c-f) mirna hit verification. hek293 cells were transfected with a mirna plasmid or its control vector (mir-con) together with the topflash (wild-type tcf binding sites) or fopflash (mutated tcf binding sites) luciferase reporter vector and were stimulated with 50% wnt3a-conditioned medium. topflash or foplash firefly luciferase activity was normalised to prl-tk renilla luciferase activity. the results are expressed as the ratio of mirna/mir-con (c,d) and ratio of topflash/fopflash (e,f). a top/fopflash ratio of ≥1.5 was used as a cutoff value infection. overexpression of mir-193b also decreased the mrna levels of np, ns1, and pb1 by 30% to 43% ( figure 3f ) and progeny virus production in the culture medium by more than one log ( figure 3g ). these results indicated that the anti-iav activity of mir-193b was not cell type dependent. we then assessed whether the effects of mir-193b on iav infection were dependent on the viral strain using an iav luciferase reporter assay (more et al., 2018 effects of mirnas that up-regulate wnt/β-catenin signalling on iav infection. (a,b) hek293 cells were transfected with a pentr-mirna or its control vector (mir-con) for 72 hr. the transfection efficiency was monitored by the gfp signal and the cells were counterstained with hoechst 33342 (blue). mirna expression was determined by real-time pcr, normalised to β-actin, and expressed as a ratio to mir-con. (c-f) hek293 cells were transfected with pentr-mirna or mir-con for 24 hr, followed by infection with a/pr/8/34 at an moi of 0.01 for 48 hr. np and ns1 protein levels were determined by western blotting, densitometrically quantified and normalised to β-actin. np, ns1, and pb1 mrna expression was determined by real-time pcr and normalised to β-actin. virus yields in the medium were titrated by the tcid 50 assay. (g) hek293 cells were transfected with a pentr-mirna or mir-con vector for 24 hr, followed by infection with pr8 iav at an moi of 0.5 for 16 hr. immunofluorescent staining was performed to detect viral np (red). gfp images show cells overexpressing the mirnas. the cells were counterstained with hoechst 33342 (blue). data are expressed as the mean ± se of three independent experiments. *p < 0.05 versus mir-con, **p < 0.01 versus mir-con, ***p < 0.001 versus mir-con, and ****p < 0.0001 versus mir-con (b and f: one-way anova, bonferroni's multiple comparisons test; d and e: two-way anova, bonferroni's multiple comparisons test) h1n1 virus, and h3n2 is a virus strain that causes seasonal flu annually and endemic outbreaks. to obtain a proper readout of the luciferase activities, we first optimised the dose of the viruses with mois of 0.001, 0.005, 0.01, 0.05, and 0.1. the dose that resulted in a ratio (firefly/renilla) between 10 and 100 was selected (data not shown). the results showed that overexpression of mir-193b inhibited iav infection of all tested strains by 45-73% in hek293 cells and by 25-76% in a549 cells (figure 4a ,b), indicating that the antiviral effect of mir-193b was not strain dependent. to determine the effects of endogenous mir-193b on iav infection, we knocked out mir-193b with crispr/cas9 and determined their effects on iav infection. two single guide rnas (sgrnas) were designed to target two regions of pre-mir-193b ( figure 5a ). a549 cells were infected with a sgrna-lenticrispr virus or a control virus, and one clone was chosen from each sgrna after puromycin selection. the surveyor mutation assay showed that the mutations were generated in both mir-193b knocked-out clones (figure 5b ). dna sequencing revealed that one base was inserted in the sgrna1 clone, and two bases were deleted in the sgrna2 clone ( figure 5c ). the results further support that mir-193b negatively regulates iav infection. to determine how mir-193b regulates iav replication by altering the wnt/β-catenin signalling pathway, we used pictar, target scan (version 5.2), and diana-microt-4.0 to predict the target genes of mir-193b in the pathway. because mir-193b inhibits wnt/β-catenin signalling, we narrowed down the target selection to the positive regulators of the signalling. three positive regulators of the wnt/β-catenin signalling pathway, lef1, ctnnb1, and fzd4, were predicted to contain binding sites for mir-193b. fzd4 encodes frizzled 4 protein, which acts as a receptor for wnt ligands (h. c. huang & klein, 2004) . lef1 and ctnnb1 encode the wnt pathway transcription factor lef1 and coactivator β-catenin, respectively. accumulation of β-catenin in the cytoplasm and its eventual translocation into the nucleus act as a transcriptional coactivator of the transcription factor lef1 (komiya & habas, 2008) . the potential binding sites between mir-193b and the 3′-utr of the targets are shown in figure 6a . to determine whether mir-193b binds these targets, we cloned the 3′-utrs of these genes into a luciferase reporter and transfected them into hek293t cells along with the mir-193b effects of mir-193b on iav replication in a549 cells. (a,b) verification of mirna overexpression. a549 cells were infected with the mir-193b or mir-con lentivirus at an moi of 100 for 48 hr. the infection efficiency was monitored by the gfp signal, and the cells were counterstained with hoechst 33342 (blue). mir-193b expression was determined by real-time pcr and normalised to mir-con. (c) cell viability at 72 hr after lentivirus infection determined by a celltiter blue assay and normalised to non-infected cells (blank). (d-g) effects of mir-193b on iav infection. a549 cells were infected with mir-193b or mir-con lentivirus (moi 100) for 48 hr, followed by infection with a/pr/8/34 (moi 0.01) for 48 hr. np and ns1 protein expression was detected by western blotting, densitometrically quantified, and normalised to β-actin. viral np, ns1, and pb1 mrna expression was determined by real-time pcr and normalised to β-actin. the virus yield in the medium was titrated by the tcid 50 assay. (h,i) mir-193b or mir-con lentivirus-infected a549 cells were infected with iav at moi 0.5 for 8 hr. np and ns1 protein levels in the infected cells were detected by western blotting and quantified. data are expressed as the mean ± se of three independent experiments. *p < 0.05 versus mir-con and **p < 0.01 versus mir-con (b and g: student's t-test; c: one-way anova, bonferroni's multiple comparisons test; e, f, and i: two-way anova, bonferroni's multiple comparisons test) effects of mir-193b on different strains of iav replication. hek293 or a549 cells were transfected with a pentr-mir-193b or control vector (100 ng), the iav firefly luciferase reporter plasmid vnp-luc/phh21 (20 ng) and prl-tk renilla plasmid (5 ng) for 24 hr. the cells were then infected with different strains of iav, a/pr/8/34, ok/09, a/wsn/33, and a/wisconsin/67/05 at an moi of 0.01, 0.05, 0.005, and 0.01, respectively, for 48 hr. firefly luciferase activity was normalised to prl-tk renilla luciferase activity in (a) hek293 and (b) a549 cells. the results are expressed as a ratio to mir-con-transfected cells. data shown are the mean ± se of three independent experiments. *p < 0.05 versus mir-con, **p < 0.01 versus mir-con, and ***p < 0.001 versus mir-con (two-way anova, bonferroni's multiple comparisons test) expression plasmid. mir-193b reduced the luciferase activities by 35-50% of all targets (figure 6b ), suggesting that mir-193b was able to bind the 3′-utrs of lef1, ctnnb1, and fzd4. we further examined whether mir-193b reduced endogenous target proteins. hek293 cells were transfected with the mir-193b plasmid for 48 hr, and western blotting was then performed to evaluate the lef1, β-catenin, and fzd4 protein levels. the lef1 and β-catenin protein levels were reduced by 71 ± 6% and 30 ± 2%, respectively, in mir-193b-overexpressing hek293 cells. however, endogenous fzd4 was not affected by mir-193b overexpression ( figure 6c ,d). these results suggest that lef1 and β-catenin, but not fzd4, are the targets of mir-193b in hek293 cells. hereafter, we further examined whether mir-193b also targeted lef1 and β-catenin in a549 cells. we infected a549 cells with an moi of 100 of mir-193b lentivirus or the control for 48 hr and detected lef1 and β-catenin by western blotting. β-catenin was reduced by overexpression of mir-193b in both of hek293 and a549 cells (figure 6e ,f). we were unable to detect lef1 in a549 cells due to its low basal expression. furthermore, the endogenous β-catenin was elevated in the mir-193b knockout a549 cells, but only sgrna1 reached a significant level (figure 6g ,h). to determine whether lef1 and β-catenin were involved in the anti-iav activities of mir-193b, rescue experiments were performed by overexpressing target genes using expression vectors that do not contain the 3′-utr of the target genes. hek293 cells were cotransfected with mir-193b alone or along with lef1 or δgsk-βto test whether mir-193b was able to affect the cell cycle progression, mir-193b-infected a549 cells were synchronised by cultivation in serum-free medium for 48 hr and then allowed to re-enter the cell cycle by culturing them in medium containing 10% fbs for 18 and 30 hr. the cells were stained with propidium iodide and analysed by (e-g) sgrna1, sgrna2, or control vector (con)-treated a549 cells were infected with a/pr/8/34 at moi 0.01 for 48 hr. viral np levels were determined with western blotting (e), and densitometrically quantified and normalised to β-actin (f). the progeny virus was determined by tcid 50 assay (g). *p < 0.05 versus con, **p < 0.01 versus con, and ***p < 0.001 versus con (one-way anova, bonferroni's multiple comparisons test) flow cytometry. approximately 72% of mir-193b-infected cells were in the g0/g1 phase compared with 60-62% of mir-con-treated cells iav infection involves a complex series of nuclear import and export events. thus, we examined whether mir-193b was able to inhibit vrnp entry into the nucleus. we infected mir-193b-overexpressing a549 cells with a/pr/8/34 for various times and immunostained the cells with np antibodies. at 2 hpi, 2% of control cells showed a np signal in the nucleus compared with almost no signal in the mir-193b-overexpressing cells. at 3 hpi, 57 ± 9% of control cells displayed np nuclear localisation compared with only 13 ± 4% of the mir-193b-overexpressing cells. at 5 hpi, the proportions of cells with np nuclear localisation increased to 93 ± 2% in control cells and 55 ± 9% in mir-193b-overexpressing cells (figure 9a,b) . at 8 hr after infection, most of control and mir-193b-overexpressing cells displayed a np signal centred in the nuclei. these data suggested that mir-193b delayed vrnp entry into the nucleus. because mir-193b induces g0/g1 arrest, we investigated the effect of cell cycle arrest on vrnp nuclear import. g0/g1-arrested a549 cells were obtained by serum starvation in medium containing 0.2% fetal bovine serum (fbs) for 72 hr. control cells were initially serumstarved for 48 hr and then cultured in complete medium for 24 hr, which allowed the cells to re-enter the normal cell cycle. the cells were then infected at an moi of 10 of a/pr/8/34 virus for 3 hr, and immunofluorescent (if) staining was performed to detect viral np. ki-67 staining was performed to examine cell cycle arrest. as the 3′-utr reporter assay. hek293t cells were transfected with a mir-193b expression vector and a 3′-utr luciferase reporter plasmid for 48 hr. firefly luciferase activity was normalised to prl-tk renilla luciferase activity and then expressed as a ratio to the vector control (mir-con). (c,d) effect of mir-193b on endogenous target proteins in hek293 cells. hek293 cells were transfected with a mir-193b overexpression plasmid for 48 hr. lef1, β-catenin, and fzd4 protein expression was detected by western blotting, densitometrically quantified, and normalised to β-actin. (e,f) effect of mir-193b on endogenous β-catenin in a549 cells. a549 cells were infected with a mir-193b lentivirus for 48 hr. β-catenin protein expression was detected by western blotting, densitometrically quantified, and normalised to β-actin. (g,h) endogenous βcatenin in mir-193b-knockout a549 cells. β-catenin protein expression in sgrna1, sgrna2, or control vector-treated a549 cells was determined by western blotting and quantified by normalisation to β-actin. data are expressed as the mean ± se of three independent experiments. *p < 0.05 versus mir-con, **p < 0.01 versus mir-con, ***p < 0.001, and ****p < 0.0001 versus mir-con (b and d: two-way anova, bonferroni's multiple comparisons test; f: student's t-test; h: one-way anova, bonferroni's multiple comparisons test) shown in figure 10a ,b, the fluorescence signal of ki-67, a marker for cell proliferation, was significantly reduced in serum-starved cells compared with the control. by contrast, np nuclear translocation was 2.3-fold higher in control cells than in the cells that were in g0/g1 cell cycle arrest. these data indicated that g0/g1 cell cycle arrest indeed suppressed vrnp entry into the nucleus. the cells were then infected with a/pr/8/34 (moi 0.01) for 48 hr. np protein expression was detected by western blotting (a,d), quantified and normalised to β-actin (b,e). progeny virus was determined by tcid 50 assay (c,f). data are expressed as the mean ± se of three independent experiments. *p < 0.05 and **p < 0.01 (one-way anova, tukey's multiple comparison test). ns: not significant figure 8 mir-193b suppresses cyclin d1 and induces g0/g1 cell cycle arrest. (a-c) hek293 cells were transfected with a mir-193b expression plasmid for 48 hr, or a549 cells were infected with a mir-193b lentivirus (moi 100) for 48 hr. the mrna (a) and protein (b) levels of cyclin d1 were detected by real-time pcr and western blotting, respectively. protein expression was densitometrically quantified and normalised to β-actin (c). (d-f) hek293 cells were seeded in 12-well plates and transfected with a mir-193b plasmid (0.5 μg) alone or along together with p-cmv-ccnd1 (0.5 μg, addgene #19927) expression plasmid. the cells were then infected with a/pr/8/34 (moi 0.01) for 48 hr. np protein expression was detected by western blotting, quantified, and normalised to β-actin. progeny virus was determined by tcid 50 assay. (g) mir-193b lentivirus-infected a549 cells were serum-starved for 24 hr and then cultured in medium containing 10% fetal bovine serum for 18 and 30 hr. the cells were stained with propidium iodide. the dna content was measured by flow cytometry. the cell cycle phase distribution was examined. data are expressed as the mean ± se of three independent experiments. (a,c, and g) *p < 0.05 versus mir-con, **p < 0.01 versus mir-con (two-way anova, bonferroni's multiple comparisons test). (e and f) *p < 0.05 and **p < 0.01 (one-way anova, tukey's multiple comparison test) to assess the effect of iav infection on mir-193b expression, a549 or and hek293 cells (figure 11) , suggesting that iav infection may counteract the mir-193b expression. to determine whether mir-193b inhibited iav replication in vivo, we progeny virus production in the lungs was also decreased at 7 dpi although it did not reach a significant level (figure 12e ). taken together, these results confirm the antiflu activities of mir-193b in vivo. figure 9 mir-193b suppresses iav vrnp nuclear import. (a) mir-193b or mir-con lentivirus-infected a549 cells were infected with a/pr/8/ 34 at an moi of 10 for 2, 3, 5, and 8 hr. viral np (red) was immunostained, and the nuclei (blue) were counterstained. (b) cells with intense np signal localised (centred) in the nucleus were considered to exhibit nuclear localisation and counted. the percent of cells with np nuclear localisation at different times was quantified. data are expressed as the mean ± se of three independent experiments. ***p < 0.001 versus mir-con (two-way anova, bonferroni's multiple comparisons test) as influenza pathogenesis is determined in part by the host response, understanding the key host modulators of viral infection and virus-induced disease is a promising approach to host-oriented drug development for preventing the disease. the wnt/β-catenin signalling pathway has been shown to play an important role in virus replication (more et al., 2018; shapira et al., 2009 ) and virus-induced immune responses (hillesheim, nordhoff, boergeling, ludwig, & wixler, 2014) . identification of mirnas that interfere with iav replication by modulating wnt/β-catenin will provide new insights into the mirna-wnt/β-catenin signalling-iav interaction network. in the present study, using mirna library screening, we identified 15 and five mirnas that up-and down-regulated wnt/β-catenin signalling, table s1 ). furthermore, four mirnas (mir-193b, mir-548f-1, mir-1-1, and mir-509-1) that down-regulated the wnt/β-catenin signalling pathway were shown to suppress iav replication and virus production. mir-193b, which reduced all of the four mirnas that inhibit wnt/β-catenin signalling reduced viral protein levels. it is noted that to the extent that a mirna inhibits wnt/β-catenin signalling does not exactly correlate with its anti-influenza virus activities (figures 1f and 2d) . it is likely that, in addition to wnt/β-catenin signalling, these mirnas also target other pathways. among the down-regulating mirnas, mir-193b was the most effective suppressor of the wnt/β-catenin signalling pathway. mir-193b is considered to be a biomarker of various cancers because it is down-regulated in lung cancer (h. hu, li, liu, & ni, 2012) , hepatocellular carcinoma , prostate cancer (rauhala et al., 2010) , and melanoma (chen et al., 2010) . by contrast, mir-193b has been demonstrated to suppress carcinoma cell proliferation, migration, invasion, and metastasis (leivonen et al., 2009; wu, lin, zhuang, & liang, 2009) , suggesting that mir-193b may function as a tumour suppressor. excluding hepatitis b virus-associated hepatocellular carcinoma (mao et al., 2014) , the role of mir-193b in modulating viral infection has rarely been studied to date. in this study, we found that mir-193b strongly suppressed iav infection. we further figure 12 mir-193b suppresses viral infection in mice challenged with a sublethal dose of a/pr/8/34. two days after receiving 10 9 ifu of adenoviral mir-193b or mir-con, c57bl/6j mice were infected with a/pr/8/34 (250 pfu/mouse). mice were euthanised at day 3 or 7 post infection. (a) percent of body weight normalised to the initial body weight (n = 7). (b) mir-193b expression in the lungs of mice at day 3 or 7 post infection was determined by real-time pcr, normalised to β-actin, and expressed as a ratio to mir-con on the corresponding days (n = 7). (c) cyclin d1 mrna expression was determined by real-time pcr and normalised to β-actin (n = 7). (d) viral mrna levels were detected by real-time pcr and normalised to β-actin (n = 7). (e) virus progeny production in lung homogenate was determined by tcid 50 assay (n = 7). the data are presented as the mean ± sd. *p < 0.05 versus mir-con and **p < 0.01 versus mir-con on the corresponding days (nonparametric friedman's twoway anova, bonferroni's multiple comparisons test) demonstrated that mir-193b inhibited iav infection by targeting and repressing β-catenin (figures 6 and 7) . to the best of our knowledge, this is the first report to show that mir-193b possesses antiviral activities. during virus infection, various cellular signalling cascades are activated, which may support or inhibit viral replication. one important signalling pathway that modulates viral replication is the canonical wnt/β-catenin pathway. two recent genome-wide sirna screens identified multiple components of the wnt pathway that interfere with rift valley fever virus and flavivirus infection (harmon et al., 2016; smith, jeng, mcweeney, & hirsch, 2017) . previous studies have also shown that the deletion of wnt pathway components significantly impacts iav replication and interferon production (shapira et al., 2009; watanabe et al., 2014) . β-catenin is a part of adherens junctions at the cell membrane and acts as a transcription factor that interacts with lef/tcf to regulate the wnt/β-catenin pathway. increasing evidence has suggested the importance of β-catenin in viral replication, but some discrepancies exist. hillesheim et al. (2014) showed that β-catenin accumulates in the nucleus after iav infection and that β-catenin supports irf3-dependent transcription of genes responsible for the cellular innate immune response, such as ifn-β and isgs. by contrast, baril et al. (2013) reported that infection by sev, another negative-strand rna virus, induces secretion of wnt ligands and stabilisation of β-catenin and decreases expression of ifnb1 via a feedback mechanism. our study showed that the reduction of β-catenin by mir-193b indeed hampered iav replication and that compensating β-catenin by β-catenin ectopic expression restored iav infection (figure 7) . similarly, watanabe et al. showed that downregulation of β-catenin significantly decreased the relative viral rna polymerase activity by more than 50% (watanabe et al., 2014) . lef1 is also a target of mir-193b. however, lef1 co-overexpression with mir-193b plasmid did not rescue the mir-193b-mediated reduction of iav infection. lef1 forms a complex with β-catenin to activate the wnt/β-catenin responsive genes. two reports have shown that whereas overexpressing lef1 alone does not activate the wnt/β-catenin signalling pathway, coexpression of lef1 and β-catenin results in 50-to 100-fold increases in the topflash activity (ahrens, romereim, & dudley, 2011; ishitani, matsumoto, chitnis, & itoh, 2005) . it has also been reported that drosophila tcf activates gene transcription in the presence of armadillo, the drosophila homologue of β-catenin. however, drosophila tcf actually functions as a repressor in the absence of armadillo (cavallo et al., 1998; de lau & clevers, 2001) . this may explain why lef1 overexpression alone even resulted in a decrease of iav np protein expression and virus production (figure 7d-f) . furthermore, β-catenin has a transactivation domain but not lef-1 (vleminckx, kemler, & hecht, 1999) . these studies suggest that lef1 requires β-catenin for its transcription activation activity. because mir-193b reduces the protein levels of both β-catenin and lef1, overexpression of lef1 alone cannot rescue the mir-193b effects on iav infection. because mir-193b inhibits both wnt/β-catenin signalling and influenza virus infection, we focused our target identification of mir-193b on positive regulators of the wnt/β-catenin pathway. however, we cannot rule out the possibility that mir-193b may also act on other targets and pathways. there are several reported targets of mir-193b in different cells. for example, mir-193b targets ets1 transcription factor to cause cell cycle arrest and to inhibit the invasion and migration of hepatoma cells . upregulation of c-kit oncogene frequently occurs in subsets of acute myeloid leukaemia (aml). overexpression of mir-193b in the kasumi-1 acute myeloid leukaemia cell line decreases c-kit expression and inhibits the downstream pdk1/akt signalling pathway (gao et al., 2011) . mir-193b also targets the urokinase-type plasminogen activator (upa), an enzyme involved in the degradation of extracellular matrix proteins, resulting in the repression of tumour progression and invasion in human breast cancer (x. f. li, yan, & shao, 2009 ). last but not the least, mir-193b induces g1-phase arrest in pancreatic ductal adenocarcinoma by targeting kras, through which the akt and erk pathways were modulated (jin et al., 2015) . a good number of studies suggest that crosstalk occurs between the cell cycle and wnt signalling. it has been well demonstrated that wnt/β-catenin signalling stimulates the g1/s transition and promotes cell cycle progression and therefore proliferation through transcriptional up-regulation of target genes that encode, for example, c-myc and cyclin d (niehrs & acebron, 2012) . by contrast, the β-catenin levels increase during the g1/s transition and peak during g2/m in normal and transformed epithelial cells (olmeda, castel, vilaro, & cano, 2003) . it was recently found that the mitotic cdk14/cyclin y complex promotes wnt/β-catenin signalling through phosphorylation of the lrp6 coreceptor (davidson & niehrs, 2010) . cyclin d1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle and is necessary for cells to enter s phase (baldin, lukas, marcote, pagano, & draetta, 1993) . down-regulation of cyclin d1 induces g0/g1 arrest (masamha & benbrook, 2009; radu, neubauer, akagi, hanafusa, & georgescu, 2003) . we observed that cyclin d1 is markedly down-regulated in mir-193boverexpressing cells. this is likely due to the combined effect of a reduced wnt/β-catenin signalling activity and a direct repression by mir-193b because cyclin d1 is a well-known down-stream target of wnt/β-catenin signalling (shtutman et al., 1999) , and cyclin d1 has been previously shown to be a direct target of mir-193 in cancer cells (chen et al., 2010) . based on the observations that mir-193b reduced cyclin d1 protein levels and induced cell cycle g0/g1 arrest, and that mir-193b and cell cycle g0/g1 arrest suppressed viral vrnp nuclear import, we speculate that mir-193b may inhibit vrnp nuclear import harrison, dove, reed, and hiscox (2007) demonstrated that the nucleocapsid protein of coronavirus is more mobile in proliferative cells and localises to the nucleolus in a cell cycle-dependent manner. the trafficking of viral proteins may depend on the dynamics of the nucleolar proteome in response to the metabolic profile of the cell. it is noted that two studies suggest that cell cycle g0/g1 arrest facilitates iav infection (he et al., 2010; jiang et al., 2013) . in summary, herein, we identified several mirnas as novel negative regulators of wnt/β-catenin signalling. mir-193b possesses potent anti-influenza virus activities. the inhibitory effect of mir-193b on iav infection occurs through inhibition of β-catenin, likely by inducing cell cycle arrest. this discovery may facilitate the identification of potential targets for anti-influenza therapeutics and drug discovery. human lung epithelial a549 cells (american type culture collection, atcc, manassas, va, usa) were maintained in f-12k medium supplemented with 10% fbs and 1% penicillin and streptomycin (p/s). cell viability was determined by using a celltiter blue cell viability assay kit (promega, madison, wi, usa) according to the manufacturer's instructions. cells were cultured in 96-well plates and were incubated with the celltiter blue reagent at 37°c for 2 hr. fluorescence was measured using an excitation at 560 nm and an emission at 590 nm on a plate reader. the mirna overexpression vectors contained the cmv promoter, followed by an egfp tag, a mature mirna with flanking sequences (~200 bp at each end), and the sv40 poly(a) terminal sequence. mature mirnas plus flanking sequences were amplified from human genomic dnas and cloned via xho i and ecor i sites into a modified plvx-puro lentiviral vector (clontech, mountain view, ca, usa) or a modified pentr vector between egfp and sv40 poly(a) terminal sequence, as previously described (bhaskaran et al., 2009; c. huang et al., 2017) . inserted vector with a similar size of genomic dna that did not contain any mirnas or stem loop structures was used as a control (mir-con). a topflash reporter vector containing three copies of tcf/lef binding sites upstream of the thiamine kinase minimal promoter and firefly luciferase gene (upstate biotechnology, inc., lake placid, ny) was used to monitor the wnt/β-catenin activity. hek293t cells were seeded in antibiotic-free culture medium in 96-well plates (2 × 10 4 cells per well). at 20 hr after seeding, the cells were transfected with a topflash vector (20 ng), a plvx-puro lentiviral mirna or control plasmid (mir-con; 100 ng), and a normalisation prl-tk vector the bioinformatics prediction tools pictar (http://pictar.mdc-berlin. de/), target scan (version 5.2; http://www.targetscan.org/), and diana-microt-4.0 (http://diana.imis.athena-innovation.gr/) were used to identify potential targets of mir-193b. the 3′-utrs of predicted target genes of the mirnas were pcramplified and inserted into the pmirglo dual-luciferase mirna target expression vector (promega, madison, wi) at the nhei and sali sites. to detect iav infection with a luciferase reporter assay, we constructed an iav reporter vector encoding firefly luciferase under control of the 3′-utrs of the influenza a/wsn/33 np segment as previously described (lutz, dyall, olivo, & pekosz, 2005) . briefly, the 3′-and 5′-utrs of the influenza a/wsn/33 nps were amplified by pcr and inserted into the rna polymerase i promoter/terminator cassette of the phh21 vector (a kind gift from dr. yoshi kawaoka, u. wisconsin) at bsmb i sites (phh21-np-3′-utr-luc-np-5′-utr). all inserts were confirmed by dna sequencing. virus titrations were performed according to a protocol adapted from de vleeschauwer, van poucke, karasin, olsen, and van reeth (2011) . briefly, mdck cells were seeded in a 96-well cell culture plate at a density of 20,000 cells per well. one day after seeding, the cells were inoculated with 50 μl of 10-fold serially diluted samples. after 1 hr, the inoculum was removed, followed by the addition of serum-free dmem containing 1-μg/ml tpck-trypsin. the cells were observed daily for 5 days for the appearance of cytopathic effects. virus titres were calculated according to the method of reed and muench (reed & hugo, 1938) . to measure mrna expression levels, total rna was extracted using tri reagent® (molecular research center, cincinnati, oh, usa). . briefly, a 10-μl reaction containing 1 μg total rna, 1 μl of 10 × reaction buffer, 1 μl of 10 mm atp and 5 units of escherichia coli poly(a) polymerase was incubated at 37°c for 20 min, followed by enzyme inactivation at 65°c for 20 min. after polyadenylation, reverse transcription was performed using 10 μl of the polyadenylated reaction product, 1 μl of 0.5 μm poly(t) adapter, 1 μl of 10 mm dntp, and 1 μl of m-mlv reverse transcriptase. the reaction was incubated at 37°c for 50 min and then terminated by heating at 70°c for 15 min. real-time pcr were performed using sybr green pcr master mix with the above-described conditions. the expression of a mirna was analysed according to the comparative ct method by normalisation to u48 or u6 small nuclear rna using the 2 −δct method. for the 3′-utr reporter assay, hek293t cells were transfected with 5 ng of 3′-utr reporter vector, 100 ng of mirna or mir-con vector using lipofectamine 2000. two days after transfection, the cells were harvested for the dual luciferase activity assay. to determine the effect of mir-193b on different strains of iav replication, a viral luciferase reporter assay was performed. briefly, hek293 or a549 cells were transfected with an iav reporter vector phh21-np-3′-utr-luc-np-5′-utr (20 ng), a mir-193b pentr or mir-con vector (100 ng), and a normalisation prl-tk vector (2 ng) the cells were lysed with an appropriate volume of protein lysis buffer the total number of np nuclear-centred cells and the total number of cells were counted. data were then presented as percentages of np nuclear-localised cells over total cells in mir-193b-infected cells versus mir-con infected cells. two single guide rnas (sgrnas) were designed by using online software (http://chopchop.cbu.uib.no/). the pam sequence near the mir-193b seed sequence and the 20-bp sequence upstream were chosen. we constructed the lentiviral crispr/cas9 mediated mir-193b gene editing vectors by subcloning each annealed sgrna oligonucleotide pair into the sgrna scaffold of lenticrispr v2 vector (addgene, #52961) via the bsmii sites. a control vector was constructed by inserting an egfp sgrna sequence into the same vector. lentivirus was prepared by transfecting sgrna-lenticrispr v2 vector and package plasmids pspax2 and pmd2g into hek293t cells using pei reagent. stable cell lines were generated by infecting a549 cells with the crispr/cas9 lentivirus containing sgrna and selected with 1-μg/ml puromycin. to detect indels, genomic dna was isolated from the crispr/cas9-edited cells, and the targeted locus that encompasses the mature mir-193b region was pcr-amplified using the following primers: forward 5′agctttggtcatcaaaatagga3′, reverse 5′ attggagtttatcggcaactgt3′. the pcr products were denatured, annealed and digested with t7 endonuclease i (neb, ipswich, ma, usa), which recognises and cleaves nonperfectly matched dna, at 37°c for 60 min. digested products were separated on a 1% agarose gel for imaging and cloned into the pgem-t vector (promega, madison, wi, usa) for dna sequencing. a549 cells were infected with a mir-193b lentivirus or control virus (moi 100) for 48 hr. the cells were trypsinised and then seeded in a 12-well plate (10 5 cells per well). at 12 hr after seeding, the medium was replaced with serum free f-12k and cultured for 48 hr. the medium was then replaced with f-12k containing 10% fbs and cultured for 18 and 30 hr. the cells were trypsinised and fixed with ethanol at −20°c for at least 2 hr. at the time of analysis, the cells were centrifuged and stained with a freshly made solution containing propidium iodide provided with the cell cycle phase determination kit (cayman, ann arbor, mi, usa), according to the manufacturer's instructions. the cell cycle distribution was measured using a flow cytometer (accuri c6, bd biosciences) and analysed with bd sampler software. the animal procedures were approved by the institutional animal a re-evaluation of two key reagents for in vivo studies of wnt signaling bioinformatic tools for microrna dissection a systematic screen for micro-rnas regulating the canonical wnt pathway cell cycle regulation during viral infection cyclin d1 is a nuclear protein required for cell cycle progression in g1 genome-wide rnai screen reveals a new role of a wnt/ctnnb1 signaling pathway as negative regulator of virusinduced innate immune responses the emergence of adamantane resistance in influenza a(h1) viruses in australia and regionally in 2006 solid phase assay for comparing reactivation rates of neuraminidases of influenza wild type and resistant mutants after inhibitor removal t cell factor-activated transcription is not sufficient to induce anchorage-independent growth of epithelial cells expressing mutant β-catenin microrna-127 modulates fetal lung development incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern drosophila tcf and groucho interact to repress wingless signalling activity cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein crispr/cas9, a novel genomic tool to knock down microrna in vitro and in vivo microrna-193b represses cell proliferation and regulates cyclin d1 in melanoma emerging links between cdk cell cycle regulators and wnt signaling lef1 turns over a new leaf cross-protection between antigenically distinct h1n1 swine influenza viruses from europe and north america. influenza and other respiratory viruses the cell cycle and virus infection microrna-193b regulates c-kit proto-oncogene and represses cell proliferation in acute myeloid leukemia influenza virus and cell signaling pathways wnt3a mitigates acute lung injury by reducing p2x7 receptormediated alveolar epithelial type i cell death a genome-wide rna interference screen identifies a role for wnt/beta-catenin signaling during rift valley fever virus infection influenza a virus replication induces cell cycle arrest in g0/g1 phase β-catenin promotes the type i ifn synthesis and the ifn-dependent signaling response but is suppressed by influenza a virus-induced rig-i/nf-κb signaling influenza vaccines: challenges and solutions microrna-193b modulates proliferation, migration, and invasion of non-small cell lung cancer cells microrna-142-3p negatively regulates canonical wnt signaling pathway microrna-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation the frizzled family: receptors for multiple signal transduction pathways microrna roles in beta-catenin pathway microrna-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer emergence and spread of oseltamivir-resistant a(h1n1) influenza viruses in oceania, south east asia and south africa nrarp functions to modulate neural-crest-cell differentiation by regulating lef1 protein stability influenza a virus ns1 induces g0/g1 cell cycle arrest by inhibiting the expression and activity of rhoa protein deregulation of the mir-193b-kras axis contributes to impaired cell growth in pancreatic cancer influenza treatment and prophylaxis with neuraminidase inhibitors: a review. infection and drug resistance tumour suppressors mir-1 and mir-133a target the oncogenic function of purine nucleoside phosphorylase (pnp) in prostate cancer wnt signal transduction pathways protein lysate microarray analysis to identify micrornas regulating estrogen receptor signaling in breast cancer cell lines mir-185-3p regulates nasopharyngeal carcinoma radioresistance by targeting wnt2b in vitro downregulation of mir-193b contributes to enhance urokinase-type plasminogen activator (upa) expression and tumor progression and invasion in human breast cancer mir-195 suppresses non-small cell lung cancer by targeting chek1 downregulation of mir-200a induces emt phenotypes and csc-like signatures through targeting the β-catenin pathway in hepatic oval cells cyclophilin a restricts influenza a virus replication through degradation of the m1 protein influenza viruses and the nf-κb signaling pathway-towards a novel concept of antiviral therapy virus-inducible reporter genes as a tool for detecting and quantifying influenza a virus replication the tumor suppressive role of mirna-509-5p by targeting foxm1 in nonsmall cell lung cancer restoration of mir-193b sensitizes hepatitis b virus-associated hepatocellular carcinoma to sorafenib cyclin d1 degradation is sufficient to induce g1 cell cycle arrest despite constitutive expression of cyclin e2 in ovarian cancer cells regulation of influenza virus replication by wnt/β-catenin signaling the nucleoprotein of influenza a virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase rnf43 down-regulation of micro-rna-1 (mir-1) in lung cancer. suppression of tumorigenic property of lung cancer cells and their sensitization to doxorubicin-induced apoptosis by mir-1 mitotic and mitogenic wnt signalling β-catenin regulation during the cell cycle study of oseltamivir and zanamivir resistance-related mutations in influenza viruses isolated from wild mallards in sweden hypersusceptibility to vesicular stomatitis virus infection in dicer1-deficient mice is due to impaired mir24 and mir93 expression pten induces cell cycle arrest by decreasing the level and nuclear localization of cyclin d1 mir-193b is an epigenetically regulated putative tumor suppressor in prostate cancer a simple method of estimating fifty per cent endpoints a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection downregulated mir329 and mir410 promote the proliferation and invasion of oral squamous cell carcinoma by targeting wnt-7b the cyclin d1 gene is a target of the β-catenin/lef-1 pathway a microrna screen identifies the wnt signaling pathway as a regulator of the interferon response during flavivirus infection current and next generation influenza vaccines: formulation and production strategies cellular micrornas inhibit replication of the h1n1 influenza a virus in infected cells diallyl disulfide suppresses proliferation and induces apoptosis in human gastric cancer through wnt-1 signaling pathway by up-regulation of mir-200b and mir-22 regulation of hepatitis c virus genome replication by xrn1 and microrna-122 binding to individual sites in the 5′ untranslated region microrna-200 (mir-200) cluster regulation by achaete scute-like 2 (ascl2): impact on the epithelial-mesenchymal transition in colon cancer cells influenza virus infection increases p53 activity: role of p53 in cell death and viral replication the c-terminal transactivation domain of β-catenin is necessary and sufficient for signaling by the lef-1/β-catenin complex in xenopus laevis inducible microrna-155 feedback promotes type i ifn signaling in antiviral innate immunity by targeting suppressor of cytokine signaling 1 microrna: past and present microrna-200b suppresses arsenic-transformed cell migration by targeting protein kinase cα and wnt5b-protein kinase cα positive feedback loop and inhibiting rac1 activation influenza virus-host interactome screen as a platform for antiviral drug development cellular networks involved in the influenza virus life cycle expression profile of mammalian micrornas in endometrioid adenocarcinoma caspase 3 activation is essential for efficient influenza virus propagation microrna-193b regulates proliferation, migration and invasion in human hepatocellular carcinoma cells interactome analysis of the influenza a virus transcription/replication machinery identifies protein phosphatase 6 as a cellular factor required for efficient virus replication ribo-gnome: the big world of small rnas p38mapk, rho/rock and pkc pathways are involved in influenzainduced cytoskeletal rearrangement and hyperpermeability in pmvec via phosphorylating erm tumor suppressive mir-509-5p contributes to cell migration, proliferation and antiapoptosis in renal cell carcinoma upregulated mir-155 in papillary thyroid carcinoma promotes tumor growth by targeting apc and activating wnt/β-catenin signaling mir-26a inhibits prostate cancer progression by repression of wnt5a control of apoptosis in influenzavirus-infected cells by up-regulation of akt and p53 signaling additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article we thank dr. gillian air (university of oklahoma health sciences center) for providing the a/oklahoma/3052/09 h1n1, a/wsn/ 1933 h1n1 and a/oklahoma/309/2006 h3n2. we also thank drs.yoshi kawaoka (u. wisconsin) and angela barth (stanford u.) for providing the phh21 vector and δgsk-β-catenin construct, respectively. lenticrispr v2 was a gift from dr. feng zhang (addgene plasmid #52961). the authors have no conflict of interest to declare. lin liu https://orcid.org/0000-0002-4811-4897 key: cord-347053-m5m4zgfy authors: pharo, elizabeth a.; williams, sinéad m.; boyd, victoria; sundaramoorthy, vinod; durr, peter a.; baker, michelle l. title: host–pathogen responses to pandemic influenza h1n1pdm09 in a human respiratory airway model date: 2020-06-24 journal: viruses doi: 10.3390/v12060679 sha: doc_id: 347053 cord_uid: m5m4zgfy the respiratory influenza a viruses (iavs) and emerging zoonotic viruses such as severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) pose a significant threat to human health. to accelerate our understanding of the host–pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (nhbe) cell culture models has gained prominence as an alternative to animal models. nhbe cells were differentiated under air-liquid interface (ali) conditions to form an in vitro pseudostratified epithelium. the responses of well-differentiated (wd) nhbe cells were examined following infection with the 2009 pandemic influenza a/h1n1pdm09 strain or following challenge with the dsrna mimic, poly(i:c). at 30 h postinfection with h1n1pdm09, the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens-1 (zo-1) from the cell cytoskeleton. wdnhbe cells produced an innate immune response to iav-infection with increased transcription of proand anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding muc5b, which may impair mucociliary clearance. poly(i:c) produced similar responses to iav, with the exception of muc5b expression which was more than 3-fold higher than for control cells. this study demonstrates that wdnhbe cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses. over the past 30 years, there has been a rapid rise in emerging infectious diseases both of zoonotic and human origin [1] . the recent emergence and worldwide spread of the severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) virus which produces the coronavirus disease 2019 (covid-19) respiratory illness demonstrates the risk to human health and the economy posed by respiratory pathogens [2] . for novel viruses, often no diagnostics or therapeutics exist, hence they present an enormous risk of a catastrophic global pandemic [3, 4] . respiratory rna viruses, particularly influenza a viruses (iav) of the orthomyxoviridae family are a major pandemic risk as they replicate rapidly, lack a proof-reading mechanism, have a high mutation rate and are readily transmissible [3, [5] [6] [7] . over 100 years ago, the 1918 h1n1 "spanish flu" pandemic killed up to 50 million people worldwide [8] [9] [10] . three global influenza pandemics have occurred since, "asian flu" h2n2 undifferentiated nhbe and wdnhbe cells were fixed in 10% neutral buffered formalin and embedded in paraffin, sectioned (5 µm; microtome), mounted and stained with hematoxylin and eosin. for immunofluorescence assays, airlifted nhbe cells were fixed in 4% w/v paraformaldehyde for at least 20 min, rinsed with and then stored in dpbs at 4 • c. fixed cells were permeabilized for 15 min at rt with 1% v/v triton x-100/dpbs (sigma-aldrich, st. louis, mo, usa), blocked for 1 h in 1% w/v bovine serum albumin (bsa; sigma-aldrich, st. louis, mo, usa)/0.5% v/v triton x-100/dpbs. apoptotic dna fragmentation which occurs in the last phase of apoptosis (programmed cell death) was detected by terminal deoxynucleotidyl transferase dutp nick end labelling (tunel) staining using the in situ cell death detection kit, tmr red (cat# 12 156 792 910; roche, mannheim, germany) according to the manufacturer's instructions. cells were then stained with primary antibody overnight at 4 • c, washed three times with dpbs and then incubated with the appropriate species-specific secondary antibody diluted at 1:200 in 1% w/v bsa/0.5 % v/v triton x-100/dpbs for at least 1 h at rt. transwell membranes were rinsed three times with dpbs, stained with phalloidin (a22287, alexa fluor 647 phalloidin, thermofisher scientific, usa) which stains f-actin filaments for 20 min at rt, washed three times with dpbs, stained with the nuclear stain 4',6-diamidino-2-phenylindole (dapi, d1306; thermofisher scientific, usa) for 10 min at rt. transwells were washed twice with sterile water, excised and mounted on glass slides with prolong gold antifade mountant (p10144, thermofisher scientific, usa). primary antibodies were used at the following dilutions 1:50 mucin 5ac (muc5ac, ma5-12178, thermofisher scientific, usa), 1:200 mucin 5b (muc5b, hpa008246, sigma-aldrich, usa), 1:50 zo-1 (zo-1-1a12; 33-9100, thermofisher scientific, usa), 1:100 acetylated tubulin (tubac, t7451, sigma-aldrich, usa) and 1:100 claudin 4 (cldn4, ab53156, abcam, cambridge, uk). a monoclonal antibody specific to the nucleoprotein (np) of type a specific influenza (iav np) was produced using hybridoma technology in mice, as previously described [72] . the hybridoma iav np monoclonal antibody h16-l10-4 used at 1:50 dilution was kindly provided by paul selleck (australian centre for disease preparedness, csiro, geelong, australia). secondary antibodies were purchased from thermofisher scientific, usa and included goat anti-mouse igg (h+l), alexa fluor 488 and alexa fluor 568 (a11029 and a11031 respectively), goat anti-rabbit igg (h+l), alexa fluor 488 (a11034) and donkey anti-rabbit igg (h+l) alexa fluor 568 (a10042). cells were imaged using the zeiss lsm 800 confocal microscope (zeiss, oberkochen, bw, germany) using a 40× oil immersion objective unless specified otherwise. images were captured as z-stacks and maximum intensity projections generated. images were captured and processed using zen 2.5 blue software (zeiss, germany). the presence of α2-3and α2-6-linked sialic acids (sas) on the surface of wdnhbe cells was analyzed by fluorescence activated cell sorting (facs). wdnhbe cells were detached from transwell membranes by two 30 the 2009 pandemic strain of influenza virus a/california/7/2009 (h1n1)pdm09, kindly provided by the who collaborating centre for reference and research, melbourne, vic, australia was propagated by allantoic cavity inoculation of 10 day old embryonated chicken eggs at 37 • c for 48 h. the virus was passaged once in mdck cells in the presence of 2 µg/ml l-1-tosylamide-2-phenylethyl chloromethyl ketone (tpck)-treated trypsin (sigma-aldrich, usa) at 37 • c for 72 h or until cytopathic effect was observed. virus was aliquoted and titrated to determine the median tissue culture infectious dose (tcid 50 ) on mdck cells in the presence of 2 µg/ml tpck-treated trypsin. briefly, virus was serially diluted 10-fold and applied in quadruplicate to cell monolayers. cytopathic effect was assessed five days post infection. viral titres were calculated according to the method of reed-muench as tcid 50 /ml [73] . 2.6. immune challenge of wdnhbe cells with influenza a virus and poly (i:c) wdnhbe cells were washed three times with ali media to remove excess mucus from the upper surface and inoculated with influenza a/h1n1pdm09 (100 µl) at a multiplicity of infection (moi) of 1.0 for 1 h at 37 • c. the virus inoculum was removed, the cells washed three times with ali media to remove unbound virus and the cells incubated at 37 • c for 30 h postinfection (experiment endpoint). at each time point (6, 18, 24 and 30 h postinfection), the virus was harvested by the addition of ali media to the upper surface for 30 min at 37 • c, with both apical and basolateral media collected and stored at −80 • c for endpoint analyses. in addition to iav challenge, wdnhbe cells were inoculated with varying concentrations of poly(i:c) dsrna viral mimic [poly(i:c) high molecular weight, cat: tlrl-pic, invivogen, san diego, ca, usa]. briefly, airlifted cells were washed in prewarmed dpbs for 15 min at 37 • c to remove excess mucus. poly(i:c) (20 µg and 30 µg) in ali media was added to the apical surface of wdnhbe cells and ali media added to the basolateral compartment. cells were incubated for 24 h at 37 • c, then washed three times with ali media to remove excess poly (i:c). basolateral media was replaced and the cells incubated for an additional 48 h at 37 • c. transepithelial electrical resistance (teer) across the epithelium of undifferentiated and wdnhbe cells was measured using an epithelial voltohmmeter 2 (evom2) with an stx2 chopstick electrode (world precision instruments, sarasota, fl, usa) [74] . ali media was added to the apical and basolateral compartments, equilibrated for at least 15-20 min at rt and the teer measured. teer readings were membrane-corrected by subtraction of measurements from control transwells (no cells) and expressed in units of ω or ω × cm 2 . dextran conjugated to fluorescein isothiocyanate (fitc-dextran, 4 kda; sigma-aldrich, usa) was used to determine the paracellular permeability of wdnhbe cells, i.e., the transport of fitc-dextran from the apical to the basolateral compartment of the transwell via extracellular space in the pseudostratified airway epithelium [74] . fitc-dextran diluted in ali media (0.5 mg/ml) was added to the upper transwell compartment and ali media alone added to the basolateral compartment and the cells incubated for 3 h at 37 • c. basolateral media was mixed, sampled in triplicate in a microtiter plate and fitc-dextran analyzed on a biotek synergy ht microtitre plate reader. assay standards and sample solutions were prepared concurrently. lactate dehydrogenase (ldh) release was quantified using the cytotox 96 non-radioactive cytotoxicity assay (g1780, promega corporation, madison, wi, usa) according to the manufacturer's instructions. controls including no cells, untreated cells and maximum ldh release (triton x-100-treated cells, 100% cytotoxicity) were analyzed together with the apical samples using a biotek synergy ht microtitre plate reader. cytotoxicity percentage was expressed as experimental ldh release compared to maximum ldh release for triton x-100 treated cells. quantitative reverse transcription pcr (rt-qpcr) was performed on total rna extracted from cells. total rna was isolated from wdnhbe cells using the rneasy mini kit (qiagen, hilden, nw, germany) with on-column dnase i digestion. total rna was quantified using the ds-11 fx spectrophotometer (denovix, wilmington, de, usa) and first strand cdna made using the superscript iii first-strand synthesis system (thermofisher scientific, usa), total rna (500 ng) and oligo(dt) 20 . gene expression was determined by rt-qpcr using first strand cdna (1:100 dilution), taqman gene expression master mix and taqman inventoried probes (applied biosystems, foster city, ca, usa), all of which were exon-spanning apart from the single exon interferon beta 1 (ifnb1) probe (table s1 ). rt-qpcr reactions for the iav h1n1pdm09 and poly(i:c) experiments were performed on the quantstudio 3 system and quantstudio 6 flex real-time pcr system (applied biosystems, usa) respectively with the appropriate no template controls included on each plate. samples were assayed in triplicate. conditions used were 50 • c for 2 min, denaturation at 95 • c for 10 min followed by 40 cycles of 95 • c for 15 s, 60 • c for 1 min. a cycle threshold (ct) of 0.1 was applied to all gene probes. gene expression levels were normalized to the human glyceraldehyde 3-phosphate dehydrogenase viruses 2020, 12, 679 6 of 26 (gapdh) housekeeping gene and expressed as fold over detectable (fod), as described [75] . minimum detectable ct was set at 40 cycles. to assess the effect of iav h1n1pdm09 infection in wdnhbe cells on the transcription of twelve innate immune genes and two mucin-encoding genes quantified by rt-qpcr, we constructed a gene coexpression network [76] . we used the normalized gene copy number in n = 12 transwells at 30 h postinfection with iav h1n1pdm09, i.e., the number of gene copies per 1000 copies of gapdh (see above) to produce the network. for each pair of genes, the pearson correlation coefficient (pcc) was calculated to estimate the strength of the linear relationship between gene expression levels. a pcc value exceeding ±0.80 was used as the coexpression threshold. for each gene-pair, a network was constructed with the expressed genes forming the network's nodes and the pcc values exceeding the 0.80 threshold forming an edge. the pcc values were estimated using the "corr" function in the r stats package, and the network was constructed and visualized using the igraph package [77] . statistical analyses for most experiments were performed in graphpad prizm 8.2.1 (graphpad software, inc., ca, usa). data from three independent experiments for each of the iav and poly(i:c) challenges was pooled and analyzed as described below. for iav h1n1pdm09 experiments, growth kinetics of iav h1n1pdm09 (tcid 50 /ml) were analyzed by one-way anova using tukey's multiple comparison test; percentage of preinfection teer, fitc-dextran transported across the epithelium and percentage cytotoxicity were analyzed by multiple t-tests using the two-stage linear step-up procedure of benjamini, krieger and yekutieli, with a false discovery rate (fdr) of 1%. teer resistance in iav h1n1pdm09-inoculated and mock-inoculated wells at 30 h postinfection was also analyzed with r using a mixed effects model using the "aov" and the "lme" functions in the r stats and nlme packages respectively [78] . poly(i:c) teers were analyzed by one-way anova with brown-forsythe and welch anova tests and dunnett's t3 multiple comparison test. poly(i:c) fitc-dextran transported and cell cytotoxicity were analyzed by one-way anova and tukey's multiple comparisons test. iav muc5ac and muc5b gene expression was analyzed by unpaired t-tests and for poly(i:c), by one-way anova using tukey's multiple comparison test. cytokines secreted apically form iav-infected cells were analyzed by unpaired t-tests. the use of human cells in all experiments was approved by the csiro health and medical human research ethics committee (ethics approval #2019_26_lr, 14th may, 2019). prior to innate immune system challenge studies, we characterized our commercially sourced nhbe cells. cells cultured at ali on collagen i-coated transwells formed a polarized, pseudostratified epithelium around four weeks postairlift. wdnhbe cells exhibited a mucociliary phenotype, characterized by the secretion of mucus on the apical surface and the coordinated beating of cilia on the surface of ciliated cells (video s1). histological staining confirmed that wdnhbe cells formed a columnar epithelium 2-3 cells thick, consisting of basal, goblet, club and ciliated cells, representative of the human airway epithelium in vivo (figure 1a ). immunofluorescence assays confirmed the expression of muc5ac, a goblet cell marker and muc5b, produced in secretory cells and the partial colocalization of muc5ac and muc5b (figure 1b) . immunostaining of wdnhbe cells also confirmed the presence of ciliated cells, characterized by the expression of acetylated tubulin on cell-surface cilia ( figure 1c ). in order to confirm that airlifted cells formed an intact epithelial barrier, we measured the teer of undifferentiated and differentiated nhbe cells. the teer indicates the degree of polarization and differentiation of nhbe cells and hence the formation of an intact airway epithelial barrier [74, 79] . for undifferentiated nhbe cells, the average teer was <130 ω × cm 2 . in contrast, teer values ≥350 ω × cm 2 were recorded for a majority of wdnhbe cells with average values consistently ≥400-500 ω × cm 2 ( figure s1 ; table s2; table s3 ). therefore, our cells provide an excellent model to study the integrity of the airway epithelium in vitro before, during and after immune system challenges. iav infects cells by binding to cell-surface sialic acid receptors. therefore, we characterized the iav receptors present on the exterior of wdnhbe cells by facs using the lectins sna i and maa ii which bind to α2-6-linked sialic acids (sa) and α2-3-linked sas respectively. while hemagglutinins of human iavs preferentially bind α2-6-sa, those from avian iavs recognize α2-3 sa [61, 80] . almost all (99.2%) wdnhbe cells had α2-6-linked sialic acids on the cell surface, while 73.6% had cell-surface α2-3-linked sialic acids ( figure s2 ), demonstrating the susceptibility of wdnhbe cells to iav infection in this study. wdnhbe cells were infected with h1n1pdm09 at moi of 1 and apical virus titre monitored over a 30 h time period postinfection for three independent replicate experiments ( figure 2a ). h1n1pdm09 replicated efficiently in wdnhbe cells exhibiting peak viral titre of 10 8 tcid 50 /ml at 18 h postinfection. the titre decreased by 0.5 logs at 30 h postinfection (5.8 × 10 7 ; p < 0.0001). the virus titre at 1 h and 6 h postinfection (10 5 tcid 50 /ml) when the virus binds and penetrates the cells is slightly higher than expected, which may be due to incomplete removal of unbound virus, despite numerous washes, and also the binding of the virus to mucus on the transwell surface. this was consistent across all three experiments. in preliminary experiments, no virus was detected in basolateral media at 72 h postinfection of wdnhbe cells with h1n1pdm09. this indicated that the virus is shed on the apical surface of polarized epithelial cells, consistent with previous studies [81, 82] . immunofluorescence assays confirmed that at 30 h postinfection, the majority of wdnhbe cells were infected with h1n1pdm09, as indicated by staining with the influenza a nucleoprotein (iav np, figure 2b ). teer indicates the degree of polarization and differentiation of nhbe cells and hence the formation of an intact airway epithelial barrier [74, 79] . for undifferentiated nhbe cells, the average teer was <130 ω x cm 2 . in contrast, teer values ≥350 ω x cm 2 were recorded for a majority of wdnhbe cells with average values consistently ≥400-500 ω x cm 2 ( figure s1 ; table s2; table s3 ). therefore, our cells provide an excellent model to study the integrity of the airway epithelium in vitro before, during and after immune system challenges. to understand the effect of h1n1pdm09 on the integrity of the barrier formed by wdnhbe cells, the effect of the virus on the airway epithelium was determined by the change in teer, paracellular permeability and ldh release (percent cytotoxicity) (figure 3 ). at 6-18 h postinfection, there was no difference in percentage teer values. during this period, the virus is undergoing virus assembly and early release of virus particles. however, at 24 h and 30 h postinfection, dramatic reductions in teer values of 53.6 and 69.4% respectively occurred, significantly lower values than the teers of mock-infected cells (p < 0.0001; refer to figure s3 for a graph of the individual teer values (ω x cm 2 ) for all three independent h1n1pdm09 experiments). this significant decrease in teer resistance was also confirmed by mixed effects modelling (p < 0.0001). from 18-30 h postinfection, higher viral replication (observed in figure 2a ) correlates with a reduction in barrier integrity of wdnhbe cells. in contrast to virus-infected cells, teer values for to understand the effect of h1n1pdm09 on the integrity of the barrier formed by wdnhbe cells, the effect of the virus on the airway epithelium was determined by the change in teer, paracellular permeability and ldh release (percent cytotoxicity) (figure 3 ). at 6-18 h postinfection, there was no difference in percentage teer values. during this period, the virus is undergoing virus assembly and early release of virus particles. however, at 24 h and 30 h postinfection, dramatic reductions in teer values of 53.6 and 69.4% respectively occurred, significantly lower values than the teers of mock-infected cells (p < 0.0001; refer to figure s3 for a graph of the individual teer values (ω × cm 2 ) for all three independent h1n1pdm09 experiments). this significant decrease in teer resistance was also confirmed by mixed effects modelling (p < 0.0001). influenza a viruses damage apical junctional complexes and alter the cell cytoskeleton and morphology of airway epithelial cells [34, 35, 83] . to investigate these characteristics in our model we performed immunofluorescence assays on mock and h1n1pdm09-infected wdnhbe cells 30 h postinfection ( figure 4) . we characterized the distribution of the zo-1 adapter protein that links tight junctions and adherens junctions to the cell cytoskeleton [88] . phalloidin was used to stain f-actin filaments of the cytoskeleton. in mock infected wdnhbe cells, zo-1 forms an intact apical to further characterize airway barrier integrity in response to iav infection, the paracellular transport of fitc-dextran (4 kda) across the epithelium was measured. paracellular transport is regulated by ajcs, hence increased fitc-dextran transport from the apical to basolateral compartment of the transwell is an indicator of "leaky" or damaged junctions [83] . at 30 h postinfection, fitc-dextran transported across iav-infected cells was 11.4-fold higher than for mock-treated cells (p < 0.001; figure 3b ), indicative of increased epithelium permeability. influenza a virus induces cell death [84, 85] . hence, we determined the cytotoxicity of h1n1pdm09 to wdnhbe cells by measuring the apical release of ldh 30 h postinfection. ldh is a soluble cytoplasmic enzyme that is released from the cytoplasm upon damage to the plasma membrane and is directly proportional to the number of cells undergoing apoptosis or necrosis [86, 87] . at 30 h postinfection, cell death due to h1n1pdm09 was 7.1-fold greater for iav-infected cells compared to cells treated with media alone (p < 0.001; figure 3c ). tunel staining also showed a greater number of apoptotic cells in wdnhbe cells infected with pandemic influenza compared to mock-infected cells ( figure s4 ). therefore, h1n1pdm09 damage to the in vitro airway epithelium is characterized by a reduced teer, increased paracellular flux and increased cell death. in order to compare the immune response of wdnhbe cells to different immunostimulants, we challenged our cells with poly(i:c) at 20 µg and 30 µg and evaluated barrier integrity as for iav-infected cells. at 48 h poststimulation, teer values were significantly reduced in a dose-dependent manner in comparison to mock-treated cells (p < 0.0001; figure 3d ). similar to the h1n1pdm09 treatment, a 72.5% reduction in teer was recorded for 30 ug poly(i:c) and a 58.4% reduction for the 20 µg treatment. in contrast, the teer of mock-treated cells increased by 14.5%. a graph of the individual teer values (ω × cm 2 ) for all three, independent poly(i:c) experiments is also provided ( figure s5 ). the effect of poly(i:c) on paracellular flux was not as great as for h1n1pdm09. at 30 µg and 20 µg poly (i:c), fitc-dextran transported across the airway epithelium was 3-fold and 2-fold greater respectively compared to control cells (p < 0.0001 and p < 0.01 respectively; figure 3e ). similarly, poly(i:c) was not as cytotoxic as h1n1pdm09. at the higher, 30 µg poly(i:c) dose, cell death was 3-fold higher than for control cells (p < 0.01; figure 3f ). however, there was no difference in cytotoxicity between the 20 µg poly(i:c) and mock treatments. in summary, both h1n1pdm09 and poly(i:c) had a detrimental effect on the integrity of the in vitro pseudostratified airway epithelium. influenza a viruses damage apical junctional complexes and alter the cell cytoskeleton and morphology of airway epithelial cells [34, 35, 83] . to investigate these characteristics in our model we performed immunofluorescence assays on mock and h1n1pdm09-infected wdnhbe cells 30 h postinfection ( figure 4) . we characterized the distribution of the zo-1 adapter protein that links tight junctions and adherens junctions to the cell cytoskeleton [88] . phalloidin was used to stain f-actin filaments of the cytoskeleton. in mock infected wdnhbe cells, zo-1 forms an intact apical circumferential belt (also known as the perijunctional actomyosin ring) around the plasma membrane of each cell (figure 4a ). in sharp contrast, zo-1 was disrupted and discontinuous in many iav h1n1pdm09-infected cells, suggestive of damage to ajcs and the epithelium (figure 4b) . these results were consistent with phalloidin staining of f-actin filaments of the cell cytoskeleton. f-actin filaments are intact in mock-infected cells (figure 4a ) but disrupted in iav-infected cells (figure 4b) . merged images show that the disruption of zo-1 and f-actin of the cytoskeleton occurs in the same cells in the in vitro airway epithelium (merge, figure 4b) . furthermore, iav-infected cells show evidence of cell distension. while h1n1pdm09 disrupted zo-1 and f-actin in wdnhbe cells, this was not observed in cells treated with 30 µg poly(i:c) ( figure s6 ). results were consistent with phalloidin staining of f-actin filaments of the cell cytoskeleton. f-actin filaments are intact in mock-infected cells (figure 4a ) but disrupted in iav-infected cells (figure 4b) . merged images show that the disruption of zo-1 and f-actin of the cytoskeleton occurs in the same cells in the in vitro airway epithelium (merge, figure 4b) . furthermore, iav-infected cells show evidence of cell distension. while h1n1pdm09 disrupted zo-1 and f-actin in wdnhbe cells, this was not observed in cells treated with 30 µg poly(i:c) ( figure s6 ). the mucosal barrier is a major component of the innate immune system in the lungs [89, 90] . muc5ac and muc5b are the major secreted mucins and play a critical, though poorly understood role in airway defense and mucociliary clearance [91, 92] . therefore, we analyzed the effect of h1n1pdm09 on the expression of these genes by rt-qpcr (figure 5a) . iav had no effect on the expression of the muc5ac goblet cell marker in wdnhbe cells (figure 5a ). in contrast, muc5b was suppressed more than 2-fold in h1n1pdm09-inoculated cells (p < 0.01; figure 5a ). poly(i:c) treatment also had no effect on muc5ac expression when compared to mock-treated cells (ns, figure 5b) , consistent with pandemic iav. in contrast, poly(i:c) had a dose-dependent effect the mucosal barrier is a major component of the innate immune system in the lungs [89, 90] . muc5ac and muc5b are the major secreted mucins and play a critical, though poorly understood role in airway defense and mucociliary clearance [91, 92] . therefore, we analyzed the effect of h1n1pdm09 on the expression of these genes by rt-qpcr (figure 5a) . iav had no effect on the expression of the muc5ac goblet cell marker in wdnhbe cells (figure 5a ). in contrast, muc5b was suppressed more than 2-fold in h1n1pdm09-inoculated cells (p < 0.01; figure 5a ). mock, n = 11; iav, n = 11 for muc5b; poly(i:c) data n = 9 biological replicates per treatment. statistical significance: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. we used rt-qpcr to determine whether wdnhbe cells produced an innate immune response to iav h1n1pdm09 in vitro. genes were selected based on their response to iav infection with poly(i:c) treatment also had no effect on muc5ac expression when compared to mock-treated cells (ns, figure 5b) , consistent with pandemic iav. in contrast, poly(i:c) had a dose-dependent effect on muc5b expression with 2-fold and 4-fold higher expression in wdnhbe cells treated with 20 µg and 30 µg poly(i:c) respectively compared to mock-treated cells (20 µg poly(i:c): p < 0.05; 30 µg: p < 0.0001; figure 5b ). we used rt-qpcr to determine whether wdnhbe cells produced an innate immune response to iav h1n1pdm09 in vitro. genes were selected based on their response to iav infection with h1n1pdm09 and/or h5n1 subtypes as reported for in vitro and/or in vivo studies [58, 62, [93] [94] [95] [96] [97] [98] . genes assayed included: the chemokine-encoding c-x-c motif chemokine ligand 10 (cxcl10), also known as interferon γ-induced protein 10 (ip10), c-c motif chemokine ligand (ccl5), alias regulated on activation, normal t cell expressed and secreted (rantes), c-c motif chemokine ligand 2 (ccl2), also known as monocyte chemoattractant protein-1 (mcp1), c-c motif chemokine ligand 2 (ccl3), alias macrophage inflammatory protein 1-α (mip1α), and c-x-c motif chemokine ligand 8 (cxcl8), also known as interleukin 8 (il8). proinflammatory cytokine-encoding genes characterized included: tumor necrosis factor (tnf), known as tnf-α, interleukin 1 β (il1b), colony stimulating factor 2 (csf2), also referred to as granulocyte-macrophage colony stimulating factor (gmcsf) and the potent proinflammatory interleukin 6 (il6). expression of the anti-inflammatory-encoding interleukin 10 (il10) and antiviral-encoding interferon β 1 (ifnb1) and radical s-adenosyl methionine domain containing 2 (rsad2), commonly known as virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible; viperin [99] was also characterized. all genes assayed were upregulated in h1n1pdm09-infected versus mock-inoculated cells (table 1 ; figure s7 ). muc5ac and muc5b values ( figure 5 ) have also been included in table 1 . notably, there was a difference in the magnitude of the response to pandemic influenza with four different levels of fold-change observed. the highest fold-increases in expression were recorded for cxcl10 (> 13,000-fold) and the antiviral-encoding ifnb1 (> 5500-fold). ccl5 and rsad2 expression were also significantly upregulated in response to iav (> 650 and >350 fold respectively). il10, tnf, mip1α and il6 exhibited a 26-53-fold increase in expression compared to mock-inoculated cells. in contrast, the magnitude of response to iav-infection was lower for the ccl2, cxcl8, csf2 and il1b genes (2-8-fold increases). the increased expression of these cytokine, chemokine and antiviral genes suggests that innate immune responses are activated in our wdnhbe cells in response to pandemic influenza. this relationship was explored by the generation of a gene coexpression network ( figure 6 ) based on gene copy numbers (table s4 ). the relationship between pro-and anti-inflammatory cytokines and chemokines genes is highlighted by the network connectivity. similarly, association between mucin gene expression is shown. in contrast, the expression of the antiviral ifnb1 and rsad2 were not linked to other genes. the induction of innate immune response genes in wdnhbe cells was also investigated by the stimulation of wdnhbe cells with 20 µg or 30 µg poly(i:c) ( figure s8 ). in contrast to iav, poly(i:c) had no effect on csf2 expression. while the lower, 20 µg dose of poly(i:c) was sufficient to upregulate il6 and cxcl8 expression, tnf and il1b were only induced by 30 µg of poly(i:c) compared to mock-treated cells. to determine whether gene expression was correlated with protein secretion, multiplex immunoassays for il-6, tnf-α, cxcl8 (il-8) and csf2 (gm-csf) were performed on media harvested from the apical (figure 7 ) and basolateral compartments ( figure s9 ) at 30 h postinfection of wdnhbe cells with h1n1pdm09. apical secretion levels of il-6 and tnf-α after a 15 min period were >8-12-fold higher in iav-infected versus mock cells (figure 7a, b) , with il-8 more than 2-fold higher (figure 7c ). in contrast, while csf2 trended lower in iav-infected cells, it was not significantly different to mock levels (figure 7d ). il-1β was secreted at the minimum limit of detection (data not shown). cytokine and chemokine secretion into the lower compartment of transwells over a 6 h period from 24 to 30 h postinfection exhibited similar trends to apical supernatants, although protein levels were generally reduced apart from il-8 ( figure s9 ). multiplex immunoassays for il-6, tnf-α, cxcl8, csf2 and il-1β were also performed for apical and basolateral media from the 20 µg and 30 µg poly(i:c) treatments ( figure s10 ). trends were analogous to the response to iav, apart from csf2, which was stimulated, rather than suppressed by poly(i:c). the apical secretion of il-1β, il-6 and csf2 was only higher for 30 µg poly(i:c) compared to mock-treated cells. in contrast, tnf-α and cxcl8 levels were greater than mock cells for both poly(i:c) doses. cytokine and chemokine secretion in basolateral media over a 48 h period the induction of innate immune response genes in wdnhbe cells was also investigated by the stimulation of wdnhbe cells with 20 µg or 30 µg poly(i:c) ( figure s8 ). in contrast to iav, poly(i:c) had no effect on csf2 expression. while the lower, 20 µg dose of poly(i:c) was sufficient to upregulate il6 and cxcl8 expression, tnf and il1b were only induced by 30 µg of poly(i:c) compared to mock-treated cells. to determine whether gene expression was correlated with protein secretion, multiplex immunoassays for il-6, tnf-α, cxcl8 (il-8) and csf2 (gm-csf) were performed on media harvested from the apical (figure 7 ) and basolateral compartments ( figure s9 ) at 30 h postinfection of wdnhbe cells with h1n1pdm09. apical secretion levels of il-6 and tnf-α after a 15 min period were >8-12-fold higher in iav-infected versus mock cells (figure 7a,b) , with il-8 more than 2-fold higher (figure 7c ). in contrast, while csf2 trended lower in iav-infected cells, it was not significantly different to mock levels ( figure 7d ). il-1β was secreted at the minimum limit of detection (data not shown). cytokine and chemokine secretion into the lower compartment of transwells over a 6 h period from 24 to 30 h postinfection exhibited similar trends to apical supernatants, although protein levels were generally reduced apart from il-8 ( figure s9 ). multiplex immunoassays for il-6, tnf-α, cxcl8, csf2 and il-1β were also performed for apical and basolateral media from the 20 µg and 30 µg poly(i:c) treatments ( figure s10 ). trends were analogous to the response to iav, apart from csf2, which was stimulated, rather than suppressed by poly(i:c). the apical secretion of il-1β, il-6 and csf2 was only higher for 30 µg poly(i:c) compared to mock-treated cells. in contrast, tnf-α and cxcl8 levels were greater than mock cells for both poly(i:c) doses. cytokine and chemokine secretion in basolateral media over a 48 h period exhibited similar trends to apical media. therefore, airlifted nhbe cells secrete cytokines and chemokines in response to both h1n1pdm09 and poly(i:c) immune system challenges. however, under experimental conditions used and properties measured, iav elicits a much stronger inflammatory response than poly(i:c) in wdnhbe cells. exhibited similar trends to apical media. therefore, airlifted nhbe cells secrete cytokines and chemokines in response to both h1n1pdm09 and poly(i:c) immune system challenges. however, under experimental conditions used and properties measured, iav elicits a much stronger inflammatory response than poly(i:c) in wdnhbe cells. and mock csf2 (n = 11); ns, not significant; *, p < 0.05; **, p < 0.01. we show that well-differentiated nhbe cells grown at the air-liquid interface are an anatomically and physiologically relevant in vitro model to investigate changes in the airway epithelium in response to h1n1pdm09 and the dsrna viral analogue, poly(i:c). our model we show that well-differentiated nhbe cells grown at the air-liquid interface are an anatomically and physiologically relevant in vitro model to investigate changes in the airway epithelium in response to h1n1pdm09 and the dsrna viral analogue, poly(i:c). our model recapitulated the in vivo response of the human airway epithelium to influenza in vitro. the cytopathic effect of h1n1pdm09 included damage to the airway epithelium, the induction of innate immune responses including the expression of pro-and anti-inflammatory cytokines and chemokines and antiviral genes and proteins, consistent with pulmonary host defense. to our knowledge, this is the first study that has shown zo-1 damage in wdnhbe cells treated with h1n1pdm09. additionally, the downregulation of muc5b, which plays an important role in mucociliary clearance suggests another mechanism of pathogenesis induced by the virus. these results form the basis for using the ali model for studying host-pathogen interactions and for testing therapeutics against newly emerged and re-emerging respiratory pathogens. the striking effect of h1n1pdm09 on zo-1 and the disruption of the apical perijunctional actomyosin ring of the cytoskeleton highlights iav damage to the 3d architecture of cells within the pseudostratified epithelium and is consistent with reduced teer, increased paracellular flux across the epithelium and increased apoptosis. the pattern of zo-1 damage in our wdnhbe cells infected with pandemic iav is similar to that of the immortalized human bronchial epithelial cell line (16hbe14o-) infected with tissue culture adapted influenza virus a (h1n1) [83] . it also mimics zo-1 dissociation from tjs caused by rhinovirus infection of 16hbe14o-cells, human airway epithelial cells grown at ali and in mice in vivo [83, 101] . zo-1 dissociation is also consistent with the in vivo airway epithelium of asthmatic patients [32] . while poly(i:c) disrupted zo-1 in cell membranes of immortalized 16hbe14ocells [71] , this was not observed in our primary wdnhbe cells. this may reflect differences between primary and immortalized cells and/or different concentrations and preparations of poly(i:c). airway protection is provided by a broad-spectrum of antimicrobial factors in mucus, e.g., lysozyme, β-defensins, cathelicidin, lactoferrin, elafin, secretory leukocyte peptidase inhibitor (slpi) and mucins [102] [103] [104] . however, the precise roles of mucins, in particular, muc5ac and muc5b, the major secretory mucins in the human airway epithelium are still to be determined. optimal airway defense requires a balance between mucus production and clearance [92] . in our wdnhbe model, mucus removal does not occur due to the closed nature of the transwell system. however, differences in mucin gene expression for h1n1pdm09 and poly(i:c) compared to mock-inoculated cells demonstrated that wdnhbe cells respond to these immune system challenges. differences in mucus composition and the relative abundance of muc5ac and muc5b can alter mucus gel viscosity, promoting pathogen growth, rather than their removal [92, 105] . hence, the significant increase in muc5b expression in response to poly(i:c) but decrease in response to h1n1pdm09 and unchanged muc5ac for both immune system stimulants was intriguing. whilst the increase in muc5b in response to poly(i:c) in our study was consistent with lever and colleagues, we did not observe an increase in muc5ac expression [70] . additionally, different iav strains (seasonal and pandemic) and different subtypes (h1n1 and h3n2) induced different levels of muc5ac expression in an nci-h292 human pulmonary mucoepidermoid carcinoma cell line [106] . seasonal strains were stronger inducers of muc5ac expression than pandemic strains and h3n2 generally had a greater effect on expression than h1n1 [106] . the differences observed in muc5ac expression in our study may reflect the use of normal primary cells versus immortalized cancer lines, the use of different virus preparations and mois, different concentrations and/or preparations of poly(i:c) and a difference between cell donors. once the mucus layer of the airway epithelium has been penetrated, pathogens such as iav attach to cell-surface receptors (α2,3-and/or α2,6sialic acids), enter and replicate in epithelial cells. the virus egresses and spreads to other nonimmune and immune cells [56] . although our model lacks immune cells, wdnhbe cells mount an innate immune response to both iav and poly(i:c), consistent with separate studies of these stimulants [62, 70] . the body's natural defense mechanism of inflammation which promotes cell repair and healing [107] was mimicked in our wdnhbe model, with the increased expression of proinflammatory cytokines and chemokines such as tnf, il1b, il6, cxcl8 and cxcl10. this suggests that wdnhbe cells recognize iav and poly(i:c) through binding to pattern recognition receptors (prrs) such as the toll-like receptors (tlr3, tlr7, and tlr8), retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein-5 (mda-5), triggering innate immune response signaling cascades as occurs in vivo [22, 54, 55, [108] [109] [110] [111] [112] [113] [114] . antiviral, pro-and anti-inflammatory cytokines and chemokines are then upregulated in the host [27, 56, 115] . key genes upregulated in our h1n1pdm09 in vitro challenge model, mimic the innate immune and inflammatory response in human patients in vivo infected with the 2009 pandemic iav. these include ifnb1, cxcl10, il10, tnf and rsad2. the more than 5500-fold increase in ifnb1, which encodes the antiviral type i interferon, ifn-β, confirms the host recognition of iav and the host-defense response to the virus in the airway epithelium in vitro, as observed by chan et al., [58] . interestingly, the kinetics and magnitude of ifn-β secretion and hence the early innate and antiviral responses can vary for different influenza a subtypes [116] . ifn-α/β stimulates the activation of hundreds of ifn-stimulated genes (isgs, e.g., cxcl10, rsad2, etc.), the first line of defence against viral infection [116] [117] [118] [119] [120] . this is observed in our model with the dramatic, 13,000-fold increase in cxcl10 expression in response to h1n1pdm09 infection, consistent with differentiated human airway epithelial cells infected with a hong kong isolate of the a/h1n1 2009 pandemic strain [59] , wdnhbe cells infected with pandemic iav strains from a fatal (a/ky/180/10) and nonfatal (a/ky/136/09) case [62] and in patients infected with pandemic iav [93, [121] [122] [123] . upregulation of cxcl10 also occurs in vitro in the a549 human lung alveolar adenocarcinoma response to iavs [124] . cxcl10 is a key chemokine responsible for early response to viral infection, is associated inflammation, e.g., asthma [125] and is a biomarker that predicts disease severity and pathogenesis [126] [127] [128] . the impact of cxcl10 is reflected in the gene coexpression network, with the increased expression of a number of other chemokines, cytokines and the anti-inflammatory il10 gene. cxcl10 is upregulated in response to other respiratory viruses, including rsv, rhinovirus and h5n1 [24, 77, 129] . however, while cxcl10 is protective in sars-cov infection [126] , its precise role in h1n1pdm09 infection is unclear. increased levels of cytokines and chemokines are detected in the sera of patients with pandemic influenza [130] , consistent with our model. in particular, increased levels of the proinflammatory il-6 and the anti-inflammatory il-10 which protects host tissue from damage during acute inflammatory responses are observed [93, 95, 121, [131] [132] [133] . elevated levels of tnf and il1b [134] are also detected [93, 122, 123] . tnf-α stimulates il-1β which exacerbates lung injury during severe influenza but may also have a vital role in lung repair after infection [130] . other cytokines and chemokines elevated in sera of patients include the chemoattractants cxcl8 [93, 131] and ccl2 [121] [122] [123] . increased expression and/or secretion of these and other chemoattractants such as ccl5 and ccl3 were observed in our wdnhbe model, consistent with other in vitro lung cell culture studies [62, 96, 135] . hence, cell-mediated immunity is triggered in wdnhbe cells, as occurs in vivo [116, 119] . the more than 350-fold induction of the antiviral-encoding rsad2 (viperin) gene in our pandemic iav-infected wdnhbe cells confirms the antiviral response of the airway epithelium in vitro. it is consistent with increased rsad2 expression in wdnhbe cells infected with the pandemic influenza strains a/ky/180/10 and a/ky/136/09 [62] and in immortalized nci-h441 [96] and a549 cells [124] . rsad2 has multiple modes of antiviral activity. it catalyzes the conversion of cytidine triphosphate (ctp) to 3 -deoxy-3 ,4 -didehydro-ctp (ddhctp) which prematurely terminates rna-dependent rna polymerase (rdrp) of selected viruses but does not interfere with host rna and dna polymerases [136] . hence, it reduces the replication of a range of rna and dna viruses including iavs, rabies virus, hiv, west nile virus, zika virus, dengue virus and hepatitis c virus [136] [137] [138] . rsad2 also restricts the release (budding) of iav h1n1 and other viruses by disrupting lipid raft microdomains on the plasma membrane of host cells [99, 139, 140] . rsad2 also plays a role in the activation of t-cells and t-cell-receptor-mediated activation of nf-κb and activating protein 1 (ap-1), key transcription factors in the expression of proinflammatory cytokines [140] . hence, rsad2 is a multifunction antiviral that has a vital role in combatting viruses such as iav. airlifted primary nhbe cells grown on transwells at the air-liquid interface are the gold standard for bronchial epithelial cell culture [141] . this model provided an excellent system to investigate innate immune responses to h1n1pdm09 and poly(i:c) in the airway epithelium in vitro. future studies could investigate the addition of other cell types including fibroblasts, endothelial smooth muscle cells and immune cells, e.g., neutrophils, to better reproduce the human airway in vitro and will undoubtably provide further information on host-pathogen interactions in the lung. airlifted nhbe cells recapitulated the pseudostratified airway epithelium in vivo, with the formation of ajcs, mucus secretion and the coordinated beating of ciliated cells. the disruption of the airway epithelium by iav h1n1pdm09 and poly(i:c), plus the induction of the innate immune response and antiviral, and pro-and anti-inflammatory genes demonstrated the viability of this model to investigate pandemic influenza. the disassembly of the ajc by h1n1pdm09 as shown by damage to zo-1 suggests that the virus can penetrate the epithelium and hence produce systemic infection. the use of multiple immune system challenges enabled the identification of differential responses of wdnhbe cells to h1n1pdm09 and poly(i:c). the reduction of muc5b in response to iav is indicative of impaired mucociliary clearance of iav which may contribute to the severity of pandemic influenza in vivo. future studies will focus of the use of this model to investigate zoonotic, emerging infectious viruses with pandemic potential including the sars-cov-2 coronavirus currently sweeping the world today. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/6/679/s1, table s1 : taqman probes used for rt-qpcr analyses, video s1: beating of cilia on the surface of ciliated cells of the wdnhbe epithelium, figure s1 :'transepithelial electrical resistance (teer) readings of wdnhbe cells, table s2 : teer readings (ω & ω × cm2) wdnhbe cells -iav h1n1pdm09, table s3 : teer readings (ω & ω × cm2) wdnhbe cells -poly(ic), figure s2 : facs analysis of α-2-3 and α-2-6-linked sialic acids on the surface of wdnhbe cells, figure s3 : transepithelial electrical resistance (teer) readings for three independent experiments of mock vs iav h1n1pdm09 inoculated wdnhbe cells, figure s4 : apoptosis in wdnhbe cells infected with pandemic iav h1n1pdm09, figure s5 : transepithelial electrical resistance (teer) readings for three independent experiments of wdnhbe cells treated with 20 µg and 30 µg poly(i:c) vs mock-treated cells, figure s6 : immunofluorescence assays of poly(i:c)-treated wdnhbe cells, figure s7 : 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response patterns in severe pandemic 2009 h1n1 and seasonal influenza among hospitalized adults immunologic changes during pandemic (h1n1) il-1 b and il-6 upregulation in children with h1n1 influenza virus infection avian influenza virus a/hk/483/97(h5n1) ns1 protein induces apoptosis in human airway epithelial cells a naturally occurring antiviral ribonucleotide encoded by the human genome the role of viperin in the innate antiviral response viperin is an important host restriction factor in control of zika virus infection the interferon inducible gene: viperin in vivo and in vitro studies on the antiviral activities of viperin against influenza h1n1 virus infection engineering airway epithelium this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors gratefully acknowledge the assistance of dianne green and jean payne for histology, john bingham and jemma bergfeld for pathology expertise, jenni harper for assistance with immunofluorescence assays, matt bruce for facs analysis, leanne davis for assistance with rt-qpcr and ryan farr for advice on rt-qpcr analysis. we acknowledge the insightful advice and assistance from dr kirsty short in establishing the transwell models at acdp. we thank microscopy australia for supporting the confocal microscopy capability utilized in this study through national collaborative research infrastructure strategy (ncris) funding. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-310004-h9ixhhzz authors: yuan, shuofeng; chu, hin; huang, jingjing; zhao, xiaoyu; ye, zi-wei; lai, pok-man; wen, lei; cai, jian-piao; mo, yufei; cao, jianli; liang, ronghui; poon, vincent kwok-man; sze, kong-hung; zhou, jie; to, kelvin kai-wang; chen, zhiwei; chen, honglin; jin, dong-yan; chan, jasper fuk-woo; yuen, kwok-yung title: viruses harness yxxø motif to interact with host ap2m1 for replication: a vulnerable broad-spectrum antiviral target date: 2020-08-28 journal: sci adv doi: 10.1126/sciadv.aba7910 sha: doc_id: 310004 cord_uid: h9ixhhzz targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current covid-19. here, we found a common yxxø-motif of multiple viruses that exploits host ap2m1 for intracellular trafficking. a library chemical, n-(p-amylcinnamoyl)anthranilic acid (aca), was identified to interrupt ap2m1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza a viruses (iavs), zika virus (zikv), human immunodeficiency virus, and coronaviruses including mers-cov and sars-cov-2. yxxø mutation, ap2m1 depletion, or disruption by aca causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of iav nucleoprotein and diminished endoplasmic reticulum localization of zikv-ns3 and enterovirus-a71-2c proteins, thereby suppressing viral replication. our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target. virus-host interactions drive mutual evolutionary changes which result in the marked diversification of viruses and host antiviral responses (1) . the emergence of viruses including coronaviruses [severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome-related coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus 2 (sars-cov-2)], hiv, zika virus (zikv), avian influenza a (h5n1), a (h7n9), and pandemic 2009 influenza a (pdmh1n1) viruses and enteroviruses [enterovirus a71 (ev-a71) and ev-d68] emphasizes the need to investigate for a conserved and broadly shared mechanism of virus-host interaction with potential therapeutic implications (1) (2) (3) (4) . viruses live briefly but perpetually. they invade cells and manipulate host machinery to replicate, transmit, and cause disease. host signal transduction in response to virus invasion activates transcription factors that determine the gene expression characteristics and signaling mechanisms of the cell fate. these signaling mechanisms have been well studied in the fields of embryonic development and cancer biology but less well studied in the context of virus infection (5) . a small set of evolutionarily conserved signaling pathways is associated with the cell fates of apoptosis, differentiation, or virus elimination during virus-host interactions. they include the transforming growth factor- (tgf-)/smad, wnt/-catenin, notch, and phosphatidylinositol 3-kinase/thymoma viral proto-oncogene (pi3k/akt) signaling pathways. here, we show that tgf- signaling is commonly altered by vastly different viruses. modulating the tgf- pathway is primarily mediated by mislocalization of tgf- cytokines, its receptors tgf-r, and smad transcriptional factors, which are largely executed through membrane and intracellular trafficking pathways (6) . therefore, we hypothesize that one approach to determine the cell fate, to die or to support virus replication, is through virusmanipulated membrane and intracellular trafficking. in this study, we performed integrative chemical and genetic screens that identified host ap2m1 protein as a critical player affecting tgf- signaling and facilitating intracellular trafficking of different viruses. ap2m1 is the mu (2) subunit of ap2 adaptor complex, which functions as the major heterotetramer (, 2, 2, and 2 subunits) that orchestrate clathrin-mediated endocytosis (cme) (7) . the direct binding between tgf-r and ap2 has been demonstrated in vitro and in vivo (8) . functionally, ap2m1 recognizes the yxxø sorting motifs present in the cytosolic tail of different cargo proteins, whereas x refers to any amino acid and ø indicates hydrophobic residues including l/m/f/i/v (9) . here, we identify a previously unrecognized role of ap2m1, which is an intracellular cargo molecule that cotraffics with different internal viral proteins for proper subcellular localizations, in addition to its role in endocytosis. the cotrafficking is mediated through protein-protein interaction (ppi) between host ap2m1 and specific viral proteins harboring a yxxø motif. our initial pharmacological screening identifies a tool compound, n-(p-amylcinnamoyl)anthranilic acid (aca), that disrupts ap2m1/yxxø interaction without affecting 1 the ap2m1 phosphorylation. aca exhibits broad-spectrum antiviral efficacy in cell cultures and mouse models. substitutions made in the influenza a nucleoprotein yxxø motifs affect viral fitness in vitro and in vitro, indicating a critical role of ap2m1/yxxø interaction during virus life cycle. our study reveals ap2m1/yxxømediated intracellular trafficking of diverse virus families, which represents a previously unidentified intervention target for a broad spectrum of emerging viral diseases. to determine the virus-induced signaling associated with cell fate pathways, we examined whether virus infection could potentiate host cell signaling by tgf-, wnt, notch, and pi3k/akt pathways. reporter gene assays with high multiplicity of infection (moi) and time-course monitoring of luciferase activity were performed in gene-transfected human primary cells including the influenza a pdmh1n1-infected human primary bronchial/tracheal epithelial cells (hbtecs), zikvinfected human primary fibroblasts hfl1 and ev-a71-infected human neural progenitor cells. tgf- signaling exhibited distinct patterns of marked changes when compared with the marginally changed wnt, notch, and pi3k/akt pathways (fig. 1a) . infection by either pdmh1n1 or zikv triggered tgf- activation, whereas ev-a71 infection suppressed its signaling, indicating that tgf- signaling is commonly exploited in the cell fate determination pathway by different rna viruses. proper control of tgf-r via membrane and intracellular trafficking is documented to mediate tgf- signaling (10) . to explore the dependence of viral fitness on these host trafficking genes, we performed a loss-of-function screen to determine the degree of pdmh1n1 replication after individual gene silencing. among the 142 cellular trafficking genes, knockdown of 17 genes reduced virus titers by more than one-log 10 unit ( fig. 1b and fig. s1 ). of the 17 influenza a virus (iav) screen hits, 4 had been previously implicated in hiv-1 assembly, release, and budding: adaptor related protein complex 1 subunit mu 1 (ap1m1), caveolin 1 (cav1), ras-related protein rab-5a (rab5a), and rho-associated protein kinase 1 (rcok1) (11); depletion of copa or copb2 profoundly restricted human cytomegalovirus replication (12) ; copg and ap2m1 were demonstrated to be important for iav production in a genome-wide small interfering rna (sirna) screening (13) . the results suggested that the selected membrane trafficking genes may serve as proviral factors with broad relevance to a number of virus infections. in view of the vulnerability of intracellular trafficking within virus life cycle, we screened a small-molecule compound library of trafficking inhibitors for finding drug hits with promising antiviral potency, which may also serve as a tool compound enabling the identification and characterization of putative targets within intracellular trafficking pathways. the library consists of 420 inhibitors targeting membrane transporters such as p-glycoprotein, exportin 1, and ion channels including cystic fibrosis transmembrane conductance regulator, proton pump, calcium pump, etc. multiple rounds of selection were performed using cell protection (supplementary data sheet) and viral load reduction assays, which identified 20 hits that suppressed virus replication for >3 logs at 10 m and another 5 candidates achieving >2 logs inhibition at 1 m (table s1). to prioritize these five compounds, we evaluated their antiviral efficacy against other emerging viruses and identified aca as the only inhibitor that exhibited a broad-spectrum antiviral effect against influenza a h1n1, zikv, hiv-1, sars-cov-2, ev-a71, human adenovirus 5 (adv5), and severe fever with thrombocytopenia syndrome virus (sftsv) (fig. 1c and fig. s2a ). the tool compound aca is a broad-spectrum antiviral in vitro, ex vivo, and in vivo the 50% cytotoxic concentration of aca ranged from 20 to 120 m in different cell lines, while its half maximal effective concentration (ec 50 ) was at or below micromolar levels ( fig. s2a ). aca (10 m) potently suppressed sars-cov-2 replication for >2 logs in both supernatants and cell lysates in caco2 cells (ec 50 = 0.59 m), indicating a good therapeutic potential for the current covid-19 pandemic ( fig. s2b ). because aca displayed the highest selectivity index of 219 against pdmh1n1 infection, aca was tested against different iav subtypes in the subsequent antiviral evaluations. flow cytometry showed that the percentage of pdmh1n1-infected madin-darby canine kidney (mdck) cells after 10 m aca treatment decreased by 83.2% at 24 hours post-infection (hpi) ( fig. 2a ). aca exhibited cross-protection against h5n1, h7n7, h7n9, and h9n2 in a dose-dependent manner (fig. 2b) . notably, aca treatment reduced supernatant viral titer by >4 logs hbtecs (fig. 2c ). using our previously established proximal differentiated threedimensional (3d) human airway organoids (aos) for predicting the infectivity of influenza viruses in humans (14) , we confirmed that aca reduced virus replication by >4 logs (fig. 2d) , with markedly decreased expression of viral nucleoprotein (np) antigen (fig. 2e) . collectively, aca robustly inhibited iavs replication in vitro and ex vivo. to determine aca's toxicity, we intraperitoneally inoculated the maximal phosphate-buffered saline (pbs)-soluble aca (6.75 mg/kg) into balb/c mice for 10 days. no body weight loss or decreased activity was observed for a consecutive monitoring of 14 and 28 days, respectively ( fig. s2c ). to evaluate the in vivo antiviral protection of aca, we challenged mice with 1000 plaque-forming units (pfu) of mouse-adapted h1n1 virus. all mice after a single intranasal (i.n.) dose of aca (0.2 mg/kg) survived (n = 8), whereas all dimethyl sulfoxide (dmso)-treated and zanamivir-treated (2 mg/kg) mice died (fig. 2f ). using the same experimental regimen, aca conferred substantial better survival against avian iav h7n9 (100% versus 0%; fig. 2f ), notably less body weight loss (fig. 2g) , and undetectable lung tissue virus titers at days 2 and 4 after challenge (fig. 2h ). on 4 days post-infection (dpi), histopathologic examination showed substantially less pulmonary alveolar damage and interstitial inflammatory infiltration in aca-treated mice (fig. 2i ). together, aca effectively protected mice challenged by two iav subtypes by reducing virus replication and pneumonia. the broad-spectrum antiviral activity of aca in cell cultures warrants further evaluation in other virus disease models (fig. 3) . type i interferon receptor-deficient a129 mice were infected with zikv-pr (a strain of the zika virus originally isolated from a traveler to puerto rico) and treated with either aca (1 mg/kg) or dmso by subcutaneous administration. mice receiving one dose of aca therapy showed a remarkably better survival rate (100% versus 0%) and mean body weight (fig. 3, a and b) . moreover, zikv titer was undetectable in the brains of aca-treated group, whereas that of dmso group was generally 4 logs higher (fig. 3c ). less histopathologic changes of meningoencephalitis and zikv-ns1 antigen expression were observed (fig. 3d) . furthermore, intranasal aca (0.2 mg/kg, i.n.) provided good protection against lethal challenge with 500 pfu of mers-cov in human dipeptidyl peptidase 4 (hdpp4)transgenic mice, whereas all dmso-treated mice died on or before day 8 after challenge (100% versus 0%; fig. 3e ). body weight loss in the dmso-treated mice began since 5 dpi, while that of the aca-treated group continued to increase (fig. 3f ). about 2 logs lower lung tissue virus titer was detected in the aca group (fig. 3g) . inflammatory infiltration and mers-cov-np antigen expression in the lung tissues were substantially reduced after aca treatment (fig. 3h) . thus, aca also exhibited broad-spectrum antiviral efficacy in vivo. small-molecule compound library screening identified aca as a broad-spectrum antiviral in vitro. a membrane transporter/cellular trafficking library was primarily screened in pdmh1n1-infected madin-darby canine kidney (mdck) cells (0.01 moi and 48 hpi) through cell protection assays (box 1, blue dots indicated >90% cell viability), followed by secondary screening using viral load reduction assays (box 2, magenta dots indicated >3 logs of viral load reduction applying 10 m drug concentration) and tertiary screening (box 3, yellow dots indicated >2 logs of viral load reduction using 1 m drug concentration). aca was prioritized due to its broad-spectrum antiviral efficacy. shown in the last panel are the antiviral effects against six different viruses as indicated. shown are the plaque-forming unit (pfu) or 50% tissue culture infectious dose (tcid 50 ) or od 450 (optical density at 450 nm) value of indicated concentrations relative to controls in the absence of compound (%). aca targets host ap2m1 protein aca is known as a phospholipase a2 (pla2) inhibitor and transient receptor potential (trp) channel blocker (15) , but such mechanisms for suppressing viral replication were excluded ( fig. s3 ). to explore its actual mechanism of antiviral action, we performed time-of-addition assays to investigate how aca interferes with different phases of the viral replication cycle. aca did not inactivate virus particles nor affect virus attachment to host cell surface ( fig. s4 ). to dissect the postvirus attachment steps, we quantified three types of influenza virus rna [vrna, complementary rna (crna), and mrna] at different time points after virus internalization. distinct dynamics of vrna, crna, and mrna synthesis were observed in the control groups, while all viral rna types in aca groups remained at the baseline level within 0 to 5 hpi of aca addition (fig. 4a) . the result suggested that aca functions within 1.5 hours after internalization before crna synthesis and therefore may inhibit virus endocytosis and nuclear import or blocking viral ribonucleoprotein (vrnps) activity directly. because lysotracker red assay indicated that the acidification of endosomal compartments was not affected, the uncoating of vrna for release into cytoplasm upon virus fusion was unlikely to be blocked by aca ( fig. s3c ). hence, we directly tracked the location of the vrna within the incoming vrnps ( fig. 4b ). at indicated time points, cells were processed and stained for the negative-stranded np vrna of pr8 using a specific rna probe set (red). in the dmso-treated cells, the dominant nuclear import of vrna was detected by 1 hpi, followed by predominant cytosol location between 1.5 and 2 hpi. by comparison, the nuclear vrna signal was rarely observed after aca addition throughout the time course. therefore, aca may inhibit iav replication by blocking vrna nuclear import. to ascertain the target of aca, we developed an unbiased drug target-elucidating platform that integrates click-chemistry, waterlogsy, and protein id techniques using liquid chromatographytandem mass spectrometry (lc-ms/ms), namely, cwid (fig. 4 , c and d). first, click-chemistry was applied to introduce an azido group to aca (azido-aca), without compromising its antiviral efficacy (fig. 4c, middle) . with an azido-reactive fluorescent dye (dylight 488), azido-aca but not aca was visualized in both the cytosol and nucleus (fig. 4c , right). next, waterlogsy, a ligandbinding determination method through observing the nuclear overhauser effect, was applied for primary nuclear magnetic resonance (nmr) screening of cellular fragments that exhibited high binding affinity to aca. virus-infected mdck cells were fractionated by gel filtration chromatography, followed by waterlogsy to capture the aca-interacting signals, individually (fig. 4d , middle). iterative rounds of subfractionation and waterlogsy were performed to obtain the maximally separated fraction with detectable aca signals. subsequently, the bound form of fraction-azido-aca mixture was stained by dylight 488 and subject to electrophoresis in a native polyacrylamide gel electrophoresis. a specific band indicating the (c) five mice in each group were euthanized at 6 dpi, and brain tissues were harvested for vial titer determination by plaque assay. the dotted line indicates the lower detection limit of plaque assay (***p < 0.001; for the purpose of statistical analysis and clarity, a value of 10 to 15 pfu/ml was assigned for any titer below the detection limit). (d) histopathologic and immunohistochemistry (ihc) analyses of the brain samples at 6 dpi indicated less severe meningitis (by h&e, ×200) and less virus infected cells as indicated by zikv ns1 antigen staining (red arrows, by ihc, ×200 magnification) after aca treatment. (e to h) aca protected human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection. the hdpp4 mice were intranasally inoculated with 500 pfu of mers-cov and intranasally treated by aca or 0.5% dmso for one dose starting 1 hpi. shown are (e) survival rate, (f) mean body weight, (g) lung viral titer at 2 dpi (n = 5 per group), and (h) representative lung tissues stained by h&e and anti-mers-cov-np immunofluorescence. the staining suggested less inflammatory cell filtration (by h&e, ×200 magnification) and less virus infected cell antigens (by immunofluorescence (if) staining, green fluorescence) as detected in aca-treated mouse lungs. results are presented as mean values ± sd. differences in survival rates were compared using log-rank (mantel-cox) tests and viral titer by student's t test. ***p < 0.001, **p < 0.01, *p < 0.05. cells were fixed at the indicated time points and hybridized with rna probes against the iav negative-stranded np vrna (red) and stained for dna (blue), examined by confocal microscopy. images are representative of three independent experiments. scale bars, 10 m. (c and d) click chemistry/waterlogsy/protein id (cwid) platform for identification of drug-binding targets. (c) click-chemistry: chemical structure of azido-aca showing the location of azido group (green circle) on aca. cellular distribution of azido-aca is shown (green), whereas aca was used as a negative control due to the lack of phosphine-reactive azido group. scale bars, 50 m. (d) waterlogsy-guided cellular fractionation was subjected to analysis for aca-featured nmr spectra. "*" and "***" indicate mild and strong binding signals, respectively. the native polyacrylamide gel electrophoresis gel photo shows the selected cell fraction as detected by a fluorescent image analyzer. red arrow indicates the specific azido-aca-binding fragment. (e) mutagenesis analysis of ap2m1 to rescue pdmh1n1 virus replication against aca. full-length ap2m1 (full), longin-like domain (lld), mhd, and mutant ap2m1 were transfected to mdck cells before virus infection and aca treatment. oneway anova. **p < 0.01; n.s, not significant. (f) partial sequence alignment of human, mouse, and dog ap2m1 is shown. n217 and k410, the key residues for aca binding, are highlighted with a box. the predicted interaction surfaces on ap2m1 (red) are shown, while aca (green) is displayed in stick and mesh representation. protein-compound complex was visualized in the azido-aca group, while it was absent in the aca or cell lysate-only group (fig. 4d , right). gel plug of the target band was collected for lc-ms/ms, which identified eight candidate proteins that were physically associated with azido-aca. to validate their biological function besides binding, individual open reading frame (orf) clone was overexpressed to overcome the inhibition of virus replication by aca. apparently, only ectopic expression of ap2m1 notably rescued pdmh1n1 growth despite the presence of aca, indicating ap2m1 being one of the most likely targets that accounts for aca's mode of action ( fig. s5 ). ap2m1 consists of an n-terminal (~150 amino acids) longin-like domain (lld) and a c-terminal (170 to 434 amino acids) mu homology domain (mhd). functionally, we demonstrated that overexpression of either full-length ap2m1 or mhd enhanced pdmh1n1 replication for about 1.5 logs despite adding aca, whereas overexpressed lld did not antagonize aca's antiviral activity (fig. 4e , left bar charts). thus, mhd harbors sites of aca interaction. amino acid residues of mhd are conserved across the human, dog, and mouse ap2m1, which is in line with the broad-spectrum antiviral coverage of aca in different species of cells and mouse models. molecular docking predicts that aca interacts mainly with ap2m1 through four amino acids, m216, n217, k400, and k410 (fig. 4f ). our mutational experiment showed that substitution in n217a or k410a failed to antagonize the aca's antiviral activity when compared with that of the m216a, k400a, or wild-type (wt) ap2m1 (fig. 4e , right bar charts). collectively, aca targets host ap2m1 by interacting with its n217 and k410 residues. the antiviral spectrum of aca spans across enveloped (zikv) and nonenveloped (ev-a71), retro (hiv-1), and nonretro (iav), as well as dna (adv-5) and rna (mers-cov) viruses ( fig. s2a ). thus, ap2m1 must be broadly exploited during life cycles of many viruses. first, we excluded the possibility that aca affected phosphorylation of ap2m1 ( fig. s6 ), which has been approved to be antiviral effective by bekerman et al. (16) . subsequently, to find the specific virus protein interacting with host ap2m1, we performed an immunoprecipitation (ip) screening of viral orf clones. previous host-iav interactome suggested m1, hemagglutinin (ha), pb2, and np as the potential ap2m1-binding proteins without individual validation (17) . after coexpression of each viral gene and ha-tagged ap2m1 in human embryonic kidney (hek) 293t cells, only np could be detected in the ip complex, but adding aca markedly diminished the target np band ( fig. s7a ). using the same approach, we identified the zikv-ns3, mers-cov-np, and ev-a71-2c as the interacting partners of ap2m1 ( fig. s7 , b to d). ap2m1 is known to mediate sorting of cargo proteins harboring yxxø or dileucine motifs (18) . using bioinformatic methods, we found that yxxø but not dileucine motif was consistently present in the implicated ap2m1-binding viral proteins. the yxxø motifs are highly conserved in specific proteins across many virus families including the np of orthomyxoviridae, gag of retroviridae, np of bunyaviridae, ns3 of flaviviridae, np of coronaviridae, 2c of picornaviridae, and core protein v of adenoviridae by bioinformatics (fig. 5a ). to determine whether aca blocked the ap2m1 yxxøbinding site, we developed a competitive enzyme-linked immunosorbent assay (elisa). as positive controls, addition of either nonlabeled yxxø motif peptide dyqrln or the low-affinity mutant d176a ap2m1 (19) resulted in diminished binding signal during binding of biotin-yxxø substrate to the immobilized his-ap2m1. aca pretreatment notably reduced ap2m1/biotin-yxxø interaction (fig. 5b ). to explore whether the yxxø-binding pocket is a druggable target for antiviral therapy, we also tested tyrphostin a23, which blocks the tyrosine-binding pocket of ap2m1 (20) . at nontoxic concentrations, tyrphostin a23 suppressed a panel of viruses including pdmh1n1, mers-cov, ev-a71, and zikv (fig. 5c) . last, the effect of ap2m1 gene depletion on viral replication was investigated. ap2m1 knockout led to dramatic viral load reduction in pdmh1n1-infected 293t cells, ev-a71-infected rhabdomyosarcoma (rd) cells, zikvinfected huh7 cells, and mers-cov-infected huh7 cells (fig. 5d) . together, ap2m1/yxxø interaction is a critical step in the viral replication cycle and druggable for antiviral intervention. next, we asked the functional roles of ap2m1/yxxø interaction during viral replication cycles using iav, ev-a71, and zikv-ns3 as three representative viruses. nuclear import of iav np through the nuclear pore complex is a prerequisite for efficient vrnp translocation and subsequent genome replication, while our data clearly showed that aca impaired vrna nuclear import and ap2m1/np binding ( fig. 4b and fig. s7a ). thus, we postulated that ap2m1 facilitated np import from cytoplasm to the nucleus via recruiting the np-yxxø motif. to quantify the retardation of np import in ap2m1 −/− 293t cells, cells were infected with 10 moi iav, and cycloheximide (chx) was added to inhibit protein synthesis. crude cell lysate was separated into nuclear (nuc) and cytoplasmic (cyto) fractions at 2 hpi (fig. 5e ). in wt cells, ap2m1 protein was mainly detected in cytoplasm, whereas considerably more viral nps were found in nucleus. however, in ap2m1 −/− cells, viral np was predominantly found in the cytoplasmic fraction instead. since the only source of np protein was from the incoming vrnps after chx treatment, the finding suggested that ap2m1 facilitates the nuclear import of incoming vrnps. to provide direct evidence for a role of ap2m1 in mediating intracellular np trafficking that beyond endocytosis, we monitored the cotrafficking of np-green fluorescent protein (gfp) with ap2m1-mcherry using live cell imaging. gfp/mcherry signal was found largely in the host nucleus (movie s1). upon aca treatment, np resides predominantly in the cytosol, and the mobility of the ap2m1-associated np puncta (yellow) was reduced remarkably, which suggested diminished np trafficking via ap2m1 (fig. 5f and movie s2). nevertheless, most positive-stranded rna viruses replicate their genomes on the cytoplasmic endoplasmic reticulum (er) membrane without entering the nucleus. for example, ev-a71-2c and zikv-ns3 proteins induce the formation of viral rna replication complex by trafficking predominantly to the er membrane (21) . in the context of synchronized viral infection, we demonstrated by confocal imaging the extensive localization of iav-np and nucleus, so were the ev-a71-2c and zikv-ns3 in er, respectively. after aca treatment, however, reduced rates of colocalization were revealed in all three representative viruses (89% versus 12% for iav-np/nucleus; 63% versus 21% for ev-a71-2c/er; 95% versus 56% for zikv-ns3/er; fig. 5g ). in summary, these results confirmed an important role of ap2m1 in trafficking viral proteins beyond endocytosis. to bridge the ap2m1-mediated trafficking and virus replication, we rescued the recombinant iav with a series of point mutations on two np-yxxø locations, i.e., y296-v299 (yslv) and y385-i388 (e) ap2m1 −/− and wt 293t cells were treated with chx before virus infection (10 moi). nuclear (nuc) and cytoplasmic (cyto) fractions were separated and detected at 2 hpi by western blotting. (f) a549 cells transfected with gfp influenza-np and mcherry-ap2m1 were incubated with dmso or aca for 24 hours. live cell imaging was performed, and motile ap2m1/np puncta were tracked (movies s1 and s2). shown is the average velocity of trackable puncta within the overall distance traveled. student's t test. (g) ap2m1 facilitates the viral protein localization. synchronized infections were used throughout the experiments. colocalization was quantified using imagej (jacop) colocalization software and manders' colocalization coefficients (mccs). bar charts indicate mean mcc values represented as percent colocalization (the fraction of green intensity that coincides with blue intensity in the case of iav-np/nucleus and the fraction of green intensity that coincides with red intensity in the case of ev-a71-2c/er and zikv-ns3/er) ± sd (error bars, n = 10 to 15). scale bars, 10 m. ***p < 0.001 by student's t test. (ywai) (fig. s8 ). mutagenesis includes two y/a substitutions (y296a and y385a), two ø/a substitutions (v299a and i388a), and two ø/l substitutions (v299l and i388l). the growth rates of the y296a, y385a, and ø299a mutant viruses were attenuated for >1-log in each time point, while ø388l mutant virus exhibited similar replication kinetics as that of the wt (fig. 6a) . the results not only indicated the critical role of np-yxxø in determining the virus fitness but also that yxxø is functionally interchangeable within homologous ø signals (i.e., i388l substitution). however, we were unable to rescue the recombinant viruses containing ø299l or ø388a mutation for three independent experiments. to validate whether the reduction of iav replication was due to the decreased np nucleus entry, a step which is beyond ap2m1-mediated endocytosis, the successfully rescued wt and mutant iav were used to infect a549 cells, followed by measuring np in both the cytosol and nucleus at 2 hpi. apparently, np was not detectable in the host nucleus after infection of iavs carrying y296a, y385a, and ø299a np substitutions, while ø388l mutant virus exhibited similar amount of np as that of the wt (fig. 6b ). as expected, influenza polymerase luciferase reporter activity from both ø/l mutants (ø299l and ø388l) was similar to that of wt, whereas all y/a (y296a and y385a) and ø/a (ø299a and ø388a) mutants displayed reduced polymerase activity. moreover, ap2m1 gene depletion reduced the polymerase activity by >75% (fig. 6c) . the results suggested that amino acid residues of np-yxxø motif determined the iav polymerase activity via modulating the np nuclear import. furthermore, we extended the analysis in vivo and selected the most attenuated y296a substitution for a full comparison with the wt. balb/c mice were challenged with two doses of wt-h5n6-gfp (wt) and y296a-h5n6-gfp (y296a) viruses, respectively. survival of y296a-challenged (10 5 pfu) group (100% versus 0% versus 60%) was obviously better than those of wt-challenged (10 5 pfu) group and wt-infected (10 4 pfu) group (fig. 6d) . mice in the 10 5 pfu y296a group displayed similar weight loss to those of the 10 4 pfu wt-infected group but rebounded after 7 dpi, whereas the 10 4 pfu y296a group displayed mild body weight loss (<5%) throughout the infection (fig. 6e ). taking advantage of the gfp reporter feature of the recombinant iav for in vivo dynamic analysis, we examined lung and brain samples from infected mice at different dpi. apparently, spreading of wt virus deeper into the bronchioles and possibly alveoli were detected since 3 dpi, while gfp signal from y296a mutant virus was confined to regions around the initial sites of infection around the trachea and bronchi, indicating limited virus spread (fig. 6f, left) . in line with the reported occurrence of neurological symptoms in highly pathogenic h5-infected animals, extensive gfp signals could be visualized in wt virus-infected mouse brain as early as 3 dpi. in contrast, the y296a group illustrated reduced gfp intensity throughout the time points (fig. 6f, right) . these data provided evidence that a single y296a substitution in np-yxxø motif notably restricted iav replication in vivo. together, host ap2m1/virus yxxø interaction is critical for intracellular virus trafficking to the sites of replication/ transcription beyond the step of endocytosis, thereby facilitating viral replication (fig. 6g) . viruses exploit distinct receptors to facilitate cell entry and to evade a hostile extracellular environment that would otherwise abrogate infection. within the intracellular settings, we demonstrated a conserved host ap2m1/virus yxxø interaction that is commonly har-nessed by different viruses during intracellular trafficking but beyond the well-defined cme process (fig. 6g) . using aca as a tool compound, we developed the cwid platform and identified the yxxøbinding pocket of the host ap2m1 as a previously unidentified target for broad-spectrum antiviral development, which is different from the previous antiviral strategy to block ap2m1 phosphorylation ( fig. s6 ). although the ap2m1 −/− mice is lethal (22) , the ap2m1 −/− cell line is not susceptible to the infections of iav, ev-a71, mers-cov, and zikv (fig. 5d ). on the virus side, yxxø motif determines the capacity of np nuclear translocation and therefore affects iav fitness (fig. 6, a to f) . the outcome of a viral infection with respect to cellular fate is a fundamental aspect of viral biology. we find that tgf- signaling is a cell fate determinant pathway with broad relevance of multiple viruses, and these viruses use ap2m1 as a common intermediate for intracellular trafficking but beyond endocytosis: first, by using live iav infection, vrna was visualized in the perinuclear region but within the cytosol, indicating that the iav entry process has not been affected by aca (fig. 4b) ; after endocytosis, however, np were predominantly excluded from the nucleus of ap2m1 −/− cells, suggesting that ap2m1 was indispensable for iav-np nuclear localization (fig. 5e ). the role of ap2m1 associated with hepatitis c virus (hcv) entry and assembly has been defined (23, 24) . our study further demonstrated the versatility and broadness of ap2m1 to cotraffic with several other internal viral proteins for the completion of their replication cycles. represented by iav (enveloped and negativestranded), ev-a71 (nonenveloped), and zikv (enveloped and positivestranded), we demonstrated that recruitment of yxxø-harboring iav-np, ev-a71-2c, and zikv-ns3 proteins by ap2m1 was functionally related in their correct localization as to facilitate viral genome replication. strategically, ap2m1 was harnessed by viral np for efficient nuclear entry, thereby promoting polymerase activity. substitutions introduced in the viral yxxø motif as y to a or ø to a greatly diminished virus growth in vitro and in vivo. since the virulence of np y296a mutant virus was attenuated, strategic usage of iav strains containing this or other mutants as vaccines might be evaluated in the future. in the case of ev-a71 and zikv, ap2m1 was required for efficient transportation of 2c and ns3 proteins to the er membrane so that their genome replication can occur. furthermore, the essentiality of ap2m1 during virus replication was validated in the context of multiple viruses including pdmh1n1, ev-a71, zikv, and mers-cov infections (fig. 5d) . together, ap2m1 might be universally exploited by different viruses to complete their replication cycle after cell entry. the architecture of ap2 has to undergo a large conformational change from a "closed," cargo-inaccessible state to an "open" structure so as to expose the yxxø binding site, which is regulated by ap2associated protein kinase 1 and cyclin g-associated kinase (7) . although it has been reported that inhibitors of this kinase (e.g., sunitinib and erlotinib) can inhibit rna viruses including dengue, ebola, and hcv, the in vivo antiviral potency of aca (100% survival in iav, zikv, and mers-cov mouse models) is much better than that of sunitinib [37% protection in dengue virus (denv) and 30% in ebola virus (ebov) mouse models] or erlotinib (conferring no protection in either the denv or ebov mouse model) (16) . targeting directly the yxxø-binding pocket instead of the t156 ap2m1 phosphorylation site, our study not only extends the therapeutic window beyond the conformational change of ap2m1, which is transient and mediated by kinase activity, but also opens up another synergistic antiviral approach by combining aca with other inhibitors including sunitinib or erlotinib. a major roadblock to translating protein kinase inhibitors into clinical development is the doubt about their poor selectivity, which is largely a consequence of the highly conserved atp-binding site shared by all protein kinases (25) . disruption of ppis, as exemplified in our study, however, is usually highly specific against the binding interface, which has less concern about cytotoxicity. occupation of ap2m1 yxxø-binding pocket by using tyrphostin a23 blocked the replication of multiple viruses (fig. 5c) . because aca interacts with n217 and k410 that lie outside the yxxø-binding cavity formed by residues f174, d176, k203, and r423 (26) , an allosteric activation mechanism of ap2 may exist (fig. 4e) . besides ap subunit genes (ap1m1, ap2a1, ap2m1, and ap3s2), several syntaxin-relevant genes (stx5, stx10, and stxbp2) were identified to play a proviral role (fig. 1b and fig. s1 ). four pharmacological inhibitors including imperatorin, pyr6, zd7288, and ethosuximide exhibited potent anti-influenza activity with an ec 99 at nanomolar range (fig. 1c) . further characterization of their underlying mechanisms is warranted. establishment of the cwid platform enables identification of drug-binding target(s) in an unbiased manner, which addresses a common difficulty that challenges all phenotypic forward chemical screening (fig. 4, c and d) . this platform may be adopted for studies using host-targeting strategies to accelerate the progress of drug target discovery. overall, we demonstrate that the ap2m1/yxxø interaction is a druggable target for broad-spectrum antiviral therapy, and virus yxxø mutant could be tested as attenuated vaccines. these approaches may provide additive and possibly synergistic antiviral effects when aca or its analogs are combined with other antiviral agents for tackling emerging viral infections. the main goal of this study was to identify a next generation of broad-spectrum antiviral with elucidated machinery. first, we comprehensively evaluated the antiviral potency of the selected drug aca in cell cultures, ex vivo and in vivo models. second, we established an integrative platform, named cwid, to identify the host ap2m1/ yxxø interaction as the aca drug target and in an unbiased manner. third, we investigated the biological importance of ap2m1/yxxø interaction in the viral replication cycle using three representative viruses. last, we characterized the effect of virulence of the substitutions in influenza a np yxxø motif. hbtecs were cultured with airway epithelial cell basal medium according to the manufacturer's protocol. human-induced pluripotent stem cell-derived neural progenitor cells (npcs) were cultured and differentiated according to a previous protocol (27) . human lung fibroblast hfl1 was cultured in f-12k medium. human t lymphoblast molt-4 ccr5 + cells were cultured in rpmi 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (fbs) and g418 (1 mg/ml). human embryonic kidney (hek) 293t cells, human lung carcinoma (a549) cells, human hepatoma (huh7) cells, human rd cells, human epithelial type 2 cells, mdck cells, and african green monkey kidney (vero) cells were maintained in dulbecco's modified eagle's medium (dmem) medium. all culture medium was supplemented with 10% heat-inactivated fbs, penicillin (50 u/ml), and streptomycin (50 g/ml). all cells were confirmed to be free of mycoplasma contamination by the plasmo test (invivogen). the iav strains a/hong kong/415742/2009(h1n1)pdm09, a/anhui/1/ 2013(h7n9), a/vietnam/1194/2004(h5n1), a/netherlands/219/ 2003(h7n7), and a/hk/1073/1999 (h9n2) were cultured in embryonated chicken eggs. the sars-cov-2 hku-001a was isolated from the nasopharyngeal aspirate specimen of a laboratoryconfirmed covid-19 patient in hong kong (28) . the mers-cov (hcov-emc/2012, a gift from r. fouchier) and sars-cov (gz50) were propagated in vero-e6 cells. the hiv-1 jr-fl virus (#4984, national institutes of health aids) was propagated in molt-4 ccr5 + cells in the presence of interleukin-2 (1 ng/ml) for 14 days as we previously described (29) . enterovirus a-71 (sz/hk08-5) was cultured in rd cells. the zikv (puerto rico strain prvabc59, a gift from b. russell and b. johnson, cdc, usa) was amplified in vero cells. a clinical isolate of human adv5 was propagated in a549 cells. the sftsv hb29 strain (a gift from m. liang, cdc, china) was propagated in vero cells. all cultured viruses were titrated by pfu assays and/or 50% tissue culture infectious dose (tcid 50 ) assay and/or quantitative reverse transcription polymerase chain reaction (rt-qpcr) assays and/or p24 elisa as indicated. all virus stocks were kept at −80°c in aliquots. all experiments with live viruses were conducted using biosafety level 2 or 3 facility as we previously described (30) . wnt signaling reporter containing t cell factor/lymphoid enhancerbinding factor was a gift from r. moon (addgene plasmid #12456). tgf- signaling reporter containing four copies of the smad binding site was shared by b. vogelstein (addgene plasmid #16495). notch signaling reporter containing centromere-binding protein 1 (cbf1)responsive element was obtained from n. gaiano (addgene plasmid #26897). pi3k/akt signaling reporter containing forkheadresponsive element was a gift from m. greenberg (addgene plasmid #1789). all primer sequences used are provided in table s2. aca was purchased from cayman chemical (michigan, usa). other chemical inhibitors were obtained from sigma-aldrich (missouri, usa) unless specified. all peptides were synthesized from cellmano biotech (hefei, china) with >95% purity. the phosphine-activated fluorescent dye dylight 488-phosphine (invitrogen) was used for specific labeling and detection of azido-tagged molecules, i.e., azido-aca. the 4′,6-diamidino-2-phenylindole (dapi; sigma-aldrich), calnexin polyclonal ab (abbkine, abp0065), and phalloidin-atto 647n (sigma-aldrich) were used for nuclear, er, and cell membrane staining, respectively. primary antibodies against human ap2m1 (abcam, ab75995), anti--actin (abcam, ab8227), anti-influenza np (abcam, ab104870), anti-zikv ns3 (genetex, gtx133309), anti-ev-a71 2c (genetex, gtx132354), anti-his-tag (invitrogen, ma1-21315), anti-ha-tag (abbkine, a02040), anti-flag-tag (sigma-aldrich, f3165), anti-lamin a (abcam, ab26300), anti-glyceraldehyde-3-phosphate dehydrogenase (gapdh; abcam, ab8245), and anti-mers-cov np mouse serum (in house) were used in relevant experiments. alexa fluor 488 goat anti-mouse immunoglobulin g (h + l) antibody (1:500; invitrogen, a28175) and alexa fluor 594 goat-rabbit (1:500; invitrogen, a27016) were used as secondary antibodies for immunofluorescence staining. individual smartpool sirna targeting ap2m1, trpm2, trpm8, or pla2g2a was purchased from dharmacon (lafayette, usa). ne-per nuclear and cytoplasmic extraction reagents (thermo fisher scientific) was used according to the manufacturer's protocol. an ap2m1 human gene knockout kit (origene, kn401377) was used to establish the crispr knockout cell lines according to the manufacturer's protocol. immunofluorescence rna was visualized using the viewrna cell plus assay kit (invitrogen, 88-19000) containing a viewrna type 1 probe set designed against the negative stranded rna np genome (vrna) of influenza a h1n1 pr8 virus (invitrogen, vf1-10583). to identify host genes essential for h1n1pdm replication, an rna interference (rnai)-based screen was performed using a commercially available library targeting 142 cellular membrane-trafficking genes (ambion silencer, a30138). briefly, 1.5 × 10 4 per well of a549 cells were seeded in 96-well plates overnight, followed by sirna transfection once daily for two consecutive days using the lipofectamine rnaimax reagent. at 48 hours after primary sirna transfection, a549 cells were infected with 0.1 moi virus. one hour later, the infectious inoculum was aspirated and replaced with fresh dmem medium containing 2% bsa and n-tosyl-l-phenylalanine chloromethyl ketone (tpck)-treated trypsin (2 g/ml). the cell culture supernatant of individual well was collected after another 48 hours for viral load titrated by the rt-qpcr method. before virus infection, wells exhibited poor cell viability (<80%) after gene silencing were excluded. a small-molecule compound library with 420 candidates (medchemexpress), targeting membrane transporters and ion channels, was used for pharmacological screening with antiviral activity. the primary screening of pdmh1n1 inhibitors was cpe inhibition based as we previously established (30) . viability of mdck cells after 0.01 moi virus infection and compound treatment (10 m) were determined at 48 hpi using the celltiter-glo luminescent cell viability kit (promega). secondary screening was performed with viral load reduction assay. briefly, the cell culture supernatant at 24 hpi and compound treatment (10 and 1 m, respectively) were collected and applied for viral copy quantification by rt-qpcr methods. favipiravir (50 g/ml) was used as a positive control throughout the screening process. waterlogsy, a ligand-observed nmr technique, was used to screen for aca-interactive cellular fractions (31) . to fractionate the pdmh1n1-infected cell lysate, 1 ml of mdck cells (10 7 cells, 1 moi, 6 hpi) was ultrasonicated three times for 10 s on ice and then centrifuged. the clarified supernatant was applied for fast protein liquid chromatography (äktaexplorer, ge healthcare) using the 320-ml hiload 26/600 superdex 200 preparative size exclusion chromatography column to harvest each protein fraction with ultraviolet 260-nm signals. aca waterlogsy experiments were conducted on a 600-mhz bruker avance spectrometer using a 5-mm pasei probe. the pulse scheme used for waterlogsy experiment was "ephogsygpno.2" with water suppression using excitation sculpting with gradients (32) . all experiments were conducted at 298 k using 5-mm-diameter nmr tubes with a sample volume of 500 l and 17 m aca supplemented and recorded using 4 k scans. all samples were dissolved in 95% h 2 o and 5% d 2 o with final concentration of 70 m trimethylsilylpropanoic acid as internal standard. control spectrum was recorded under the same conditions without cellular fraction to confirm the absence of self-aggregated aca macromolecules. to identify the aca-binding protein target (protein ids), the cellular fraction was lyophilized before resuspended with 10 l of h 2 o and incubated with 70 m azido-aca for 1 hour. the mixtures were further incubated with 100 m dylight 488-phosphine for 3 hours to allow linkage of fluorescent dye with the azido group, before loading to a 10% nondenaturing native gel for electrophoresis. subsequently, a typhoon fla 9500 laser scanner using alexafluor 488 filter was used to visualize the fluorescent bands that were associated with azido-aca other than aca. the photomultiplier tube voltage was set as 300 to 350 v and the resolution as 50 m. target bands were excised and subjected to lc-electrospray ionization ms/ ms analysis by q exactive as previously established (shanghai applied protein technology co. ltd.) (33) . samples with or without aca were also incubated with dylight 488-phosphine to act as controls. to validate the target protein essential for aca-dependent mode of action, eight protein ids as revealed by the cwid platform were overexpressed individually and screened for their capacity to antagonize the aca's antiviral activity. orf clone of each protein was obtained from mission trc3 human orf collection (merck). in a 24-well plate, mdck cells were transfected with 500 ng of each plasmid and incubated for 48 hours before pdmh1n1 infection. cell culture supernatants of the infected cells, with or without aca (5 m), were collected for viral titer determination by standard plaque assay. under the protocol approved by the institutional review board (uw 13-364) of university of hong kong/hospital authority hong kong west cluster, normal human lung tissue from a patient was obtained surgically. informed consent was obtained from the human participant, and the experiments were performed in compliance with the approved standard operating procedures. anti-influenza activity of aca was also evaluated in 3d human aos, as we previously reported (14) . briefly, the 3d aos were sheared mechanically to expose the apical surface to the virus inoculum. the sheared organoids were then incubated with viruses at a moi of 0.01 for 2 hours at 37°c. after washing, the inoculated organoids were re-embedded in matrigel and cultured in the medium containing aca (10 m) or dmso (0.1%). at the indicated times, aos were harvested for the quantification of intracellular viral load or fixed for immunofluorescence staining. balb/c mice, hdpp4 transgenic c57bl/6 mice, and interferon receptor / knockout (ifnar −/− ) a129 mice were kept in biosafety level 2 or 3 housing and given access to standard pellet feed and water ad libitum, as we previously described (34) (35) (36) . all experimental protocols were approved by the animal ethics committee (culatar 4057-16, 4371-17, 4511-17) in the university of hong kong and were performed according to the standard operating procedures of the biosafety level 2 or 3 animal facilities. to evaluate the cross-subtype anti-influenza virus efficacy of aca in vivo, balb/c mice (20 mice per group) were inoculated intranasally with 200 pfu of influenza a h7n9 virus or 1000 pfu of mouse-adapted influenza a h1n1 virus in 20-l pbs. treatment was performed 1 hour after challenge by intranasal administration. one group of mice was inoculated with 20 l of aca (0.2 mg/kg). a second group was treated with 20 l of intranasal zanamivir (2 mg/kg) (30) . a third group was given intranasally 0.5% dmso in pbs as an untreated control. animal survival and clinical disease were monitored for 14 days or until death. lung tissues (five mice per group) were collected for viral load detection and hematoxylin and eosin (h&e) histopathologic analyses on days 2 and 4 after challenge, respectively. to evaluate the anti-zikv efficacy of aca in vivo, 4-to 6-weekold a129 mice were randomly divided into two groups to receive aca treatment or sham treatment through the subcutaneous route (n = 13 per group). the mice were inoculated subcutaneously with 1 × 10 6 pfu (in 100-l pbs) of zikv-pr under anesthesia. each mouse was then received one dose of subcutaneous-administered aca (1 mg/kg) or 0.5% dmso in pbs at 1 hpi. the mice were monitored daily for body weight change and clinical signs of disease. five mice in each group were euthanized at 6 dpi, and brain tissues were harvested for vial titers and histopathologic and immunohistochemistry analyses. the survival of the other mice was monitored until 14 dpi. to examine the anti-mers-cov activity of aca, a total of 26 mice (n = 13 per group) were evaluated. after anesthesia, mice were intranasally inoculated with 20 l of virus suspension containing 500 pfu of mers-cov. intranasal therapeutic treatments were initiated at 1 hpi. one group of mice was inoculated with 20 l of aca (0.2 mg/kg). the other group was given intranasally 0.5% dmso in pbs as an untreated control. animal survival and clinical disease were monitored for 14 days or until death. five mice in each group were euthanized randomly on 2 dpi, respectively. mouse lungs were collected for virus titration and h&e histopathologic and immunofluorescence staining. the whole-organ lungs and brains of the gfp virus-infected mice were excised at the indicated time after challenge. after fixation in 4% paraformaldehyde for overnight, the images were acquired in a pe ivis spectrum in vivo imaging system fitted with gfp excitation/ emission filters. live imaging was used to visualize the cotrafficking of ap2m1 and influenza a np according to a previous report with some modifications (37) . time-lapse images were taken using the nikon ti2-e widefield microscope with a 100× 1.46 oil objective in a heated (37°c) chamber. gfp-labeled np protein and mcherry-fused ap2m1 protein were tracked by sequential imaging every 2 s with 50-and 200-ms exposures for each channel, respectively. individual colocalized puncta run lengths and transport velocities were calculated using the track points for metamorph analysis software, measuring the distance traveled (in any direction) between frames for each respective puncta. the movies were made using the stack function for metamorph analysis software via accumulating the relative frames in order. the recombinant influenza virus carrying a gfp reporter gene in the ns segment (ns1-gfp virus) was rescued using standard reverse genetics techniques (38) . the 8-plasmid system, with a/duck/ hubei/wh18/2015(h5n6) background, was a gift from j. meilin (huazhong agricultural university, china). np constructs harboring yxxø mutations, i.e., phwnp-y296a, phwnp-v299a, phwnp-v299l, phwnp-y385a, phwnp-i388a, and phwnp-i388l, were performed by site-directed mutagenesis (stratagene) based on the wt phwnp plasmid. titers of viral stocks were determined by plaque assay on mdck cells. luciferase reporter plasmids reflecting the up-or down-regulation of cell fate determination pathways were used. experimentally, individual reporter plasmid (100 ng), together with a transfection efficiency control plasmid (pnl1.1.tk, promega) construct (5 ng), was cotransfected into the indicated cells for 24 hours. subsequently, 10 moi of each virus was used to infect the transfected cells, followed by luminance detection at 0, 1, 3, 6, or 12 hpi according to the manufacturer's protocol (dual-luciferase reporter assay system, promega). the transfected cell with mock infection was taken as a baseline control for normalization. influenza a virus mini-genome reporter assays were performed as described previously with some modifications (39) . rnp complexes composed of pa, pb1, pb2, and np or their mutants were mixed with a luciferase reporter plasmid (50 ng each) and pnl1.1.tk construct (5 ng) and then cotransfected into hek293t cells. luminescence was determined at 24 hours after transfection. the assay was designed to detect ap2m1 and yxxø binding. free or biotin-labeled yxxø motif, with a peptide sequence of dyqrln, was synthesized. to expose the ap2m1 binding site, calyculin a, an inhibitor of ap2m1 dephosphorylation which "locks" ap2m1 in its yxxø binding active conformation, was added according to a previous report (24) . hek293t cells in six-well plates were transfected with his-ap2m1 or its low binding affinity mutant d176a for 48 hours. next, 10 g per well transfected cell lysate containing the overexpressed proteins was incubated with the ni-nta hissorb 96-well plate (qiagen) for overnight at 4°c. after washing, drugs were added 1 hour before incubation, followed by input of biotin-yxxø probe (10 g/ml) and detection of binding signal using horseradish peroxidase-conjugated streptavidin (thermo fisher scientific, n100) and trimethylboron substrate (thermo fisher scientific, n301). in this experiment, the unlabeled peptide dyqrln was used as a positive control binding inhibitor, while mock-transfected cell lysate was taken as a background control. the washing and dilution buffer consisting 100 nm calyculin a, pbs, and 0.1% tween-20 was used throughout the assay. the aca (pubchem cid: 5353376) 3d structure was downloaded from pubchem database. leadfinder version 1804 was used to perform ligand-receptor docking (40) . extra precision mode (-xp) was applied to search the ligand conformational space more thoroughly. energy grid map was generated according to the binding pose of yxxø motif in the ap2m1/yxxø complex structure (protein data bank code: 2xa7) (7). default grid map spacing of 0.375a was set for a good trade-off between accuracy and performance. bond orders were assigned, hydrogens were added, and cap termini were included with the protein preparation wizard module as implemented in maestro. protonation states of side chains were predicted using propka3.1 server. partial charges over all atoms were assigned within the amber99 force field scheme as implemented in ambertools. the top-ranked pose was visualized by using pymol, while 2d intermolecular interaction was visualized with ligplot+. data were analyzed using graphpad prism 7 (graphpad software, san diego, ca, usa). the values shown in the graphs are presented as means ± sd of at least three independent experiments. statistical differences between groups were analyzed using a one-way analysis of variance (anova) statistical test with dunnett's multiple comparisons tests or two-tailed unpaired t tests. colocalization rate was quantified using imagej (jacop) colocalization software and manders' colocalization coefficients (mccs) as previously described (23) . p < 0.05 was considered statistically significant. supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/sciadv.aba7910/dc1 view/request a protocol for this paper from bio-protocol. evolutionary conflicts between viruses and restriction factors shape immunity middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease zika virus the emergence of influenza a h7n9 in human beings 16 years after influenza a h5n1: a tale of two cities signaling mechanisms controlling cell fate and embryonic patterning tollip, an intracellular trafficking protein, is a novel modulator of the transforming growth factor- signaling pathway a large-scale conformational change couples membrane recruitment to cargo binding in the ap2 clathrin adaptor complex transforming growth factor- receptors interact with ap2 by direct binding to 2 subunit regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of ap2 intracellular trafficking of transforming growth factor  receptors an sirna screen of membrane trafficking genes highlights pathways common to hiv-1 and m-pmv virus assembly and release identification of host factors involved in human cytomegalovirus replication, assembly, and egress 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treatment strategy molecular determinants and dynamics of hepatitis c virus secretion insertion of a gfp reporter gene in influenza virus the k526r substitution in viral protein pb2 enhances the effects of e627k on influenza virus replication lead finder: an approach to improve accuracy of protein−ligand docking, binding energy estimation, and virtual screening the hong kong hainan commercial association south china microbiology research fund, the jessie & george ho charitable foundation, and perfect shape medical; and funding from the theme-based research scheme (t11-707/15-r) of the research grants council, hong kong special administrative region; and the high level-hospital program, health commission of guangdong province, china. the sponsors had no role in the design and conduct of the study, in the collection, analysis, and interpretation of data, or in the preparation, review, or approval of the manuscript key: cord-309381-cb80ntxs authors: nogales, aitor; l. dediego, marta title: host single nucleotide polymorphisms modulating influenza a virus disease in humans date: 2019-09-30 journal: pathogens doi: 10.3390/pathogens8040168 sha: doc_id: 309381 cord_uid: cb80ntxs a large number of human genes associated with viral infections contain single nucleotide polymorphisms (snps), which represent a genetic variation caused by the change of a single nucleotide in the dna sequence. snps are located in coding or non-coding genomic regions and can affect gene expression or protein function by different mechanisms. furthermore, they have been linked to multiple human diseases, highlighting their medical relevance. therefore, the identification and analysis of this kind of polymorphisms in the human genome has gained high importance in the research community, and an increasing number of studies have been published during the last years. as a consequence of this exhaustive exploration, an association between the presence of some specific snps and the susceptibility or severity of many infectious diseases in some risk population groups has been found. in this review, we discuss the relevance of snps that are important to understand the pathology derived from influenza a virus (iav) infections in humans and the susceptibility of some individuals to suffer more severe symptoms. we also discuss the importance of snps for iav vaccine effectiveness. influenza a viruses (iav) belong to the orthomyxoviridae family, and they contain a single-stranded (ss) negative-sense viral (v)rna genome formed by eight segments that are encapsidated into particles with an envelope ( figure 1a) . each of the vrna segments contains a long central coding region flanked at 5 and 3 termini by non-coding regions (ncrs), which work as promoters to initiate viral rna synthesis (transcription and replication). moreover, the packaging signals playing a role in the efficient encapsidation of the viral segments into nascent virions, are located at the 3 and 5 end of the coding regions ( figure 1b ) [1] . structurally, vrnas form viral ribonucleoprotein complexes (vrnps), where vrnas are coated with multiple subunits of the viral nucleoprotein (np) and are associated with the heterotrimeric polymerase, which contains the polymerase basic 2 and 1 (pb2 and pb1, respectively) and acidic (pa) proteins ( figure 1a ) [2] [3] [4] . each vrnp acts as an independent transcription-replication unit using an uncommon mechanism among negative-sense rna viruses, given that viral rna synthesis occurs in the infected-cells nucleus. vrnas are used as templates by the viral polymerase to synthesize two positive-sense rna molecules, the complementary rnas (crnas), from which the same viral polymerase synthesizes more copies of genomic vrna, and the mrnas for viral protein synthesis [1] [2] [3] [4] [5] [6] . the small iav genome encodes for up to 16 viral proteins through the viral envelope is decorated with the two viral glycoproteins hemagglutinin (ha) and neuraminidase (na) at a ratio of approximately four to one, respectively [10, 11] . ha envelope protein mediates virus entry by binding to sialic acid-containing cell receptors, and then fusing endosomal and viral membranes during endocytosis [12, 13] , while na is required for viral release from infected host cells, and it acts as a receptor destroying enzyme, cleaving terminal sialic acid residues from glycoproteins present at the cell surface [14] [15] [16] . the matrix 2 (m2) protein is also found in the viral membrane, although in much lower abundance than ha or na glycoproteins. m2 is a small transmembrane protein that forms a proton-selective ion channel in the viral envelope. m2 promotes uncoating of the vrnps after membrane fusion and the protein has also an essential role in viral assembly and release [17] . under the viral envelop, there is an inner shell composed of the matrix 1 (m1) protein, which interacts in the virion with the vrnp and the ha and na proteins. m1 apart from being a membrane-associated scaffold factor of the virion, acts as a crucial factor for different viral processes during infection, including virion assembly and budding [18] [19] [20] . the nonstructural (ns) gene or segment 8 of iav encodes an mrna transcript that is alternatively spliced to express two viral proteins, the nonstructural protein 1 (ns1), produced from a continuous primary transcript, and the nuclear export protein (nep), which is produced by an alternatively processed transcript, using a weak 5 splice site. nep is also located in the virion and may interact with m1 in the viral particle [21] [22] [23] ( figure 1a) . during the infection, nep is responsible for the nuclear export of synthetized vrnp, ensuring that the vrnps are available for packaging [24] . moreover, nep has also other functions during iav infection, contributing to viral budding and to regulate viral rna synthesis. ns1 is a multifunctional protein and a key viral factor that counteracts the host antiviral responses. ns1 has been shown to inhibit the production of interferon (ifn), the activity and expression of multiple interferon-induced genes (isg) and the processing and nuclear transport of host mrnas causing cellular shut-off [25, 26] . segment 3 of iav also encodes two proteins, the polymerase component pa and pa-x. pa is translated directly from the pa mrna, whereas pa-x is translated using a +1 frameshift mechanism from the same open reading frame (orf) [9] . synergistically with ns1, pa-x is also able to block the cellular antiviral responses by inhibiting host protein expression. moreover, the pa-x protein has been shown to modulate host inflammation, immune responses, apoptosis, and virus pathogenesis [25] [26] [27] [28] [29] [30] . human iav infections cause contagious respiratory diseases associated with mild to severe respiratory illness or even death, and they are considered as an important public health threat worldwide, which also results in significant economic losses [31] [32] [33] . iav are divided into multiple subtypes, based on the ha and na glycoproteins. currently, there are 18 ha (h1 to h18) and 11 na (n1 to n11), but the growing iav surveillance programs and sequencing technologies could increase the number of subtypes in the following years. iav can infect a wide range of avian and mammalian species, although the natural reservoirs of iav are shorebirds and wild waterfowls [34] [35] [36] [37] . among all the ha and na subtypes, only h3n2 and h1n1 iav subtypes are circulating in human beings and they are responsible for annual recurrent epidemics that affect the entire world [38, 39] . seasonal influenza infections are prevented and controlled through annual vaccination campaigns to decrease iav infections and viral transmission as well as to reduce their negative impact in the global economy. however, although vaccination remains the most effective approach to protect the population from seasonal infections, the effectiveness of current vaccination approaches is suboptimal [16, [31] [32] [33] [39] [40] [41] [42] [43] [44] . thus, the production of improved prophylactic approaches, including universal vaccines, are highly desired. concerns associated with iav are further aggravated by the adaptive capacity of the viruses to infect new hosts or escape to the immune system, as well as their ability to transmit efficiently in the population and the limited therapeutic options to treat viral infections [14, 16, 25, 45] . because of the ability of iav to modify their genome using two main evolutionary mechanisms, antigenic drift and shift, viruses encoding novel antigenic proteins to which the population has limited or no preexisting immunity can be generated [10, 31, 37, 40] . for that reason, seasonal vaccines have to be reformulated yearly to guarantee that the viral glycoproteins (ha and na) in the vaccine match seasonal viruses circulating worldwide [38, 43, 46] . in addition, iav variability can lead to the generation of new virus strains with pandemic potential. for example, the first iav pandemic of this century occurred in 2009 and it is estimated that in approximately one year, the pandemic 2009 h1n1 (ph1n1) iav infected more than 600,000 human beings, causing near 16,000 deaths in over 200 countries [40, 41] . in addition, although only h1n1 and h3n2 are circulating in humans, the avian h5, h7, and h9 subtypes eventually cross the species barrier to infect humans, representing a new and serious public health problem [13, 37, [47] [48] [49] . the cellular defense mechanisms provided by the innate immune system are a formidable barrier to inhibit virus infections [50] and involve the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs). this recognition leads to the activation of signaling pathways and the production and secretion of ifns of type i (ifnα and ifnβ) and iii (ifnλ2 or il-28a, ifnλ3 or il-28b, and ifnλ1 or il-29) , and chemokines and cytokines involved in inflammatory processes [50] . iav rnas are mainly recognized by the endosomal, membrane-associated prr toll-like receptors (tlrs) 3 (double-stranded rnas, dsrnas) or 7/8 (ssrnas), respectively [50, 51] , by the cytoplasmic prr retinoic acid-inducible gene i (rig-i), which detects dsrna and 5 -triphosphates of the negative ssrna viral genome [50, 52] , generated during replication of multiple viruses, by the nod-like receptor family member nod-, lrr-and pyrin domain-containing 3 (nlrp3), which recognizes various stimuli (see below) [53] and by the absent in melanoma 2 (aim2) protein, recognizing not well-characterized influenza stimuli [54] . the result of prr detection of viral pamps is the activation of multiple transcription factors, such as the nuclear factor kappa β (nf-κb), the activator protein 1 (ap-1), and ifn regulatory factors (irf)-3 and irf-7, which are responsible for the transcription of ifns [50, 55, 56] and pro-inflammatory cytokines [57] . secreted type i and iii ifns signal through different receptors in a paracrine or autocrine way to induce the transcription of ifn-stimulated genes (isgs), several of which counteract viral replication [50, 56, 58] . just as an example mentioned below, ifitm3 is an isg playing antiviral roles against influenza virus infection and other viruses [59] . type i and iii ifns signaling pathways lead to the post-translational phosphorylation of the signal transducer and activator of transcription (stat) 1 and 2 transcription factors [60] , being the tyrosine kinase 2 (tyk2) and janus protein tyrosine kinase 1 (jak1) critical for the phosphorylation [61] . moreover, stat1 is phosphorylated by ikkε during ifn signaling and this step is important for the ifn-inducible innate immune response [62, 63] . upon phosphorylation, stat1 and stat2 associate with irf-9 forming the heterotrimeric isg factor 3 (isgf3) complex [60] . this heterotrimeric complex then translocates to the nucleus, and binds to ifn-stimulated response elements (isres) located in the promoters of isgs, up-regulating their expression [60, 64] . inflammatory cytokines, such as interleukins (il)-1a il-1b and tumor necrosis factor (tnf)-α contribute to the proliferation and migration of different immune cells, such as monocytes, macrophages, neutrophils, and natural killer (nk) cells, to the infected tissue. nk cells have the ability to kill virus-infected cells, are important for the activation of a protective cytotoxic t lymphocyte (ctl) response [65] , and nk-cell ifn-γ production is augmented by t-cell il-2 production in recall responses [66] . neutrophils and resident alveolar macrophages are also important for virus clearance, due to their ability to destroy infected cells [67] . in addition, cytokine signaling improves dendritic cells (dc) maturation, increasing the induction of adaptive immune responses by antigen presentation and co-stimulation [68, 69] . these adaptive immune responses initiated upon innate immune activation are required for protection and viral clearance [70] . nlrp3 is expressed by myeloid cells such as macrophages, monocytes, neutrophils, and dendritic cells [71] or by human bronchial epithelial cells [72] . upon stimulation, nlrp3 activates the inflammasome system, activating caspase-1 and leading to pro-inflammatory processes through the processing and activation of proil-1b, proil-18, and proil-33 [73] . nlrp3 senses iav dsrna [74] , and pb1-f2 protein [75] . furthermore, protein flux through the viral m2 ion channel activity in the trans-golgi network activates nlrp3, leading to inflammasome activation [76] . in addition to nlrp3 activation, iav activates the inflammasomes through aim2, increasing iav-induced lung injury and mortality [54] . the complement system is an important branch of innate immunity that plays an essential role in the clearance of pathogens. the complement system is triggered by three main pathways, the classical, the lectin, and the alternative pathways [77] . the first two pathways are activated with the help of pattern recognition molecules, whereas the alternative pathway is activated spontaneously. interestingly, it is known that viruses are recognized by the three pathways. in the classical pathway, the c1 complex recognizes antigen-antibody complexes, which are formed on the pathogen surface. c1qbp (complement c1q binding protein) can bind to the globular heads of c1q molecules, activating the classical pathway [78] . on the other hand, in the lectin pathway, the mannan-binding lectin (mbl)/ficolin/mannan-binding lectin serin protease (map) complex recognizes specific carbohydrates on the pathogen surface. complexes activated after the classical and lectin pathways, cleave c4 and c2, resulting in the generation of c4bc2a (c3 convertase). in the alternative pathway, spontaneous hydrolysis of native c3 results in the formation of c3b-like c3 that binds factor b and after cleavage by factor d forms the initial c3 convertase [77] . the three pathways converge at the cleavage of c3 into c3a and c3b by c3 convertases (c4b, 2a and c3b, bb). then, the c3b molecules formed bind covalently to the c3-convertases forming the c5-convertases that cleave c5 into c5a and c5b. cd55 blocks c3 and c5 activation by preventing the formation of new c3 and c5 convertases [79] . c5b starts the formation of c5b-9 or the membrane attack complex (mac). next, c8 binds to the membrane attached trimer and begins binding and polymerization of c9 that is inserted into the membrane, inducing virolysis [77] . unregulated complement activation could play a central role in the acute lung injury (ali) pathology induced by highly pathogenic viruses, including severe acute respiratory syndrome (sars) coronavirus and avian iav h5n1, and h7n9 [80] . in virus-induced acute lung diseases, high levels of chemotactic, and anaphylatoxic c5a can be generated as a result of excessive complement triggering and causing a "cytokine storm". accordingly, the blockade of c5a signaling has been involved in treating the ali induced by highly pathogenic viruses [80] . currently, particular attention is being paid to single nucleotide polymorphisms (snps) that are loci within the genome of an organism in which two or more alleles can exist. snps affect a single nucleotide or base pair and they are one of the most frequent types of genetic variations in the genome [81] [82] [83] . snps need to be presented into the population with a frequency equal to or greater than 1% to be considered as polymorphisms. there are multiple types of snps, depending on their location that can be in different regions of the genes such as promoters, exons, introns or utrs ( figure 2 ). snps in coding regions are classified as synonymous, when a nucleotide substitution does not change the amino acid sequence of the encoded protein, although other effects, such as changes in mrna structure or folding may account for variation in protein expression. on the other hand, non-synonymous snps are divided in missense or nonsense. in the first case, nucleotide substitution results in the change of one amino acid for another, affecting the protein sequence coded by a gene and therefore may lead to its dysfunction. in contrast, nonsense mutations are produced when instead of substituting one amino acid for another, the altered gene contains an early stop codon in the orf or a stop codon is abrogated, producing an elongated protein. this type of mutations results in shortened or elongated proteins leading typically to nonfunctional proteins. the functional role of snps in coding areas of the genome can be easily analyzed by studying the gene products. however, most snps fall within non-coding genome regions, therefore, predicting their effects is challenging. for example, snps in the promoter regions could affect their activity and regulation producing changes in gene expression levels. snps in utrs or intron regions have been related with an effect in protein translation or the production of splice variants of transcripts, leading to longer or shorter protein sequences, respectively. in summary, snps may influence gene regulation, the structure and stability of rna, the expression of rnas or proteins, the conformation and function of proteins, etc. thus, the identification of snps in genes and the analysis of their effects may lead us to better understand gene function or their impact on human health [84] . in fact, snps that are or could be important for multiple human pathologies, such as cancer, diabetes, heart disease, schizophrenia, blood-pressure homeostasis, and autoimmune or metabolic diseases, have been identified [85] [86] [87] [88] [89] [90] [91] . moreover, some described snps increase the human susceptibility to getting infected by viruses, bacteria or other pathogens [84, 86, [92] [93] [94] [95] [96] [97] . advanced sequencing and bioinformatics technologies have allowed the identification of a large number of human snps whose information is accessible in the databases. nevertheless, the biological significance and function for most of the snps found in the human genome remain unknown. currently, the scientific community recognizes the importance of this kind of genome variations that can act as biological markers and assist researchers in multiple aspects, such as: (1) locate genes associated with multiple diseases, (2) anticipate an individual's response to a specific infection, (3) predict population responses to several treatments such as drugs or vaccines, (4) design individualized therapies, (5) identify markers for medical testing, (6) perform pharmacogenetic studies, etc. this review focuses on the role of known snps on iav infection, as well as their impact on the effectiveness of vaccines against iav. instead of substituting one amino acid for another, the altered gene contains an early stop codon in the orf or a stop codon is abrogated, producing an elongated protein. this type of mutations results in shortened or elongated proteins leading typically to nonfunctional proteins. the functional role of snps in coding areas of the genome can be easily analyzed by studying the gene products. however, most snps fall within non-coding genome regions, therefore, predicting their effects is challenging. for example, snps in the promoter regions could affect their activity and regulation producing changes in gene expression levels. snps in utrs or intron regions have been related with an effect in protein translation or the production of splice variants of transcripts, leading to longer or shorter protein sequences, respectively. an snp is a variation on a single nucleotide which may occur at some specific point in the genome and that causes variations in dna sequences between members of the same species. (b) types of snps: dna variation can be located in non-coding or coding regions. snps within a coding sequence can be synonymous if they do not produce an amino acid change (silent mutation), or non-synonymous if they affect the protein sequence. nonsynonymous changes can be divided into missense (producing an amino acid change in the protein) or nonsense (producing a truncated or longer protein). an snp is a variation on a single nucleotide which may occur at some specific point in the genome and that causes variations in dna sequences between members of the same species. (b) types of snps: dna variation can be located in non-coding or coding regions. snps within a coding sequence can be synonymous if they do not produce an amino acid change (silent mutation), or non-synonymous if they affect the protein sequence. non-synonymous changes can be divided into missense (producing an amino acid change in the protein) or nonsense (producing a truncated or longer protein). risk factors, including underlying co-morbidities, age, and pregnancy, affect iav susceptibility, but do not explain all the conditions under which serious iav-associated disease can occur, making likely that snps in viral and host genes affect iav susceptibility and the outcome of the disease. in fact, there are some examples of the presence of snps in host genes affecting influenza severity (table 1) , which will be discussed in this review. snps affecting iav disease have been found in genes recognizing viral components, in transcription factors important for ifn production and signaling, in isgs with antiviral activities, and in genes involved in inflammation. tlr3 recognizes dsrna, one of the iav replication intermediate products, and in turn activates ifn production, leading to an antiviral response. a missense mutation (f303s) of the tlr3 gene was found in one out of three patients developing iav-associated encephalopathy (iae), a neurological consequence of severe viral infection [98] . assays in tissue culture cells showed that a tlr3 receptor encoding the missense f303s mutation was impaired in activating the transcription factor nf-κb, and in triggering downstream signaling via the ifnβ receptor, indicating that this genetic polymorphism could lead to increased iav replication [98] . in a study of 51 italian children diagnosed with iav h1n1 infection, an additional tlr3 snp (rs5743313, genotype c/t) was identified [99] . this tlr3 snp was found in all the children developing iav-associated pneumonia (18 cases). however, the snp was found in significantly less proportion in children with milder disease, suggesting a link between tlr3 and iav pathogenicity. furthermore, in a multicenter study involving 275 adult cases of avian h7n9 and ph1n1 iav, in mainland china and hong kong, the tlr3 cc rs5743313 snp was associated with fatal cases [100] . in addition to iav, there are other examples of snps in tlr3 or tlr3 signaling genes affecting viral infections. for instance, susceptibility to chikungunya virus (chikv) infection is highly increased in human and mouse cells with defective tlr3 molecules [101] . furthermore, tlr3 snps, rs3775292, and rs6552950, leading to unknown functional consequences, were associated with an increased risk of chikv disease occurrence [101] . patients with impaired tlr3-mediated responses show an elevated susceptibility to herpes simplex-1 virus (hsv-1)-mediated encephalitis by encoding tlr3-deficient alleles [102, 103] , or by encoding deficient traf3, tbk1 and trif molecules, leading to impaired tlr-3 signaling [104] [105] [106] . in a saudi arabian population, the tlr3 rs78726532 snp was strongly associated with hepatitis b (hbv) and hepatitis c (hcv) virus infections when compared to that in healthy control subjects [107, 108] . the tlr3 rs5743314 c allele was also associated with hcv-related liver disease progression (cirrhosis and hepatocellular carcinoma) [107] . however, the functional effects of these snps seem to be unknown. rig-i detects dsrna and 5 -triphosphates of the negative ssrna iav genome, leading to innate immune responses activation [52] . a caucasian male patient with severe iav h1n1 infection during the 2009 swine flu pandemic showed two heterozygous variants (one in each chromosome): p.r71h (snp rs72710678) and p.p885s (snp rs138425677), located, respectively, in the caspase activation and recruitment domain (card) and rna binding domains of rig-i [109] . these variants significantly decreased the recognition function of rig-i, and therefore, patient cells proved impaired antiviral responses to rig-i ligands and elevated proinflammatory responses to iav, providing evidence for dysregulation of the innate immune response and increased immunopathology [109] . these results suggest that these rig-i polymorphisms may have contributed to severe iav outcome in this patient and reinforce that rig-i variants should be evaluated in future studies of host factors affecting ssrna virus infections. irf-7 is a transcription factor that increases interferon (ifn) production in response to viruses [110] [111] [112] . a patient suffering from an unusual life-threatening disease after ph1n1 infection encodes homozygous null mutations in the irf-7 factor. both irf-7 alleles from this patient encode mutations c.1228t>g/t (f410v) and c.1261c>t/c (q421x), which are mutations decreasing the ability of irf-7 to induce the transcription of ifn genes after iav infections [113] . these findings suggest that irf-7-dependent production of type i and iii ifns is required for controlling iav infections in humans. the rare allele a of two irf-7 snps, rs12272434 and rs12290989, both located at exon/intron boundaries, were significantly associated with impaired levels of ifnα production by human plasmacytoid dendritic cells (pdcs) in response to human immunodeficiency virus 1 (hiv-1) infection [114] . therefore, these polymorphisms may affect the ability of human subjects to control hiv-1 infections, reinforcing the role of irf-7 in controlling viral infections. however, the effect of these snps should be further studied. irf-9 is a transcription factor essential for ifn signaling and the transcriptional induction of isgs [60] . stat1 and stat2, when phosphorylated, associate with irf-9 to form a heterotrimeric isg factor 3 (isgf3) complex [60] , which translocates to the nucleus, and binds isres present in the promoters of isgs, up-regulating their transcription [60, 64] . a homozygous, loss-of-function mutation in irf-9 was described in a child born to first-cousin algerian parents and living in france affected by a severe pulmonary influenza infection [115] . in particular, the homozygous mutation (c.991g>a) occurred in the final nucleotide of exon 7 and disrupted the essential splice site at the boundary of exon 7 and intron 7, leading to deleted irf-9 proteins. the consequence of this mutation was an impaired activation of irf-9, and therefore, an impaired transcription of isgs, many of which show antiviral activities [115] . similarly, a family in which several members showed a surprising susceptibility to infection by different viruses, including iav, also showed to be irf9 deficient [116] . the index patient, a boy with 10 years born at term from healthy consanguineous parents (first cousins of portuguese origin and residing in venezuela) encoded a homozygous splicing mutation in the irf9 gene. the mutation, c.577+1g>t, was located in the donor splice site of introns 5 and 6, leading to transcripts lacking exon 5. irf9 protein expression was undetectable in cells transfected with the c.577+1g>t irf9 construct, suggesting that either the protein was quickly degraded or the mrna was not translated. again, irf9-deficient cells showed a profound defect in inducing the expression of multiple isgs [116] . collectively, these findings show that human irf9-and isgf3-dependent type i and iii ifn responsive pathways are essential for controlling viral infections, including iav. the antiviral protein ifitm3 is an isg which abrogates the release of iav content from late endosomes into the cytoplasm [59] . in addition, ifitm3 promotes the survival of mouse lung-resident cd8+ t cells following iav challenge, which may help clear the infection [117] . furthermore, mice in which the expression of ifitm3 is abolished, showed severe disease after iav infection, compared to parental mice [118] . one of the clearest associations of snps in genes affecting influenza severity is located in the isg ifitm3. the human ifitm3 gene is encoded by two exons and is predicted to encode two splice variants that differ in the first amino-terminal 21 amino acids. different studies have described the effect of ifitm3 snps in influenza disease severity. northern european patients infected with iav ph1n1 2009 virus requiring hospitalization showed over-representation of the snp rs12252 in the ifitm3 gene, in which the majority t allele is replaced for a minority c allele [118] . this leads to an alteration of the first splice acceptor site, originating an ifitm3 protein lacking the first 21 amino acids (n∆21) due to the protein starting from an alternative start codon. according to these results suggesting that this snp could affect influenza disease, the minority (cc) variant rendered homozygous cells more susceptible to iav infection, and this susceptibility correlated with decreased levels of ifitm3 protein expression in comparison to the majority (tt) variant cells [118] . furthermore, cells expressing the n∆21 protein showed an impaired ability to restrict viral replication when compared to wild-type ifitm3 cells [118] . this data is consistent with previous results which show that the amino-terminal 21 amino acids of ifitm3 are relevant for attenuating vesicular stomatitis virus (vsv) replication in vitro [119] . moreover, the cc genotype was found in 25% of chinese patients showing mild disease after ph1n1 virus infection compared to 69% in patients developing a severe ph1n1 virus infection. in addition, the cc genotype was estimated to confer a six-fold increased risk for severe infection than the ct and tt genotypes [120] , reinforcing the idea that ifitm3 is a factor affecting human iav disease [121] . in another study, over-representation of the ifitm3 cc genotype was detected among fatal cases of chinese patients infected with iav ph1n1 and h7n9 viruses [100] , and in a more general study, including twelve studies published before february 2018 with more than 16,000 subjects, revealed increased risk of severe influenza in both the east asian and white populations in the subjects encoding the ifitm3 cc genotype [122] . another important snp (rs34481144) associated with risk of severe influenza in humans from the united states (us) infected with seasonal iavs is located in the 5 -utr of the ifitm3 gene [123, 124] . this snp affected ifitm3 expression being the risk allele associated with lower mrna expression. the mechanism for this lower mrna expression involves the decreased irf-3 binding and increased binding of the transcriptional repressor ccctc-binding factor (ctcf) in promoter-binding assays for the risk allele [123] . moreover, the risk allele disrupted a cpg site that becomes differentially methylated in cd8+ t cell subsets, leading to less cd8+ t cells in the airways during natural influenza infection in the carriers of the risk allele, and suggesting that a critical role for ifitm3 may be to promote immune cell persistence at mucosal sites [123] . interleukins 1a and 1b (il-1a and il-1b, respectively) are inflammatory cytokines that play critical roles in recruiting immune and inflammatory cells and developing adaptive immune responses. furthermore, accumulating evidence suggests that both cytokines play central roles in innate immunity against viral infections [125] . the frequencies of snp (allele c) located 31 base pairs upstream from the transcription start site (rs1143627), on the il-1b promoter were associated with increased risk of influenza disease in chinese subjects [126] . this nucleotide change is localized in a tata-box motif of il-1b and modulates the transcription activity of il-1b by binding to multiple transcription factors [127] . the allele t of rs1143627 enhanced il-1b protein expression, as indicated by several reports [128] . people carrying allele t showed a higher il-1b expression, which could lead to increased ifnγ production, which promotes virus clearance [129] . in contrast, expression of il-1b may be decreased in individuals who carry allele c, leading to a weaker immune response during viral infection. in addition, a t allele in il-1a gene (snp rs17561) increased the risk of iav ph1n1 susceptibility, as observed in chinese subjects [126] . the snp rs17561 introduces a nonsynonymous mutation (a114s) in il-1a protein, suggesting that this genetic variant may lead to a functional variation in host susceptibility to ph1n1. nevertheless, the molecular mechanism needs to be evaluated and the real risk of these alleles should be analyzed in larger populations. tnf-α is a pro-inflammatory cytokine which orchestrates the host´s defense. a minor allele (a) at position -238 of tnf (snp rs361525) was more frequent in greek patients infected with ph1n1 virus compared to control subjects [130] , and developing pneumonia was more uncommon in greek and mexican subjects with no copies of the minor allele compared to subjects with at least one copy of the minor allele [130, 131] , leading to the hypothesis that this snp allele could be linked with an elevated susceptibility to infection with the ph1n1 virus [124, 130] . decreased tnf-α expression was observed in subjects encoding the minor allele at position -238 [92] . this may explain how snps leading to lower production of tnf-α may predispose to more severe clinical symptoms following iav infections. however, the tnf-α rs 1800629 minor a allele, associated with higher levels of tnf-α expression, was associated with susceptibility to japanese encephalitis virus infection in an indian population [132] . the tnf-α rs 1800629 minor a allele was a risk factor to develop liver cirrhosis and hepatocellular carcinoma following hbv infection in a han chinese population [133] , suggesting that the protective or deleterious roles of tnf-α expression may vary depending on the infecting virus. chemokine receptor 5 (ccr5) is expressed mainly on macrophages, t cells, and dendritic cells. ccr5 mediates leukocyte chemotaxis in response to its ligands, including mip-1a, mip-1b, and rantes. it can help direct multiple immune cell subsets, including regulatory t cells or th17 cells to sites of infection, supporting the antiviral immune response. evidence in humans support that homozygosity for the ccr5-∆32 allele, a naturally occurring polymorphism of ccr5 encoding a 32-bp deletion, prevents its expression on the cell surface, and is linked with an elevated susceptibility to west nile virus (wnv) [134] and with increased severity of illness among patients infected with ph1n1 [135] , although this evidence is modest due to the limited number of subjects analyzed. in contrast, homozygous carriers of the ∆32 mutation are resistant to hiv-1 infection because this molecule, absent in the cell surface in subjects encoding the deletion, is a molecule normally used by hiv-1 to enter cd4+ t cells [136] . cd55 is an important complement regulatory protein which blocks c3 and c5 activation by preventing the formation of new c3 and c5 convertases, two proteases involved in inflammation and complement activation. consequently, cd55 protects cells from complement attack and decreases amplification of the complement cascade [79] . the cd55 snp (rs2564978, genotype t/t) was significantly associated with severe iav infection in chinese patients infected with ph1n1 2009 virus [137] and was associated with increased death risk in greek patients [138] . the rs2564978 snp of cd55 is located in the minimal promoter region [139] and individuals with this genotype showed significantly lower levels of cd55 expression in comparison to those with the more frequent allele [137] . therefore, patients who carry the t/t genotype may have more robust complement activation during iav infection, resulting in enhanced inflammation and disease severity [47, 79] . according to these results, the polymorphism rs2564978 in gene cd55 was linked to disease severity in adult chinese cases of avian (h7n9) and human ph1n1 iav in another study [100] . however, these findings need to be confirmed in bigger cohorts. c1qbp can bind to the globular heads of c1q molecules, activating the classical pathway of complement [78] . an increased risk of severe disease after iav infection was found in patients homozygous for the minor allele of the snp rs3786054 in european and mexican populations [138, 140] . however, the effect of this snps on gene expression and function is undescribed. soluble pattern-recognition molecules, forming part of the innate immune system, can neutralize iav infection. particularly, the serum mannose-binding lectin (mbl), several secreted human c-type lectins of the collectin family, collectin 11, and the pulmonary surfactant proteins (sp) -a1, -a2, and -d (sftpa1, sftpa2, and sftpd, respectively), may neutralize iav infectivity in vitro [141] . mice lacking sp-a or sp-d were more susceptible to iav infection, indicating that sps exert relevant roles against iav infection [142] [143] [144] . two frequent sp-a2 (sftpa2) missense alleles (rs1965708-c, leading to the mutation q223k and rs1059046-a, leading to the mutation t9n) were associated with acute respiratory failure, mechanical ventilation, and acute respiratory distress syndrome after infection with ph1n1 2009 virus in a spanish population [145] . in addition to c-type lectins, s-type lectins have been described, such as galectins, which recognize galactose-containing oligosaccharides present in the cellular plasma membranes and in viruses, such as iav. importantly, intranasal treatment of galectin-1 enhanced survival of mice infected with iav by reducing viral load, apoptosis, and inflammation in the lung [146] . moreover, galectin-1 knockout mice showed increased susceptibility to influenza virus infection than wild-type mice [146] . to study human genetic susceptibility to avian iav h7n9 infection, a genome-wide association study involving 106 heavily-exposed healthy poultry chinese workers and 102 iav h7n9 patients was performed [147] . functional variants of galectin-1 gene, including rs4820294 and rs13057866, causing increased expression levels of galectin-1 expression, may confer more protection from iav h7n9 infection to the carriers of these variants [147] . the cleavage of the iav ha by host proteases is critical for viral infectivity. tmprss2 is a type ii transmembrane serine protease family member, which was shown to activate ha proteins of multiple human iavs in tissue culture cells. furthermore, deletion of tmprss2 in mice impairs the spread of h1n1 influenza viruses, including the ph1n1 2009 swine iav [148] . in addition, bodyweight loss and survival after h3n2 iav infection were less severe in tmprss2 mutant mice compared to wild type mice [148] . the genetic predisposition to severe ph1n1 2009 influenza virus was evaluated in chinese human subjects, finding that the gg genotype of rs2070788, leading to increased expression of tmprss2, was a risk variant to severe ph1n1 influenza [149] . furthermore, rs2070788 and rs383510, both of them associated with increased gene expression, were significantly associated with the susceptibility to iav h7n9 [149] . table 1 . single nucleotide polymorphisms associated with susceptibility and severity of influenza infections. recognizes dsrna, triggering ifn production. rs not annotated; f303s (nonsyn). rs5743313 (ncr). [98] [99, 100] rig-i detects dsrna and 5 -triphosphates of the negative ssrna iav genome, leading to innate immune responses activation. rs72710678; r71h (nonsyn). rs138425677; p885s (nonsyn). [109] irf-7 transcription factor that increases ifn production in response to viruses. rs786205223; f410v (nonsyn) rs375323253; q421x (nonsyn) [113] irf-9 transcription factor essential for ifn signaling and the transcriptional induction of isgs. c.991g>a occurred in the final nucleotide of exon 7 and disrupted the essential splice site at the boundary of exon 7 and intron 7 (nonsyn). c.577+1g>t, was localized in the donor splice site of introns 5 and 6 and led to transcripts lacking exon 5 (nonsyn). [115] [116] ifitm3 isg which abrogates the release of iav content from late endosomes into the cytoplasm. ifitm3 increases the survival of mouse lung-resident cd8 + t cells after iav infection, which can help clear the infection. rs12252, leading to an alteration of the first splice acceptor site, leading to an ifitm3 protein lacking the first 21 amino acids (nonsyn). rs34481144, is located in the 5 -utr and affects ifitm3 expression with the risk allele showing lower mrna expression (ncr). [ 100, 118, 120, 122] [123] il-1b inflammatory cytokine involved in the development of adaptive immune responses. furthermore, accumulating data has suggested that il-1a and il-1b have critical roles in innate immunity against viral infections. rs1143627, located 31 base pairs upstream from the transcription start site, on the il-1b promoter. this nucleotide change is located in a tata-box motif of il-1b, affecting the transcription activity of il-1b (ncr). [128, 129] galectin-1 recognizes galactose-containing oligosaccharides present in the cellular plasma membranes and in viruses, such as iav. -rs4820294 (ncr). -rs13057866 (ncr). [147] tmprss2 type ii transmembrane serine protease family member which activates ha proteins of diverse human iav in tissue culture cells. deletion of tmprss2 in mice impairs the spread of h1n1 influenza viruses, including the ph1n1. moreover, body weight loss and survival were less severe in tmprss2 mutant mice compared to wild type mice after infection with h3n2 iav. -rs2070788, localized in an intron (ncr). -rs383510, localized in an intron (ncr). [149] syn-synonymous, nonsyn-nonsynonymous, ncr-non-coding region (intron, regulatory regions, promoter or utr). currently, iav vaccines are the main strategy to prevent iav infection, though their effectiveness is suboptimal in many cases. notably, the efficacy of vaccines against iav infections can fluctuate and there is a significant immune response variability across the population. factors such as previous exposure to iav infections or vaccines, age, and the closeness of the match between the vaccine and circulating strains are important to explain differences in vaccine effectiveness between seasons and group populations [44, 46, [150] [151] [152] . however, multiple reports have demonstrated that the host genetic background and polymorphisms on key immune response genes modulate the immune response to infection or vaccination [153] [154] [155] [156] [157] [158] [159] [160] . therefore, new insights into iav-host interaction and immune response modulating factors could allow us to design better vaccination strategies. snps may modify the humoral immune response after iav vaccination. therefore, their impact on the immune responses induced after iav vaccination are being analyzed [153] [154] [155] [156] . the major histocompatibility complex (mhc) is localized in chromosome 6 of the human genome, it includes multiple genes and exhibits considerable diversity between populations. moreover, in this genomic region, there is a higher presence of snps than in other sections of the genome. mhc class i and class ii molecules have an essential role in the adaptive immune system in response to infections. both classes of proteins bind peptide fragments derived from pathogens to be presented on the cell surface for recognition by appropriate t cells [97, 161, 162] . in those genes, the human leukocyte antigens (hla) class i and ii are important because of their role in the immune system. gelder et al. studied whether hla class ii polymorphisms modulate anti-iav antibody responses to vaccination in a united kingdom population [154] . for that, a cohort of hla-typed donors at risk was investigated, and hemagglutination-inhibition (hai) titers were evaluated before and 28 days after the administration of seasonal trivalent influenza vaccine. a correlation between hla class ii alleles and iav hai titers in the influenza risk group was found. moreover, a positive association between non-responsiveness to influenza vaccine and hla-drb1*07 and a negative association with hla-drb1*13 and hla-dqb1*0603-9/14 [154] was reported, suggesting that polymorphisms in hla class ii molecules affect antibody responses to iav vaccination. these findings are important because they could potentially identify individuals who may not be protected by current vaccination approaches. in another study, poland et al. analyzed the immunogenetic relationships between hla, cytokine and cytokine receptor gene polymorphisms in the induction of antibodies in response to inactivated seasonal vaccines [156] . authors did not find statistically significant associations between hla class ii alleles and iav hai titers. however, they established a positive association of some hla class i alleles and iav h1n1 hai titers, including hla-a*1101, a*6801, b*3503, b*1401, and c*0802. in contrast, they did not find associations between the hla-a, b or c alleles and hai antibody titers for iav h3n2. in addition, when authors evaluated a panel of 586 cytokine and cytokine receptor snps, they identified several significant associations between snps, in regulatory or coding regions of cytokine (il-6, il-12b) or cytokine receptor (il-1r, il-10rb, tnfrsf1a) genes and variations in hai antibody titers for iav h1n1 [156] (table 2) . notably, snps from three genes, il-6 (rs1800796), il-12b (rs3212227) and il-1r1 (rs3732131) revealed links with iav h1n1-induced antibody responses in an allele dose-related way. the presence of snp allele c or g in the il-12b or il-1r1genes, respectively resulted in reduced hai titers. however, high hai titers in the presence of minor snp allele g in the il-6 gene were observed [156] . snps associations between cytokine or cytokine receptor genes and iav h3n2 hai titers were also identified ( table 2) . for example, a variant ga for non-synonymous snps within the il-12 receptor gene (rs2307153; d465g) and tnf receptor 2 gene (rs5746026; k232e) displayed associations with lower hai titers, while a minor allele t variant (rs12722605) located in the 3 region of the il-2 receptor gene was related with high antibody titers ( table 2 ). these data suggest that host snps affect responses to influenza vaccine. mannose-binding lectin 2 (mbl-2) is a protein that binds n-acetylglucosamine, mannose, and fucose on different microorganisms and activates the lectin complement pathway [163, 164] . tang et al. studied the presence of snps in subjects who received an inactivated influenza vaccine. for that, authors classified the vaccine recipients in poor, normal or adverse responders. they observed that the g to a snp in the codon 54 allele (rs1800450) in mbl-2 was associated with a decreased risk for the development of adverse or poor responses (table 2 ) [165] . in addition, they did not find a significant association between responses and either tnf-α or il-10 promoter snps among the 3 response groups [165] . cytokine expressed as a response to infections or tissue injuries. it plays an important role in host defense through the stimulation of acute-phase responses. -rs1800796 (ncr). -rs2069861 (ncr). iiv [156] il-12b cytokine that serves as a crucial inducer of th1 cell development. rs3212227, located in 3´utr (ncr). iiv [156] ifn-b1 cytokine released as part of the innate immune response against infection by viruses or other pathogens. rs1364613 (ncr). iiv [156] tnfrsf1a cytokine receptor, its interaction with tnf-α control cell survival, apoptosis, and inflammation. rs4149621 (ncr). iiv [156] il-1r1 cytokine receptor involved in inflammatory and immune responses. rs3732131, located in 3´utr (ncr). iiv [156] il-10rb cytokine receptor that mediates the activation of the jak/stat signaling pathway leading to the expression of isg. rs3171425, located in 3´utr (ncr). iiv [156] il-2ra this cytokine receptor is important for the signaling pathway leading to immune cell differentiation and function. -rs2228150 (syn). -rs12722605 (ncr). iiv [156] il-10ra cytokine receptor that is involved in the inhibition of the synthesis of several proinflammatory cytokines. -rs4252249 (syn) -rs4252243 (ncr). iiv [156] il-12rb2 cytokine receptor that plays a role in th1 cell differentiation. rs2307153; d465g (nonsyn). iiv [156] il-1rn cytokine receptor which modulates a variety of immune and inflammatory responses related with il-1. -rs315952 (syn). -rs315951 located in 3´utr (ncr). iiv [156] tnfrsf1b cytokine receptor involved in the recruitment of anti-apoptotic proteins. rs5746026; k232e (nonsyn) iiv [156] mbl-2 this calcium-dependent protein that plays an important role in innate immunity, and activates the lectin complement pathway. rs1800450; g54d (nonsyn) iiv [165] il-28b (ifnl3) type iii ifn molecule, with brad functions in antiviral responses rs8099917 (ncr). iiv [153] syn-synonymous, nonsyn-nonsynonymous, ncr-non-coding region (intron, regulatory regions, promoter or utr). iiv: inactivated seasonal vaccine. il-6, interleukin 6. il-12b, interleukin 12. ifn-b1, interferon beta 1 (ifnβ). tnfrsf1a, tnf receptor superfamily member 1a. il-1r1, interleukin 1 receptor type 1. il-10rb, interleukin 10 receptor subunit beta. il-2ra, interleukin 2 receptor subunit alpha. il-10ra, interleukin 10 receptor subunit alpha. il-12rb2, interleukin 12 receptor subunit beta 2. il-1rn, interleukin 1 receptor antagonist. tnfrsf1b, tnf receptor superfamily member 1b. mbl-2, mannose binding lectin 2. il-28 b or ifnl3, interferon lambda 3. other snps that are not related with immune responses have been also linked to vaccine effectiveness. egli et al. revealed that the presence of the t/g or g/g genotype (rs8099917, minor-allele) in il-28b (ifnλ3), a type iii ifn, was linked with increased seroconversion in recipients of an inactivated influenza vaccine (table 2 ) [153] . moreover, iav-stimulated b-and t-cells from the minor-allele carriers exhibited increased hla-dr and il-4 expression, respectively. in addition, the expression of il-28b, but not il-28a or il-29, mrnas was significantly reduced in the rs8099917, minor-allele carriers. authors also reported that the il-28b rs8099917 polymorphism affected humoral responses to the iav vaccine, and had a strong outcome on cellular immune responses by modulating the th1/th2 cytokine response [153] . these findings are important because they will help to predict which individuals could not be protected by present vaccines and they can also be used to design personalized vaccine strategies to optimize the immune reaction. the sequencing of the human genome together with the development of novel bioinformatic tools have made possible the identification of multiple snps. more information is available for the scientific community in the databases. in addition, the identification and study of the human genome variability has opened the opportunity to investigate their association with the risk of developing multiple human diseases facilitating their diagnosis or the susceptibility to infections caused by viruses or other pathogens. moreover, the knowledge and analysis of genomic variability will be a valuable tool to predict the outcome of prophylactic or therapeutic interventions, including vaccines and drugs. the analysis of human snps and their association with iav infections or vaccination outcomes have just begun. however, current research and data reflect the importance to obtain a better understanding of these relations and the mechanisms underlying the effect of snps in the human immune system. in the future, this knowledge 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genetic polymorphisms on vaccine induced antibody response immunogenetics of seasonal influenza vaccine response immunological variation due to genetics of inflammatory snps and age and impact on disease manifestation genetics and vaccines in the era of personalized medicine host genetic factors can impact vaccine immunogenicity and effectiveness the genetic background influences the cellular and humoral immune responses to vaccines the major histocompatibility complex: a paradigm for studies of the human genome the nature of selection on the major histocompatibility complex a journey through the lectin pathway of complement-mbl and beyond the role of mannose-binding lectin in health and disease host single-nucleotide polymorphisms and altered responses to inactivated influenza vaccine we apologize for those publications we could not refer due to space limitations. the authors declare no conflict of interest. key: cord-342691-8jcfzexy authors: ochsner, scott a.; pillich, rudolf t.; mckenna, neil j. title: consensus transcriptional regulatory networks of coronavirus-infected human cells date: 2020-09-22 journal: sci data doi: 10.1038/s41597-020-00628-6 sha: doc_id: 342691 cord_uid: 8jcfzexy establishing consensus around the transcriptional interface between coronavirus (cov) infection and human cellular signaling pathways can catalyze the development of novel anti-cov therapeutics. here, we used publicly archived transcriptomic datasets to compute consensus regulatory signatures, or consensomes, that rank human genes based on their rates of differential expression in mers-cov (mers), sars-cov-1 (sars1) and sars-cov-2 (sars2)-infected cells. validating the cov consensomes, we show that high confidence transcriptional targets (hcts) of mers, sars1 and sars2 infection intersect with hcts of signaling pathway nodes with known roles in cov infection. among a series of novel use cases, we gather evidence for hypotheses that sars2 infection efficiently represses e2f family hcts encoding key drivers of dna replication and the cell cycle; that progesterone receptor signaling antagonizes sars2-induced inflammatory signaling in the airway epithelium; and that sars2 hcts are enriched for genes involved in epithelial to mesenchymal transition. the cov infection consensomes and hct intersection analyses are freely accessible through the signaling pathways project knowledgebase, and as cytoscape-style networks in the network data exchange repository. infection of humans by coronaviruses (cov) represents a major current global public health concern. signaling within and between airway epithelial and immune cells in response to infections by cov and other viruses is coordinated by a complex network of signaling pathway nodes. these include chemokine and cytokine-activated receptors, signaling enzymes and transcription factors, and the transcriptional targets encoding their downstream effectors [1] [2] [3] . placing the transcriptional events resulting from cov infection in context with those associated with host signaling systems has the potential to catalyze the development of novel therapeutic approaches. the cov research community has been active in generating and archiving transcriptomic datasets documenting the transcriptional response of human cells to infection by the three major cov strains, namely, middle east respiratory syndrome coronavirus (mers-cov, or mers) and severe acute respiratory syndrome coronaviruses 1 (sars-cov-1, or sars1) and 2 (sars-cov-2, or sars2) [4] [5] [6] [7] [8] [9] . to date however the field has lacked a resource that fully capitalizes on these datasets by, firstly, using them to identify human genes that are most consistently transcriptionally responsive to cov infection and secondly, contextualizing these transcriptional responses by integrating them with 'omics data points relevant to host cellular signaling pathways. we recently described the signaling pathways project (spp) 10 , an integrated 'omics knowledgebase designed to assist bench researchers in leveraging publically archived transcriptomic and chip-seq datasets to generate research hypotheses. a unique aspect of spp is its collection of consensus regulatory signatures, or consensomes, which rank genes based on the frequency of their significant differential expression across transcriptomic experiments mapped to a specific signaling pathway node or node family. by surveying across multiple independent datasets, we have shown that consensomes recapitulate regulatory relationships between signaling pathway nodes and their transcriptional targets to a high confidence level 10 . here, as a service to the research community to catalyze the development of novel cov therapeutics, we generated consensomes for infection of human cells by mers, sars1 and sars2 covs. computing the cov consensomes against those for a broad range of cellular signaling pathway nodes, we discovered robust intersections between genes with high rankings in the cov consensomes and those of nodes with known roles in the response to cov infection. integration of the cov table 1 . doi-driven links to consensomes and hct intersection networks. spp dois point to the tabular web version of the consensome, which can be downloaded as an excel file. ndex consensome dois point to the full consensome network; for ease and clarity of display, only the top 5% of the consensome is shown in the initial graphic display; in addition, a subset of the data corresponding only to the top 5% of the consensome can be reached via a link in the "description". ndex virus node family hct intersection dois point to networks containing all node families; ndex virus node hct intersection dois point to the full hct intersection network; for ease and clarity of display, only the top 5% of the hct intersection network is shown in the initial graphic display; in addition, a subset of the data corresponding only to the top 5% of the hct intersection network can be reached via a link in the "description". isgs were assigned elevated rankings across the four viral consensomes. the mean percentile of the isgs was however appreciably higher in the iav (98.7 th percentile) and sars1 (98.5 th percentile; p = 6e-1, t-test iav vs sars1) consensomes than in the sars2 (92 nd percentile, p = 5e-2, t-test iav v sars2) and mers (82 nd percentile; p = 7e-5, t-test iav v mers) consensomes. this is consistent with previous reports of an appreciable divergence between iav and sars2 infection with respect to the interferon transcriptional response 8 . other genes with known critical roles in the response to viral infection have high rankings in the cov consensomes, including ncoa7 14 (percentiles: sars1, 98 th ; sars2, 97 th ; mers, 89 th ; iav, 99 th ), stat1 15 (percentiles: sars1, 99 th ; sars2, 98 th ; mers, 89 th ; iav, 99 th ) and tap1 16 (percentiles: sars1, 99 th ; sars2, 94 th ; mers, 83 rd ; iav, 99 th ). in addition to the appropriate elevated rankings for these known viral response effectors, the cov consensomes assign similarly elevated rankings to transcripts that are largely or completely uncharacterized in the context of viral infection. examples of such genes include psmb9, encoding a proteasome 20s subunit (percentiles: sars1, 98 th ; sars2, 97 th ; mers, 98 th ; iav, 98 th ); csrnp1, encoding a cysteine and serine rich nuclear protein (percentiles: sars1, 99 th ; sars2, 94 th ; mers, 98 th ; iav, 94 th ); and ccnl1, encoding a member of the cell cycle-regulatory cyclin family (percentiles: sars1, 99 th ; sars2, 94 th ; mers, 99 th ; iav, 97 th ). finally, a preprint of a crispr/cas9 study described validation of 27 human genes as critical modulators of the host response to sars2 infection of human cells 17 . corroborating our analysis, 16 of these genes have significant (q < 0.05) rankings in the sars2 consensome, including ace2 and dyrk1a (both 97 th percentile), ctsl (96 th percentile), kdm6a, atrx, pias1 (all 94 th percentile), rad54l2 and smad3 (90 th percentile). to gather evidence for human signaling pathway nodes orchestrating the transcriptional response to cov infection, we next compared transcripts with elevated rankings in the cov consensomes with those that have predicted high confidence regulatory relationships with cellular signaling pathway nodes. we generated four lists of genes corresponding to the mers, sars1, sars2 and iav transcriptomic consensome 95 th percentiles. we then retrieved genes in the 95 th percentiles of available spp human transcriptomic (n = 25) consensomes and chip-seq (n = 864) pathway node consensomes 10 . for convenience we will refer from hereon to genes in the 95 th percentile of a viral infection, node (chip-seq) or node family (transcriptomic) consensome as high confidence transcriptional targets (hcts). we then used the r geneoverlap package 18 to compute the extent and significance of intersections between cov hcts and those of the pathway nodes or node families. we interpreted the extent and significance of intersections between hcts for covs and pathway node or node families as evidence for a biological relationship between loss or gain of function of that node (or node family) and the transcriptional response to infection by a specific virus. results of viral infection and signaling node hct intersection analyses are shown in fig. 2 ifit1 ifih1 ddx58 ifi6 ifit3 mx1 isg15 irf9 ifit2 isg20 ifitm3 ifitm1 ifi35 ifi35 irf7 ifit5 ifi35 irf7 ifit5 ifitm2 ifi16 ifi30 sars2 consensome percentile consensome q-value sars1 0 10 20 30 40 50 60 70 80 90 100 1e-55 1e-47 1e-39 1e-31 1e-23 1e-15 1e-07 ifit1 ifi6 ifi35 ifit2 mx1 ifitm1 ddx58 isg15 oas1 irf9 ifit5 ifih1 irf7 ifitm3 isg20 ifit3 ifitm2 ifi27 ifi16 ifi30 0 10 20 iav ifit2 ifi27 mx1 ifitm1 ifitm3 irf9 ifit1 ifit3 isg15 oas1 ddx58 irf7 ifih1 ifi35 ifit5 isg20 ifitm2 ifi6 ifi16 ifi30 a. b. c. d. www.nature.com/scientificdata www.nature.com/scientificdata/ transcription factors and enzymes, respectively) and figshare file f2 12 (based on chip-seq consensomes for selected co-nodes). figshare file f1 12 , sections 6 (node family transcriptomic hct intersection analysis) and 7 (node chip-seq hct intersection analysis) contain the full underlying numerical data. please refer also to table 1 for links to virus-node family and virus-node hct intersection networks in the ndex repositorysee section "visualization of the cov transcriptional regulatory networks in the signaling pathways project knowledgebase and network data exchange repository" below and the instructional youtube video (http://tiny. cc/2i56rz; strategy 6) for instructions on navigating these resources. we surveyed q < 0.05 hct intersections to identify (i) canonical inflammatory signaling pathway nodes with characterized roles in the response to cov infection, thereby validating the consensome approach in this context; and (ii) evidence for nodes whose role in the transcriptional biology of cov infection is previously uncharacterized, but consistent with their roles in the response to other viral infections. in the following sections all q-values refer to those obtained using the geneoverlap analysis package in r 18 . receptors reflecting their well-documented roles in the response to cov infection [19] [20] [21] [22] , we observed appreciable significant intersections between cov hcts and those of the toll-like (tlrs; q-values: sars1, 3e-85; sars2, 5e-49; mers, 2e-33), interferon (ifnr; q-values: sars1, 1e-109; sars2, 6e-53; mers, 1e-24) and tumor necrosis factor (tnfr; q-values: sars1, 1e-48; sars2, 1e-35; mers, 5e-32) receptor families (fig. 2) . indeed, anti-tnf therapy has been recently mooted as a potential clinical approach to sars2 infection 23 . hct intersections between cov infection and receptor systems with previously uncharacterized connections to cov infection, including epidermal growth factor receptors (egfr; q-values: sars1, 4e-21; sars2, 3e-48; mers, 1e-35), and notch receptor signaling (q-values: sars1, 6e-24; sars2, 2e-33; mers, 2e-29; fig. 2 ), are consistent with their known role in the context of other viral infections [24] [25] [26] [27] [28] . the notch receptor hct intersection points to a possible mechanistic basis for the potential of notch pathway modulation in the treatment of sars2 29 . the strong hct intersection between cov infection and xenobiotic receptors (q-values: sars1, 1e-30; sars2, 1e-44; mers, 5e-32; fig. 2 ) reflects work describing a role for pregnane x receptor in innate immunity 30 and points to a potential role for members of this family in the response to cov infection. in addition, the robust intersection between hcts for sars2 infection and vitamin d receptor (q = 2e-35) is interesting in light of epidemiological studies suggesting a link between risk of sars2 infection and vitamin d deficiency 31, 32 . consistent with a robust signature for the glucocorticoid receptor across all covs (gr; q-values: sars1, 3e-35; sars2, 1e-35; mers, 7e-32), recent studies have shown the gr agonist dexamethasone is a successful therapeutic for sars2 infection 33 . finally, independent in vitro analyses confirm our predictions of the modulation by sars2 infection of erbb/egfr 22, 34 and tgfbr 17,34 signaling systems (fig. 2) . transcription factors not unexpectedly -and speaking again to validation of the consensomes -the strongest and most significant cov hct intersections were observed for hcts for known transcription factor mediators of the transcriptional response to cov infection, including members of the nfκb (q-value ranges: sars1, 1e-7-1e-9; sars2, 9e-3-2e-3; mers, 1e-3-1e-4) [35] [36] [37] , irf (q-value ranges: sars1, 2e-2-1e-31; sars2, 2e-4-1e-17; mers, 9e-4-7e-5) 38 and stat (q-value ranges: sars1, 1e-7-1e-55; sars2, 2e-3-3e-29; mers, 5e-2-3e-5) [39] [40] [41] transcription factor families (fig. 3) . consistent with the similarity between sars1 and iav consensomes with respect to elevated rankings of isgs (fig. 2a,d) , the irf1 hct intersection was strongest with the sars1 (q = 2e-34) and iav (q = 3e-49) hcts. corroborating our finding of a strong intersection between stat2 and sars2 infection hcts (q = 3e-29), a recent preprint has shown that stat2 plays a prominent role in the response to sars2 infection of syrian hamsters 42 . hct intersections for nodes originally characterized as having a general role in rna pol ii transcription, including tbp (q-values: sars1, 2e-10; sars2, 6e-23; mers, 3e-16), gtf2b/ www.nature.com/scientificdata www.nature.com/scientificdata/ tfiib (q-values: sars1, 7e-10; sars2, 3e-23; mers, 9e-14) and gtf2f1 (q-values: sars1, 2e-4; sars2, 2e-13; mers, 5e-5) were strong across all covs, and particularly noteworthy in the case of sars2. in the case of gtf2b, these data are consistent with previous evidence identifying it as a specific target for orthomyxovirus 43 , and the herpes simplex 44 and hepatitis b 45 viruses. moreover, a recent preprint has identified a high confidence interaction between gtf2f2 and the sars2 nsp9 replicase 34 . white cells represent q > 5e-2 intersections. due to space constraints some class and family names may differ slightly from those in the spp knowledgebase. all q-values refer to those obtained using the geneoverlap analysis package in r 18 . full numerical data are provided in figshare file f1 12 , section 7; see also . this is likely due to the fact that chip-seq consensomes are based on direct promoter binding by a specific node antigen, whereas transcriptomic consensomes encompass both direct and indirect targets of specific receptor and enzyme node families. enzymes compared to the roles of receptors and transcription factors in the response to viral infection, the roles of signaling enzymes are less well illuminated -indeed, in the context of cov infection, they are entirely intersections. due to space constraints some class and family names may differ slightly from those in the spp knowledgebase. all q-values refer to those obtained using the geneoverlap analysis package in r 18 . full numerical data are provided in figshare file f1 12 , section 7; see also table 1 for links to virus-node hct intersection networks in the ndex repository. www.nature.com/scientificdata www.nature.com/scientificdata/ unstudied. through their regulation of cell cycle transitions, cyclin-dependent kinases (cdks) play important roles in the orchestration of dna replication and cell division, processes that are critical in the viral life cycle. cdk6, which has been suggested to be a critical g1 phase kinase 46, 47 , has been shown to be targeted by a number of viral infections, including kaposi's sarcoma-associated herpesvirus 48 and hiv-1 49 . consistent with this common role across distinct viral infections, we observed robust intersection between the cdk family hcts (q-values: sars1, 8e-23; sars2, 2e-31; mers, 1e-30; fig. 2 ) and the cdk6 hcts (q-values: sars1, 1e-7; sars2, 8e-8; mers, 3e-4; fig. 4 ) and those of all viral hcts. as with the tlrs, ifnrs and tnfrs, which are known to signal through cdk6 50,51 , intersection with the cdk6 hcts was particularly strong in the case of the sars2 hcts (fig. 4) . again, the subsequent proteomic analysis we alluded to earlier 34 independently corroborated our prediction of a role for cdk6 in the response to sars2 infection. consistent with a recent study 52 , the intersection of hcts for the lysine demethylase kmt2a was much stronger with iav (q-value = 2e-9) than with any of the covs (q-values: sars1, 7e-2; sars2, 2e-3; mers, 1e-2). ccnt2 is another member of the cyclin family that, along with cdk9, is a component of the viral-targeted p-tefb complex 53 . reflecting a potential general role in viral infection, appreciable intersections were observed between the ccnt2 hcts and all viral hcts (q-values: sars1, 4e-4; sars2, 6e-3; mers, 7e-5; fig. 4 ). finally in the context of enzymes, the dna topoisomerases have been shown to be required for efficient replication of simian virus 40 54 and ebola 55 viruses. the prominent intersections between dna topoisomerase-dependent hcts and the cov hcts (q-values: sars1, 3e-15; sars2, 6e-21; mers, 1e-26; fig. 2 ) suggest that it may play a similar role in facilitating the replication of these covs. hypothesis generation use cases. we next wished to show how the cov consensomes and hct intersection networks, supported by existing canonical literature knowledge, enable the user to generate novel hypotheses around the transcriptional interface between cov infection and human cellular signaling pathways. given the current interest in sars2, we have focused our use cases on that virus. figshare file f2 12 contains an additional use case linking the telomerase catalytic subunit tert to cov infection that was omitted from the main text due to space constraints. unless otherwise stated, all q-values below were obtained using the geneoverlap analysis package in r 18 . we stress that all use cases represent preliminary in silico evidence only, and require rigorous pressure-testing at the bench for full validation. hypothesis generation use case 1: transcriptional regulation of the sars2 receptor gene, ace2. ace2, encoding membrane-bound angiotensin converting enzyme 2, has gained prominence as the target for cellular entry by sars1 56 and sars2 57 . an important component in the development of ace2-centric therapeutic responses is an understanding of its transcriptional responsiveness to cov infection. interestingly, based on our cov consensome analysis, ace2 is more consistently transcriptionally responsive to infection by sars covs (sars1: 98 th percentile, consensome q value (cqv) 10 = 1e-25; sars2: 97 th percentile, cqv = 4e-7) than by iav (78 th percentile, cqv = 3e-8) or mers (49 th percentile, cqv = 2e-16; figshare file f1 12 , sections 2-5). the data points underlying the cov consensomes indicate evidence for tissue-specific differences in the nature of the regulatory relationship between ace2 and viral infection. in response to sars1 infection, for example, ace2 is induced in pulmonary cells but repressed in kidney cells (fig. 5) . on the other hand, in response to sars2 infection, ace2 is repressed in pulmonary cells -a finding corroborated by other studies 58,59 -but inducible in gastrointestinal cells (fig. 5) . these data may relate to the selective transcriptional response of ace2 to signaling by ifnrs (92 nd percentile; figshare file f1 12 , section 8) rather than tlrs (48 th percentile; figshare file f1 12 , section 9) or tnfrs (13 th percentile, figshare file f1 12 , section 10). these findings are consistent with a recent study confirming repression of induction of ace2 by interferon stimulation and by iav infection 60 . our data reflect a complex transcriptional relationship between ace2 and viral infection that may be illuminated in part by future single cell rna-seq analysis in the context of clinical or animal models of sars2 infection. hypothesis generation use case 2: evidence for antagonistic cross-talk between progesterone receptor and interferon receptor signaling in the airway epithelium. a lack of clinical data has so far prevented a definitive evaluation of the connection between pregnancy and susceptibility to sars2 infection in covid-19. that said, sars2 infection is associated with an increased incidence of pre-term deliveries 61 , and pregnancy has been previously associated with the incidence of viral infectious diseases, particularly respiratory infections 62, 63 . we were therefore interested to observe consistent intersections between the progesterone receptor (pgr) hcts and cov infection hcts (q-values: sars1, 3e-35; sars2, 5e-41; mers 5e-28), with the intersection being particularly evident in the case of the sars2 hcts ( fig. 2; figshare file f1 12 , section 6). to investigate the specific nature of the crosstalk implied by this transcriptional intersection in the context of the airway epithelium, we first identified a set of 12 genes that were hcts for both sars2 infection and pgr. interestingly, many of these genes encode members of the classic interferon-stimulated gene (isg) response pathway 13 . we then retrieved two spp experiments involving treatment of a549 airway epithelial cells with the pgr full antagonist ru486 (ru), alone or in combination with the gr agonist dexamethasone (dex). as shown in fig. 6 , there was unanimous correlation in the direction of regulation of all 12 genes in response to cov infection and pgr loss of function. these data are consistent with the reported pro-inflammatory effects of ru486 in a mouse model of allergic pulmonary inflammation 64 . interestingly, sars2-infected pregnant women are often asymptomatic 65, 66 . based on our data, it can be reasonably hypothesized that suppression of the interferon response to sars2 infection by elevated circulating progesterone during pregnancy may contribute to the asymptomatic clinical course. indeed, crosstalk between progesterone and inflammatory signaling is well characterized in the reproductive system, most notably in the establishment of uterine receptivity 67 as well as in ovulation 68 . consistent with our hypothesis, a recently launched clinical trial is evaluating the potential of progesterone for treatment of covid-19 in hospitalized men 69 . interestingly, the recently reported inhibition by progesterone of sars2 replication in www.nature.com/scientificdata www.nature.com/scientificdata/ vero 6 cells 70 indicates an additional mechanism, distinct from its potential crosstalk with the interferon response, by which progesterone signaling may impact sars2 infection. hypothesis generation use case 3: association of an epithelial to mesenchymal transition transcriptional signature with sars2 infection. epithelial to mesenchymal transition (emt) is the process by which epithelial cells lose their polarity and adhesive properties and acquire the migratory and invasive characteristics of mesenchymal stem cells 71 . emt is known to contribute to pulmonary fibrosis 72 , acute interstitial pneumonia 73 and acute respiratory distress syndrome (ards) 74 , all of which have been reported in connection with sars2 infection in covid-19 [75] [76] [77] . we were interested to note therefore that significant hct intersections for three well characterized emt-promoting transcription factors were specific to sars2 infection (q-values: snai2/slug 78 , 2e-2; epas1/hif2α 79 , 9e-9; lef1 80 , 1e-3; fig. 3 , bold symbols; figshare file f1 12 , section 7). consistent with this, intersections between hcts for tgfbrs, smad2 and smad3, known regulators of emt transcriptional programs 81 -were stronger with hcts for sars2 (q-values: tgfbrs, 2e-31; smad2, 2e-7; smad3, 5e-17) than with those of sars1 (q-values: tgfbrs, 6e-29; smad2, 2e-2; smad3, 3e-9) and mers (q-values: tgfbrs, 1e-16; smad2, 3e-3; smad3, 2e-12) -see also figs. 2 and 3 and figshare file f1 12 , sections 6 and 7). moreover, a recent crispr/cas9 screen identified a requirement for both tgfbr signaling and smad3 in mediating sars2 infection 17 . to investigate the connection between sars2 infection and emt implied by these hct intersections, we then computed intersections between the individual viral hcts and a list of 335 genes manually curated from the research literature as emt markers 82 , see also figshare file f1 12 , section 11. in agreement with the hct intersection analysis, we observed significant enrichment of members of this gene set within the sars2 hcts (q = 4e-14), but not the sars1 or mers (both q = 2e-1) hcts (fig. 7a) . consistent with previous reports of a potential link between emt and iav infection 83 , we observed significant intersection between the emt signature and the iav hcts (q = 1e-4). one possible explanation for the selective intersection between the literature emt signature and the sars2 hcts relative to sars1 and mers was the fact that the sars2 consensome was exclusively comprised of epithelial cell lines, whereas the sars1 and mers consensomes included non-epithelial cell biosamples (figshare www.nature.com/scientificdata www.nature.com/scientificdata/ file f1 12 , section 1). to exclude this possibility therefore, we next calculated airway epithelial cell-specific consensomes for sars1, sars2 and mers and computed intersections between their hcts and the emt signature. we found that significant intersection of the emt signature with the cov hcts remained specific to sars2 (q-values: sars1 & mers, 2e-1; sars2, 1e-8) in the lung epithelium-specific cov consensomes. we next retrieved the canonical emt genes in the sars2 hcts and compared their percentile rankings with the other cov consensomes. although some emt genes, such as cxcl2 and irf9, had elevated rankings across all four viral consensomes, the collective emt gene signature had a significantly higher mean percentile value in the sars2 consensome than in each of the other viral consensomes ( fig. 7b ; sars2 mean percentile = 97.5; sars1 mean percentile = 86, p = 1e-5, t-test sars2 v sars1; mers mean percentile = 63, p = 1e-9, t-test sars2 v mers; iav mean percentile = 76, p = 2e-7, t-test sars2 v iav)). a column named "emt" in figshare file f1 12 , sections 2 (sars1), 3 (sars2), 4 (mers) and 5 (iav) identifies the ranking of the emt genes in each of the viral consensomes. given that emt has been linked to ards 74 , we speculated that the evidence connecting emt and sars2 acquired through our analysis might be reflected in the relatively strong intersection between ards markers in sars2 hcts compared to other viral hcts. to test this hypothesis we carried out a pubmed search to identify a set of 88 expression biomarkers of ards or its associated pathology, acute lung injury (ali). a column named "ali/ards" in figshare file f1 12 , sections 2 (sars1), 3 (sars2) 4 (mers) and 5 (iav) identifies the expression biomarker genes using the pubmed identifiers for the original studies in which they were identified. consistent with our hypothesis, we observed appreciable intersections between this gene set and the hcts of all four viruses (sars1 odds ratio (or) = 7, q = 5e-9; sars2 or = 10.4, q = 1e-9; mers, or = 4.2, q = 2e-5; iav or = 6.8; q = 9e-8) with a particularly strong intersection evident in the sars2 hcts. although emt has been associated with infection by transmissible gastroenteritis virus 84 and iav 83 , this is to our knowledge the first evidence connecting cov infection, and specifically sars2 infection, to an emt signature. interestingly, lipotoxin a4 has been shown to attenuate lipopolysaccharide-induced lung injury by reducing emt 85 . moreover, several members of the group of sars2-induced emt genes have been associated with signature pulmonary comorbidities of cov infection, including adar 86 , cldn1 87 and sod2 88 . of note in the context of these data is the fact that signaling through two sars2 cellular receptors, ace2/at2 and cd147/basigin, has been linked to emt in the context of organ fibrosis [89] [90] [91] . finally, a recent preprint has described emt-like transcriptional and metabolic changes in response to sars2 infection 92 . collectively, our data indicate that emt warrants further investigation as a sars2-specific pathological mechanism. . based on these data, we speculated that sars2 infection might impact the expression of e2f-regulated cell cycle genes more efficiently than other covs. to investigate this we retrieved a set of sars2 hcts that were also hcts for at least three of e2f1, e2f3, e2f4 and tfdp1 (figshare file f1 12 , section 3, columns e2f1, e2f3, e2f4, tfdp1 & 95th 3/4). consistent with the role of e2f/dp-1 nodes in the regulation of the cell cycle, many of these genes -notably cdk1, pcna, cdc6, cenpf and nusap1 -are critical positive regulators of dna replication and cell cycle progression [97] [98] [99] [100] [101] and are known to be transcriptionally induced by e2fs 102-105 . strikingly, with the exception of e2f3, all were consistently repressed in response to sars2 infection (fig. 8a ). to gain insight into the relative efficiency with which the four viruses impacted expression of the e2f/dp-1 hct signature, we compared their mean percentile values across the viral consensomes. consistent with efficient repression of the e2f/dp-1 hcts by sars2 infection relative to other viruses, their mean percentile ranking was appreciably higher in the sars2 consensome (97 th percentile) than in the sars1 (76 th percentile; p = 6e-12, t-test sars2 v sars1), mers (71.2 percentile; p = 9e-6, t-test sars2 v mers) and iav (71.2 percentile; p = 2e-5, t-test sars2 v iav) consensomes (fig. 8b) . although manipulation of the host cell cycle and evasion of detection through deregulation of cell cycle checkpoints has been described for other viruses [106] [107] [108] , this represents the first evidence for the profound impact of sars2 infection on host cell cycle regulatory genes, potentially through disruption of e2f mediated signaling pathways. the sars2 infection-mediated induction of e2f3 (fig. 8a) may represent a compensatory response to transcriptional repression of other e2f family members, as has been previously observed for this family in other contexts 109, 110 . consistent with our prediction in this use case, while this paper was in revision, a study appeared showing that infection by sars2 results in cell cycle arrest 111 . our results represent evidence that efficient modulation by sars2 of e2f signaling, resulting in repression of cell cycle regulatory genes, may contribute to its unique pathological impact. to enable researchers to routinely generate mechanistic hypotheses around the interface between cov infection human cell signaling, we next made the consensomes and accompanying hct intersection analyses freely available to the research community in the spp knowledgebase and the network data exchange (ndex) repository. table 1 contains digital object identifier (doi)-driven links to the consensome networks in spp and ndex, and to the virus-node and virus-node family hct intersection networks in ndex. we have previously described the spp biocuration pipeline, database and web application interface 10 . figure 9 shows the strategy for consensome data mining on the spp website. the individual cov consensomes can be accessed by configuring the spp ominer query form as shown, in this example for the sars2 consensome (fig. 9a) . figure 9b shows the layout of the consensomes, showing gene symbol, name, percentile ranking and www.nature.com/scientificdata www.nature.com/scientificdata/ other essential information. genes in the 90 th percentile of each consensome are accessible via the user interface, with the full consensomes available for download in a tab delimited text file. target gene symbols in the consensome link to the spp regulation report, filtered to show only experimental data points that contributed to that specific consensome (fig. 9c ). this view gives insights into the influence of tissue and cell type context on the regulatory relationship. these filtered reports can be readily converted to default reports that show evidence for regulation of a specific gene by other signaling pathway nodes. as previously described, pop-up windows in the report provide experimental details, in addition to links to the parent dataset (fig. 9d) , curated accordingly to our previously described protocol 10 . per fair data best practice, cov infection datasets -like all spp datasetsare associated with detailed descriptions, assigned a doi, and linked to the associated article to place the dataset in its original experimental context (fig. 9d) . the full list of datasets is available for browsing in the spp dataset listing (https://www.signalingpathways.org/index.jsf). the ndex repository facilitates collaborative publication of biological networks, as well as visualization of these networks in web or desktop versions of the popular and intuitive cytoscape platform [112] [113] [114] . figure 10 shows examples of consensome and hct intersection network visualizations within the ndex user interface, in which transcripts or nodes, respectively, are organized using spp's category-class-family classification 10 . for ease of viewing, the initial rendering of the full sars2 (fig. 10a ) and other consensome networks shows a sample (fig. 10a, red arrow 1) containing only the top 5% of regulated transcripts; the full data can be explored using the "neighborhood query" feature available at the bottom of the page (red arrow 2). the integration in ndex of the popular cytoscape desktop application enables any network to be seamlessly be imported in cytoscape for additional analysis (red arrow 3). zooming in on a subset of the sars2 consensome (orange box) affords an appreciation of the diversity of molecular classes that are transcriptionally regulated in response to sars2 infection (fig. 10b) . transcript size is proportional to rank percentile, and edge weight is proportional to the transcript geometric mean fold change (gmfc) value. selecting a transcript allows the associated consensome data, such as rank, gmfc and family, to be examined in detail using the info table (fig. 10b, right panel) . highlighted to exemplify this feature is il6, an inflammatory ligand that has been previously linked to sars2 infection-related www.nature.com/scientificdata www.nature.com/scientificdata/ pathology 8, 115 . consensome gmfcs are signless with respect to direction of regulation 10 . researchers can therefore follow the spp link in the info table (fig. 10b, red arrow 4) to view the individual underlying viral infection data points on the spp site (fig. 9c shows the example for ifi27) . a network of the top 20 ranked transcripts in the sars2 consensome (fig. 10c) includes genes with known (oas1, mx1 116 ) and previously uncharacterized (pdzkip1, sat1, tm4sf4) transcriptional responses to sars2 infection. finally, to afford insight into pathway nodes whose gain or loss of function contributes to sars2 infection-induced signaling, fig. 10d shows the top 5% ranked nodes in the sars2 node hct chip-seq intersection network (see figshare file f1 12 , section 7; see also figs. 2 and 3 and accompanying discussion above). in this, as with all hct intersection networks, node size is proportional to the q-value, such that the larger the circle, the lower the q-value, and the higher the confidence that a particular node or node family is involved in the transcriptional response to viral infection. the ndex interface leverages the spp classification system to provide visual insights into the impact of cov infection on human cell signaling that are not readily appreciated in the current spp interface. for example, it is readily apparent from the ndex sars2 consensome network ( fig. 10c; table 1 ) that the single largest class of sars2 hcts encodes immunomodulatory ligands (or = 4.6, p = 3.8 e-24, hypergeometric test), many of which are members of the cytokine and chemokine superfamilies. in contrast, although still overabundant (or = 1.58, p = 6.8e-4, hypergeometric test), inflammatory ligands comprise a considerably smaller proportion of the sars1 hcts (table 1) . these data represent evidence that compared to sars1, sars2 infection may be relatively efficient in modulating a transcriptional inflammatory response in host cells. clicking on a data point opens a fold change information window that links to the spp curated version of the original archived dataset (d). like all spp datasets, cov infection datasets are comprehensively aligned with fair data best practice and feature human-readable names and descriptions, a doi, one-click addition to citation managers, and machine-readable downloadable data files. for a walk-through of cov consensome data mining options in spp, please refer to the accompanying youtube video (http://tiny.cc/2i56rz). www.nature.com/scientificdata www.nature.com/scientificdata/ fig. 10 visualization of viral consensomes and hct intersection networks in the ndex repository. in all panels, transcripts (consensome networks; panels a-c) and nodes (hct intersection network; panel d) are color-coded according to their category as follows: receptors (orange), enzymes (blue), transcription factors (green), ion channels (mustard) and co-nodes (grey). additional contextual information is available in the description of each network on the ndex site. red arrows are explained in the text. www.nature.com/scientificdata www.nature.com/scientificdata/ an effective research community response to the impact of cov infection on human health demands systematic exploration of the transcriptional interface between cov infection and human cell signaling systems. it also demands routine access to computational analysis of existing datasets that is unhindered either by paywalls or by lack of the informatics training required to manipulate archived datasets in their unprocessed state. moreover, the substantial logistical obstacles to high containment laboratory certification emphasize the need for fullest possible access to, and re-usability of, existing cov infection datasets to focus and refine hypotheses prior to carrying out in vivo cov infection experiments. meta-analysis of existing datasets represents a powerful approach to establishing consensus transcriptional signatures -consensomes -which identify those human genes whose expression is most consistently and reproducibly impacted by cov infection. moreover, integrating these consensus transcriptional signatures with existing consensomes for cellular signaling pathway nodes can illuminate transcriptional convergence between cov infection and human cell signaling nodes. to this end, we generated a set of cov infection consensomes that rank human genes by the reproducibility of their differential expression (p < 0.05) in response to infection of human cells by covs. using hct intersection analysis, we then computed the cov consensomes against high confidence transcriptional signatures for a broad range of cellular signaling pathway nodes, affording investigators with a broad range of signaling interests an entrez into the study of cov infection of human cells. although other enrichment based pathway analysis tools exist 117 , hct intersection analysis differs from these by computing against only genes that have the closest predicted regulatory relationships with upstream pathway nodes (i.e. hcts). the use cases described here represent illustrative examples of the types of hct-based analyses that users are empowered to carry out to illuminate principles of cov infection signaling. previous network analyses across independent viral infection transcriptomic datasets, although valuable, have been limited to stand-alone studies 118, 119 . here, to enable access to the cov consensomes and their >3,000,000 underlying data points by the broadest possible audience, we have integrated them into the spp knowledgebase and ndex repository to create a unique, federated environment for generating hypotheses around the impact of cov infection on human cell signaling. ndex provides users with the familiar look and feel of cytoscape to reduce barriers of accessibility, and provides for intuitive click-and-drag data mining strategies. incorporation of the cov data points into spp places them in the context of millions more existing spp data points documenting transcriptional regulatory relationships between human pathway nodes and their transcriptional targets. in doing so, we provide users with evidence for signaling nodes whose gain or loss of function in response to cov infection gives rise to these transcriptional patterns. the transcriptional impact of viral infection is known to be an amalgam of host antiviral responses and co-option by viruses of the host signaling machinery in furtherance of its life cycle. it is hoped that dissection of these two distinct modalities in the context of cov infection will be facilitated by the availability of the cov consensomes in the spp and ndex knowledgebases. the cov consensomes have a number of limitations. primarily, since they are predicated specifically on transcriptional regulatory technologies, they will assign low rankings to transcripts that may not be transcriptionally responsive to cov infection, but whose encoded proteins nevertheless play a role in the cellular response. for example, masp2, which encodes an important node in the response to cov infection 120 , has either a very low consensome ranking (sars1, mers and iav), or is absent entirely (sars2), indicating that it is transcriptionally unresponsive to viral infection and likely activated at the protein level in response to upstream signals. this and similar instances therefore represent "false negatives" in the context of the impact of cov infection on human cells. another limitation of the transcriptional focus of the datasets is the absence of information on specific protein interactions and post-translational modifications, either viral-human or human-human, that give rise to the observed transcriptional responses. although these can be inferred to some extent, integration of existing 34, 70, 111 and future proteomic and kinomic datasets will facilitate modeling of the specific signal transduction events giving rise to the downstream transcriptional responses. finally, although detailed metadata are readily available on the underlying data points, the consensomes do not directly reflect the impact of variables such as tissue context or duration of infection on differential gene expression. as additional suitable archived datasets become available, we will be better positioned to generate more specific consensomes of this nature. the human cov and iav consensomes and their underlying datasets are intended as "living" resources in spp and ndex that will be updated and versioned with appropriate datasets as resources permit. this will be particularly important in the case of sars2, given the expanded budget that worldwide funding agencies are likely to allocate to research into the impact of this virus on human health. incorporation of future datasets will allow for clarification of observations that are intriguing, but whose significance is currently unclear, such as the intersection between the cov hcts and those of the telomerase catalytic subunit (figshare file f2 12 ), as well as the enrichment of emt genes among those with elevated rankings in the sars2 consensome (fig. 7) . although they are currently available on the spp website, distribution of the cov consensome data points via the spp restful api 10 will be essential for the research community to fully capitalize on this work. for example, several co-morbidities of sars2 infection, including renal and gastrointestinal disorders, are within the mission of the national institute of diabetes, digestive and kidney diseases. in an ongoing collaboration with the niddk information network (dknet) 121 , the spp api will make the cov consensome data points available in a hypothesis generation environment that will enable users to model intersections of cov infection-modulated host signaling with their own research areas of interest. we welcome feedback and suggestions from the research community for the future development of the cov infection consensomes and hct node intersection networks. consistent with emerging nih mandates on rigor and reproducibility, we have used the research resource identifier (rrid) standard 122 to identify key research resources of relevance to our study. (2020) 7:314 | https://doi.org/10.1038/s41597-020-00628-6 www.nature.com/scientificdata www.nature.com/scientificdata/ dataset biocuration. datasets from gene expression omnibus (rrid: scr_005012) and array express (rrid: scr_002964) were biocurated as previously described, with the incorporation of an additional classification of peptide ligands 123 to supplement the existing mappings derived from the international union of pharmacology guide to pharmacology (rrid: scr_013077). dataset processing and consensome analysis. array data processing. to process microarray expression data, we utilized the log2 summarized and normalized array feature expression intensities provided by the investigator and housed in geo. these data are available in the corresponding "series matrix files(s)". the full set of summarized and normalized sample expression values were extracted and processed in the statistical program r. to calculate differential gene expression for investigator-defined experimental contrasts, we used the linear modeling functions from the bioconductor limma analysis package 124 . initially, a linear model was fitted to a group-means parameterization design matrix defining each experimental variable. subsequently, we fitted a contrast matrix that recapitulated the sample contrasts of interest, in this case viral infection vs mock infection, producing fold-change and significance values for each array feature present on the array. the current bioconductor array annotation library was used for annotation of array identifiers. p values obtained from limma analysis were not corrected for multiple comparisons. rna-seq data processing. to process rna-seq expression data, we utilized the aligned, un-normalized, gene summarized read count data provided by the investigator and housed in geo. these data are available in the corresponding "supplementary file" section of the geo record. the full set of raw aligned gene read count values were extracted and processed in the statistical program r using the limma 124 and edger analysis 125 packages. read count values were initially filtered to remove genes with low read counts. gene read count values were passed to downstream analysis if all replicate samples from at least one experimental condition had cpm >1. sequence library normalization factors were calculated to apply scale normalization to the raw aligned read counts using the tmm normalization method implemented in the edger package followed by the voom function 126 to convert the gene read count values to log2-cpm. the log2-cpm values were initially fit to a group-means parameterization design matrix defining each experimental variable. this was subsequently fit to a contrast design matrix that recapitulates the sample contrasts of interest, in this case viral infection vs mock infection, producing fold-change and significance values for each aligned sequenced gene. if necessary, the current bioconductor human organism annotation library was used for annotation of investigator-provided gene identifiers. p values obtained from limma analysis were not corrected for multiple comparisons. differential expression values were committed to the consensome analysis pipeline as previously described 10 . briefly, the consensome algorithm surveys each experiment across all datasets and ranks genes according to the frequency with which they are significantly differentially expressed. for each transcript, we counted the number of experiments where the significance for differential expression was ≤0.05, and then generated the binomial probability, referred to as the consensome p-value (cpv), of observing that many or more nominally significant experiments out of the number of experiments in which the transcript was assayed, given a true probability of 0.05. genes were ranked firstly by cpv, then by geometric mean fold change (gmfc). a more detailed description of the transcriptomic consensome algorithm is available in a previous publication 10 . the consensomes and underlying datasets were loaded into an oracle 15c database and made available on the spp user interface as previously described 10 . statistical analysis. high confidence transcriptional target intersection analysis was performed using the bioconductor geneoverlap analysis package 18 (rrid: scr_018419) implemented in r. briefly, given a whole set i of ids and two sets a ∈ i and b ∈ i, and s = a ∩ b, geneoverlap calculates the significance of obtaining s. the problem is formulated as a hypergeometric distribution or contingency table, which is solved by fisher's exact test. p-values were adjusted for multiple testing by using the method of benjamini & hochberg to control the false discovery rate as implemented with the p.adjust function in r, to generate q-values. the universe for the intersection was set at a conservative estimate of the total number of transcribed (protein and non protein-coding) genes in the human genome (25,000) 127 . r code for analyzing the intersection between an investigator gene set and cov consensome hcts has been deposited in the spp github account. a two tailed two sample t-test assuming equal variance was used to compare the mean percentile ranking of the emt (12 degrees of freedom) and e2f (14 degrees of freedom) signatures in the mers, sars1, sars2 and iav consensomes using the prism software package v 7.0 (rrid: scr_005375). the procedure for generating transcriptomic consensomes has been previously described 10 . to generate the chip-seq consensomes, we first retrieved processed gene lists from chip-atlas 128 (rrid: scr_015511), in which human genes are ranked based upon their average macs2 occupancy across all publically archived datasets in which a given pathway node is the ip antigen. of the three stringency levels available (10, 5 and 1 kb from the transcription start site), we selected the most stringent (1 kb). according to spp convention 10 , we then mapped the ip antigen to its pathway node category, class and family, and the experimental cell line to its appropriate biosample physiological system and organ. we then organized the ranked lists into percentiles to generate the node chip-seq consensomes. the 95 th percentiles of all consensomes (hcts, high confidence transcriptional targets) was used as the input for the hct intersection analysis. spp web application. the spp knowledgebase (rrid: scr_018412) is a gene-centric java enterprise edition 6, web-based application around which other gene, mrna, protein and bsm data from external databases such as ncbi are collected. after undergoing semiautomated processing and biocuration as described above, the data and annotations are stored in spp's oracle 15c database. restful web services exposing spp data, which are served to responsively designed views in the user interface, were created using a flat ui toolkit with a combination of javascript, d3.js, ajax, html5, and css3. javaserver faces and primefaces are the primary www.nature.com/scientificdata www.nature.com/scientificdata/ technologies behind the user interface. spp has been optimized for firefox 24+, chrome 30+, safari 5.1.9+, and internet explorer 9+, with validations performed in browserstack and load testing in loaduiweb. xml describing each dataset and experiment is generated and submitted to crossref (rrid: scr_003217) to mint dois 10 . important note on data availability: this paper refers to the first versions of the consensomes and hct intersection networks based on the datasets available at the time of publication. as additional cov infection datasets are archived over time, we will make updated versions of the consensomes and hct intersection analyses accessible in future releases. the entire set of experimental metadata is available in figshare file f1 12 , section 1. consensome data points are in figshare file f1 12 , sections 2-5. spp spp mers 129 , sars1 130 , sars2 131 and iav 132 consensomes, their underlying data points and metadata, as well as original datasets, are freely accessible at https://ww.signalingpathways.org. programmatic access to all underlying data points and their associated metadata are supported by a restful api at https://www.signalingpathways.org/docs/. all spp datasets are biocurated versions of publically archived datasets, are formatted according to the recommendations of the force11 joint declaration on data citation principles, and are made available under a creative commons cc by 4.0 license. the original datasets are available are linked to from the corresponding spp datasets using the original repository accession identifiers. these identifiers are for transcriptomic datasets, the gene expression omnibus (geo) series (gse); and for cistromic/chip-seq datasets, the ncbi sequence read archive (sra) study identifier (srp). spp consensomes are assigned dois as shown in table 1 . ndex ndex versions of consensomes (mers 133 , sars1 134 , sars2 135 and iav 136 ) and node family (mers 137 , sars1 138 , sars2 139 and iav 140 ) and node (mers 141 , sars1 142 , sars2 143 and iav 144 ) hct intersection networks are freely available in the ndex repository and assigned dois as shown in table 1 . ndex is a recommended repository for scientific data, springer nature and the plos family of journals and is registered on fairsharing. org; for additional info and documentation, please visit http://ndexbio.org. the official spp account in ndex is available at: https://bit.ly/30nn129. spp source code is available in the spp github account under a creative commons cc by 4.0 license at https:// github.com/signaling-pathways-project. toll-like receptors how cells respond to interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis cell host response to infection with novel human coronavirus emc predicts 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reveals extensive transcriptional noise chip-atlas: a data-mining suite powered by full integration of public chip-seq data middle east respiratory syndrome coronavirus (mers-cov) transcriptomic consensome, version 1. signaling pathways project sudden acute respiratory syndrome coronavirus 1 (sars-cov-1) transcriptomic consensome, version 1. signaling pathways project sudden acute respiratory syndrome coronavirus 2 (sars-cov-2) transcriptomic consensome, version 1. signaling pathways project influenza a virus (iav) transcriptomic consensome, version 1. signaling pathways project mers-cov transcriptomic consensome -full. the network data exchange sars-cov-1 transcriptomic consensome -full. the network data exchange sars-cov-2 transcriptomic consensome -full. the network data exchange iav transcriptomic consensome -full. the network data exchange -ndex https mers-cov vs pathway node family (transcriptomic) -all families. the network data exchange sars-cov-1 vs pathway node family (transcriptomic) -all families. the network data exchange sars-cov-2 vs pathway node family (transcriptomic) -all families. the network data exchange hct overlap: iav vs pathway node family (transcriptomic) -all families. the network data exchange mers-cov vs pathway node (chip-seq) -full. the network data exchange sars-cov-1 vs pathway node (chip-seq) -full. the network data exchange sars-cov-2 vs pathway node (chip-seq) -full. the network data exchange hct overlap: iav vs pathway node (chip-seq) -full. the network data exchange analysis of the dexamethasone (dex)-dependent transcriptome in a549 lung adenocarcinoma cells this work was supported by the national institute of diabetes, digestive and kidney diseases niddk information network (dk097748), the national cancer institute (ca125123, ca184427) and by the brockman medical research foundation. we acknowledge the assistance of apollo mcowiti, shijing qu and michael dehart in making the datasets and consensomes available in the spp knowledgebase. we thank all investigators who archive their datasets, without whom spp would not be possible. the authors declare no competing interests. correspondence and requests for materials should be addressed to n.j.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-000354-05lnj3w0 authors: de vries, erik; tscherne, donna m.; wienholts, marleen j.; cobos-jiménez, viviana; scholte, florine; garcía-sastre, adolfo; rottier, peter j. m.; de haan, cornelis a. m. title: dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway date: 2011-03-31 journal: plos pathog doi: 10.1371/journal.ppat.1001329 sha: doc_id: 354 cord_uid: 05lnj3w0 influenza a virus (iav) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. whereas dynamin-dependent, clathrin-mediated endocytosis (cme) is generally considered as the iav infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. by manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of iav. whereas entry of iav in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. this finding was confirmed with the use of small interfering rnas targeting dynamin-2. in the presence of serum, both iav entry pathways were operational. under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative eipa, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. the sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in iav entry. consistently, iav particles and soluble fitc-dextran were shown to co-localize in cells in the same vesicles. thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, iav enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis. influenza a virus (iav) is an enveloped, segmented negativestrand rna virus infecting a wide variety of birds and mammals. as its first step in infection iav attaches to host cells by the binding of its major surface protein, the hemagglutinin (ha), to sialic acids, which are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. where the structural requirements for this interaction have been studied in great detail, much less is known about whether and how the attachment to specific sialylated receptors (e.g. to n-linked glycoproteins, olinked glycoproteins or gangliosides or even to specific receptors within these groups) affects the subsequent endocytic steps. obviously, knowledge about the repertoire of endocytic pathways that can successfully be used by iav will increase our insights into cell and species tropism of iav. in turn, this will contribute to our understanding of the requirements for the generation of novel viruses with pandemic potential that can arise by exchange of rna segments between currently circulating human serotypes and an animal virus during occasional co-infection in a human or an animal host. clathrin mediated endocytosis (cme) has for long been identified and studied as the major route of iav cell entry [1, 2] and is, by far, the best characterized endocytic pathway. evidence obtained from live cell imaging has revealed the de novo formation of clathrin-coated pits at the site of virus attachment [3] and the requirement for the adapter protein epsin 1, but not eps15, in this process [4] . still, specific transmembrane receptors linking viral entry to epsin 1 or to other adapters have not been identified although a recent study performed in cho cells indicated the specific requirement for n-linked glycoproteins in iav entry [5] . some recent papers provided indications for the utilization of alternative entry pathways by iav. studies in which cme was obstructed by pharmacological or genetic intervention indicated the ability of iav to enter host cells via alternative endocytic routes [4, 6, 7] . also live cell imaging revealed the simultaneous availability of entry routes involving non-coated as well as clathrin-coated pits [4] . however, this alternative iav entry route has not been characterized in any detail and requirements for any specificity in receptor usage apart from the need for the proper sialic acid moiety have not been established. during the past decades quite a variety of endocytic pathways have been identified in eukaryotic cells [8, 9, 10] . their occurrence, abundance and mechanistic details appear to vary between cell types, tissues and species and their utilization by viruses as a route of entry makes them an important factor in host and cell-type permissiveness for infection [11, 12] . besides by cme, different viruses have been shown to enter cells via caveolae, macropinocytosis or other, less well described, routes [11, 12] . most often, the selection of a specific endocytic route is linked to the utilization of a specific receptor that facilitates traveling via that particular route. nevertheless, many receptors allow flexibility by their capacity to enter through multiple pathways. for iav, an additional level of complexity to the dissection of potential entry routes is added by the apparent lack of an iav-specific protein receptor. a full experimental characterization of the iav entry pathways will benefit from separation of the iav entry pathways into routes that can be studied independently. whereas co-localization with clathrin is an established marker for endocytosis via this route, the complete lack of unique markers for macropinosomes or most other endocytic compartments [13, 14] complicates such studies. furthermore, crucial to any study concerning endocytic pathways is the abundantly documented fact that such pathways are highly dependent on experimental cell culture conditions [15] [16] [17] [18] [19] . pathways that are constitutive in one cell type may be absent or inducible by specific experimental conditions in other cell types. moreover, the manipulation of specific endocytic pathways may result in up or down regulation of other specific pathways. here we have established entry assay conditions that allow dissecting cell entry of iav into a dynamin-dependent (dyna-dep) and a dynamin-independent (dyna-ind) component. dynamin is a large gtpase forming multimeric assemblies around the neck of newly formed endocytic vesicles. gtp hydrolysis is required for pinching off of the vesicles [20] . whereas cme is completely dependent on dynamin, several other endocytic routes do not require dynamin [21] . we performed an extensive characterization of the dynamin-independent iav entry route using pharmacological inhibitors as well as by expressing dominant-negative mutants and applying sirna induced gene silencing as tools. taken together the results identify a pathway that closely resembles macropinocytosis as a novel entry pathway for iav. to identify and characterize potential non-cme entry routes taken by iav, we adapted a luciferase reporter assay [22] to enable the quantitative determination of infection or entry by measuring the activity of secreted gaussia luciferase. twentyfour hours prior to infection hela cells were transfected with a plasmid (phh-gluc) allowing constitutive synthesis (driven by the human poli promoter) of a negative strand viral rna (vrna) encoding a gaussia luciferase under control of the untranslated regions (utrs) of the np segment of influenza a/wsn/33 (h1n1) (hereafter called iav-wsn) np segment. upon iav infection, the combined expression of the viral polymerase subunits and np will drive transcription of luciferase mrna from the negative strand vrna and subsequent synthesis of gaussia luciferase. a dose-response curve demonstrating the applicability of the assay to inhibitor screening (fig. 1a) was obtained for bafilomycin a1 (bafa1), a known inhibitor of iav entry [23] . bafa1 acts upon the vacuolar-type h(+)-atpase, thus preventing endosomal acidification and thereby trapping iav in peri-nuclear immature endosomes with a lumenal ph that does not permit viral membrane fusion. remarkably, dynasore, a small molecule inhibitor of the gtpase dynamin 2 that is crucial for endocytic vesicle formation in clathrin-and caveolin-mediated endocytosis [8] as well as in a poorly described clathrin-and caveolin-independent endocytic pathway [8, 19] , did not give significant inhibition (fig. 1b) . bafa1 specifically inhibits iav during the entry phase as demonstrated in fig. 1c . the continuous presence of 10 nm bafa1 (added to the cells 1 hr prior to infection) for 16 hrs completely prevents infection. in contrast the addition of bafa1 at 1 hr or 2 hrs post infection resulted in high levels of luciferase activity (again measured at 16 hrs p.i.) that were 63% or 90% respectively of the control to which no bafa1 was added, indicating that entry was essentially completed within 2 hrs. the last bar of fig. 1c shows that the inhibition by bafa1 is reversible as withdrawal of the inhibitor after 2 hrs resulted in high levels of infection. the specific effect of bafa1 on iav entry was confirmed by confocal microscopy demonstrating that bafa1, as expected, traps iav particles in a peri-nuclear location, presumably in nonacidified endosomes (fig. 1d) . bafa1 was subsequently exploited to establish a specific iav entry assay (hereafter further referred to as the gluc-entry assay). hela cells transfected with phh-gluc were inoculated with iav at a range of mois and incubated for 2 hrs after which the entry medium was replaced by complete growth medium containing 10% fcs and 10 nm bafa1 to prevent any further entry of virus. entry was indirectly quantified by determination of luciferase activity after further incubation for 14 hrs demonstrating a quantitative correlation between infection dose and luciferase activity across a wide range of mois (fig. 1e) . the indirect gluc-entry assay was next tested for its capacity to examine the effects of inhibitors on iav entry. dynasore or bafa1 (fig. 1f) were included in the medium (dmem containing 10% attachment to and entry into a host cell are the first crucial steps in establishing a successful virus infection and critical factors in determining host cell and species tropism. influenza a virus (iav) attaches to host cells by binding of its major surface protein, hemagglutinin, to sialic acids that are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. iav subsequently enters cells of birds and a wide variety of mammals via receptormediated endocytosis using clathrin as well as via (an) alternative uncharacterized route(s). the elucidation of the endocytic pathways taken by iav has been hampered by their apparent redundancy in establishing a productive infection. by manipulating the entry conditions we have established experimental settings that allow the separate analysis of dynamin-dependent (including clathrin-mediated endocytosis) and independent entry of iav. collectively, our results indicate macropinocytosis, the main route for the non-selective uptake of extracellular fluid by cells, as an alternative iav entry route. as the dynamindependent and -independent iav entry routes are redundant and independent, their separate manipulation was crucial for the identification and characterization of the alternative iav entry route. a similar strategy might be applicable to the study of endocytic pathways taken by other viruses. fcs) during entry (the first 2 h of infection) and were removed when the inoculum was replaced by growth medium containing bafa1. concentrations up to 80 mm dynasore did not inhibit entry which is in agreement with the result shown in fig. 1b . in contrast, 1.25 nm bafa1 already inhibited entry for more than 60% (fig. 1f) . as a control, dynasore was also added at 2 hrs post infection to analyze whether the drug affected iav replication during the post entry phase. as expected, 80 mm dynasore did not significantly inhibit iav replication when present from 2 to 16 hrs p.i. (fig. 1f ). thus, with the gluc-entry assay we can study the effect of specific inhibitors on iav entry in a quantitative manner, at least as long as the inhibitors do not irreversibly affect iav replication during the post entry phase. furthermore, the lack of inhibition of iav entry by dynasore demonstrates that under these experimental conditions iav is able to enter cells via a pathway that is fully redundant to any dynamindependent (dyna-dep) entry route, including the classical cme pathway. also when iav travels via this novel dynamin-independent (dyna-ind) route, iav apparently enters via low ph compartments as entry is fully sensitive to bafa1. as factors present in serum are known for their potential to induce specific endocytic pathways, we further explored the conditions required for the novel dyna-ind iav entry pathway (using the gluc-entry assay) by inoculating cells in pbs in the presence of increasing concentrations of fetal calf serum (fcs). whereas dynasore completely inhibited entry in pbs, inclusion of 5% and 10% fcs resulted in increasing levels of dynasore resistant entry ( fig. 2a) , suggesting the existence of a serum-inducible dyna-ind iav entry pathway. this effect was not caused by inactivation of dynasore during the experiment as vesicular stomatitis virus (vsv), which enters cells by cme [24, 25] , was still sensitive to 80 mm dynasore in the presence of 10% fcs (fig. 2b) . in agreement herewith, the uptake of transferin, known to occur via cme, was inhibited by dynasore regardless of the hela cells were grown on glass cover slips and infected with iav (strain wsn; moi of 10) and fixated after 30 min, 3 hrs or 6 hrs (column 1, 2 or 3 respectively). infection was performed in 0.2% dmso (upper row panels) or in the presence of 10 nm bafa1 (lower row panels). the nucleus was visualized by dna staining with topro-3 (red). iav infection was visualized by staining with monoclonal antiserum directed against np (green). in the absence of inhibitor, iav localized to the nucleus after 3 hrs, while new virus particles spread to the cytoplasm after 6 hrs. bafa1 (lower row panels) caused accumulation of incoming virus particles at a peri-nuclear location. (e) quantitative determination of iav entry by a single-cycle gluc-entry assay. hela cells (10,000 cells/well in dmem supplemented with 10% fcs) were transfected with phh-gluc 24 hrs prior to infection with a serial dilution of infectious iav particles (plotted on the x-axis). two hours after infection 10 nm bafa1 was added to block any further entry. cells were incubated for a further 14 hrs to allow expression of luciferase activity (y-axis; relative light units, rlu). (f) effect of dynasore and bafa1 on iav entry in the gluc-entry assay. dynasore (dy, dark grey bars; 20, 40 or 80 mm) or bafa1 (light grey bars; 1.25, 2.5 or 5 nm) were present from 1 hr prior to infection (strain wsn; moi 0.5) to 2 hrs p.i. after which the inhibitor-containing medium was replaced with medium containing 10 nm bafa1 to block any further entry. cells were incubated for a further 14 hrs to allow the quantitative expression of luciferase activity (y-axes; rlu relative to the control infection without inhibitor). whereas bafa1 displayed dose-dependent inhibition of iav entry, dynasore did not significantly inhibit iav entry. presence of fcs (fig. s2, panel a) . as expected, both dyna-dep entry in pbs and dyna-ind entry in the presence of 10% fcs and 80 mm dynasore required sialic acid receptors for efficient entry as pre-treatment of hela cells with neuraminidases almost completely abolished entry via either pathway (fig. 2c ). the kinetics of the dyna-dep and dyna-ind entry pathways were compared by performing a time-course experiment in which iav entry was terminated by the addition of 10 nm bafa1 at different time points (fig. 2d) . in comparison to entry via the dyna-dep pathway (the only pathway available in pbs) entry in the presence of fcs (when presumably both the dyna-dep and dyna-ind entry pathways are available) showed similar kinetics. in contrast, entry via the dyna-ind pathway (which is the only pathway that is active in the presence of 10% fcs and 80 mm dynasore) was slower. the difference was most prominent after 15 min, while after 4 hrs similar levels of entry were reached. to validate and extend these results we visualized the reduction of the number of infected cells by immunoperoxidase staining using an antibody against np (fig. 3) . a number of different cells of mammalian and avian origin were infected for 2 hours at an moi of 1 in pbs with or without serum. after 2 hours the inoculum was replaced by growth medium containing 10% fcs and 10 nm bafa1 and the expression of np was examined after 14 hours later. after incubation in pbs, staining was completely prohibited by the presence of 80 mm dynasore whereas in the presence of serum dynasore had no effect. a serum-inducible, dyna-ind route of entry was thus functional in all five cell lines, including the human epithelial airway carcinoma cell line a549. to confirm our results and to obtain further proof for the utilization of dyna-dep and dyna-ind entry routes by iav, we additionally used an iav virus-like particle (vlp) direct entry assay [26] . these vlps contain iav ha and na in their envelope and harbor a beta-lactamase reporter protein fused to the influenza matrix protein-1 (blam1), which allows the rapid and direct detection of entry, independent of virus replication. upon fusion of viral and endosomal membrane, blam1 gains access to the cytoplasmically retained fluorigenic substrate ccf-2 that, after cleavage by blam1, shifts to a shorter fluorescent emission wavelength that can be detected by flow cytometry. entry into hela cells was performed in the absence or presence of 10% fcs using vlps containing ha and na either from iav-wsn (having a strict alpha 2-3 linked sialic acid binding specificity) or from the pandemic 1918 iav (ha from a/ newyork/1/18, binding to alpha 2-3 and alpha 2-6 linked sialic acids; na from a/brevigmission/1/18). entry of vlps of both iav strains was severely inhibited by dynasore when no serum was added to the inoculum (fig. 4a, 4d) , whereas the presence of 10% fcs rendered entry completely dynasore resistant. (fig. 4b, 4e ). quantification of vlp entry is shown in fig. 4c and f. importantly, to confirm the existence of the serum-inducible entry pathway by a method that is independent of dynasore, we used sirna induced silencing of dynamin 2. fig. 4g shows that two different sirnas had a significant inhibitory effect (48 hrs after sirna transfection) on entry of the renilla luciferaseencoding pseudovirus wsn-ren [27] in hela cells in the absence of fcs, whereas the presence of 10% serum no reduction in entry levels was observed, confirming the results obtained with dynasore. knockdown of dynamin 2 protein levels (48 hrs after sirna transfection) was analyzed by western we conclude that a dyna-ind entry pathway can be induced by serum in different cell types from several species. the evidence was obtained using both replication-dependent (gluc-entry assay and immunodetection of infected cells) and replication-independent assays (entry of vlps), the latter allowing immediate detection of the fusion-mediated delivery of viral m1 protein into the cytoplasm. cascade blue). in the histograms entry is displayed by a shift to higher fluorescence (the grey area represents background fluorescence of noninfected cells). (c and f) quantification of facs results. background fluorescence was subtracted from each measurement (geometric mean) and data were normalized to vlp entry in optimem without dynasore (dy) (red curve of panel a and and d). vlp entry was not inhibited by dynasore in presence of 10% fcs whereas the access of blam1 to its ccf2 substrate in the cytoplasm was blocked by dynasore in pbs. vlp entry was more efficient in the presence of serum. (g) effect of downregulation of dynamin 2 by sirna silencing. serum-inducible dyna-ind entry was analyzed in hela cells that were transfected 48 hrs prior to infection with two different sirnas targeting dynamin-2 (dyna). sirna treated cells were infected with the pseudovirus wsn-ren in pbs (grey bars) or in pbs containing 10% fcs (black bars) and luciferase activity was determined after 16 hrs post infection (y-axis; rlu relative to infection of cells transfected with a scrambled sirna). entry of pseudovirus wsn-ren (moi 0.5) was reduced by 50% to 70% when entry was performed in pbs (grey bars) whereas entry was not significantly affected in the presence of 10% serum (black bars). (h) western blot showing the knockdown of dynamin 2 (in comparison to tubulin) at 48 hrs after transfection with sirnas. (i) quantification of the residual levels of dynamin 2 (dyna) mrna (determined by quantitative rt-pcr) and protein (determined by densitometric scanning of the western blot) 48 hrs after sirna transfection. data were normalized to 18s rna (rt-pcr) or tubulin protein levels and calculated relative to the levels obtained after transfection with a scrambled sirna that served as a control. doi:10.1371/journal.ppat.1001329.g004 inhibitors of growth factor receptor tyrosine kinases and actomyosin network dynamics reduce dyna-ind entry of iav the dyna-ind entry pathway was further characterized by inhibitor profiling using an 80-compound kinase inhibitor library. serum-induced dyna-ind entry was examined in 10% fcs using the gluc-entry assay. 80 mm dynasore was added in order to block cme and any other potential dyna-dep entry pathways. this allowed the independent inhibitor profiling of the novel pathway by avoiding the potentially masking effect of the presence of redundant entry pathways. cells were preincubated with the kinase inhibitors (10 mm) for 1 h at 37uc and then inoculated with virus (moi 0.5) in the presence of 10% fcs and 80 mm dynasore for 2 h at 37uc (dyna-ind entry). in parallel, inoculations were also done in pbs to compare the effects of the inhibitors on dyna-dep entry. after 2 hr the medium and inhibitor were replaced by full growth medium containing 10% fcs and 10 nm bafa1 to allow the subsequent expression of gluc activity under identical conditions for the dyna-ind and -dependent entry assay. six kinase inhibitors appeared to act non-discriminatively, inhibiting both dyna-dep and dyna-ind entry (fig. 5a ): the protein kinase c (pkc) inhibitors ro 31-8220, rottlerin (both displaying moderate cytotoxicity, result not shown) and hypericin, which have all three been previously identified as iav inhibitors [28, 29] ; the highly cytotoxic pan-specific serine/threonine protease inhibitor staurosporine; the irreversible pi-3 kinase inhibitor wortmannin and the receptor tyrosine kinase inhibitor tyr9. in order to investigate whether some of these inhibitors affect iav replication during the post-entry phase, we performed the same experiments but now adding the kinase inhibitors after viral entry. four of the inhibitors thus appeared to induce significant inhibition of post-entry processes (fig. 5a ). although unlikely, we cannot formally exclude that post-entry processes specific for only one of the two entry pathways are affected. interestingly, whereas no specific dyna-dep entry inhibitors were identified, 15 inhibitors (none displaying cytotoxic effects, data not shown) caused significant (p,0.05) inhibition (.5-fold) of dyna-ind entry (fig. 5b ). this included inhibitors of the calmodulin dependent kinases myosin light chain kinase (mlck) and camkii and seven inhibitors of different growth factor receptor tyrosine kinases. in contrast to the three non-specific pkc inhibitors mentioned above, the pkc inhibitors bim-1 and hbdde appeared to have a specific inhibitory effect on dyna-ind entry. the specific effect of these drugs on dyna-ind entry is not only shown by the lack of inhibition of dyna-dep entry in pbs, but also by the observation that none of the fifteen compounds induced .2-fold inhibition when added post-entry (at t = 2 hr post infection). the kinase library screen was repeated on a549 human epithelial lung carcinoma cells in order to confirm the results in a potentially more natural host cell line. the inhibition profiles obtained were very similar to those found for hela cells with the exception of the strong effect of ag879 (99% inhibition) and moderate effects of ag825 (39% inhibition) and tyr51 (68% inhibition) on dyna-dep entry. (fig. 5c) . mlck inhibitors ml-7 and ml-9 have been reported to be highly specific for their target kinase [30] . phosphorylation by mlck activates non-muscle myosin ii light chain, indicating that a functional actomyosin network might be essential for dyna-ind entry of iav. this was further examined by testing the effect of blebbistatin, an inhibitor of myosin ii heavy chain activity, and of several inhibitors that affect actin dynamics by disrupting actin microfilaments (cytochalasin b and d), by enhancing actin polymerization (jasplakinolide) or by inhibiting actin polymeriza-tion (latrunculin a). actin inhibitors were used at the minimal concentration required to induce clearly visible changes in the actin cytoskeleton as pre-determined by staining with fitcphalloidin (results not shown). whereas the inhibitors did not affect dyna-dep entry (fig. 6a) using gluc-entry assay, all inhibitors as well as ml-7 and ml-9 significantly inhibited dyna-ind entry (fig. 6b) . next, hela cells were transfected with plasmids encoding dominant negative or wildtype rab5 fused to green fluorescent protein (rab5 dn and rab5 wt in fig. 6 ) 24 h prior to infection with iav. rab5 is a small gtpase found in association with several endosomal compartments and crucial for the function and maturation of early endosomes. it is required for the trafficking of a wide range of endocytic cargo following different routes, including dyna-dep as well as dyna-ind routes [31] . entry of iav has been shown to require rab5 [32] . consistently, we found that hela cells expressing rab5 dn (as identified by gfp fluorescence, fig. 6c ) were much less susceptible to productive iav infection (as judged by indirect immunofluorescence using alexa-488 labeled np antibodies) than cells transfected with several dynamin-independent endocytic pathways have been described [8, 19] . of these, macropinocytosis has been demonstrated to be stimulated by growth factors present in serum and to depend on actin dynamics [12] [13] [14] . yet, studies on macropinocytosis are hampered by a lack of specific inhibitors, cargo, membrane markers and characteristic morphology. amiloride and the more potent derivative eipa are inhibitors of epithelial sodium channels (enac) as well as of several other na+/h+ antiporters. eipa has often been used as a hallmark inhibitor that specifically inhibits endocytosis via the macropinocytic pathway [14] . whereas dyna-dep entry of iav was not inhibited by eipa (fig. 7a) , dyna-ind entry was fully blocked eipa (fig. 7b) . the existence of redundant entry pathways in the presence of 10% fcs is clearly demonstrated by the marginal inhibition by either eipa or dynasore whereas the combination of eipa and dynasore resulted in strong inhibition both in the gluc-entry assay (fig. 7c ) and in the direct vlp entry assay ( fig. 7d and e) . supplementary fig. s1 shows that other cell lines, including the human lung epithelial cell line a549, display similar iav inhibition patterns for eipa and dynasore. consistently, virus production displayed a similar inhibitor sensitivity profile (fig.7 f and g) as virus entry indicating that the entry pathways we characterized lead to a productive infection. clearly, vlps and viral particles follow similar redundant entry pathways, distinguishable in a dyna-dep and a dyna-ind pathway, the latter being sensitive to eipa and dependent on actomyosin function. one characteristic of macropinocytosis is the nonselective uptake of large amounts of extracellular solutes [33] . therefore, the uptake of soluble fitc labeled dextran (fdx) into relatively large vesicles (0.3 to 5 mm) has often been applied as a morphological marker for macropinosomes. using this marker we found that the addition of 10% fcs to the culture medium slightly increased the uptake of fdx into hela cells (fig. 8a) . notably, the distribution of fdx changes in response to serum from a random distribution into a more granular pattern. at high magnification and at color settings adjusted to higher intensity it could be seen that these fdx granules were free of actin staining (by phalloidin) indicating that they were in the lumen of vesicles (result not shown). interestingly, in the presence of iav (moi of 10) the uptake of fdx into vesicles was clearly enhanced. at a higher magnification viral particles could be found to co-localize in fdx loaded vesicles as well as outside these vesicles (fig. 8b) . phalloidin staining of actin was used to demonstrate that many virus particles localized to actin-rich protrusions at the periphery of the cell. the uptake of fdx was studied in a quantitative manner by flow cytometry (fig. 8 c) . a moderate, but reproducible shift to higher fdx fluorescence was observed at 37uc when virus was added in presence of 10% fcs whereas such a shift was absent when no serum or virus was added. this result confirms the observations by confocal microscopy (fig. 8a) which showed that the combined presence of fcs and iav increases the uptake of fdx as compared to fcs alone. in a control experiment the uptake of fdx in 10% fcs in presence of iav was shown to be specifically inhibited by eipa, but not by dynasore (fig. s2, panel b) . in contrast, transferrin uptake, which serves as a specific marker for cme, was affected by dynasore, but not by eipa (fig. s2, panel a) . in conclusion, serum induces the uptake of fdx into large vesicles, which can be further enhanced by the addition of iav particles that, after entry, co-localize in part with these vesicles. these results indicate the utilization of a macropinocytic pathway for entry of iav, which is consistent with the observed sensitivity of the seruminducible dyna-ind entry of iav and vlps to eipa. macropinocytosis has been implicated in the entry of several viruses [12, 14] . however, differences in susceptibility to inhibitors suggest that distinct forms of macropinocytosis might be used by different viruses [34, 35] . by screening specific inhibitors in the gluc-entry assay using dyna-ind entry conditions we evaluated the possible involvement of a few signaling cascades that have been implicated in the induction of macropinocytosis. serum-inducible macropinocytosis has been shown to be activated via a myriad of signaling cascades initiated by growth factors binding to transmembrane tyrosine kinase receptors [14, 17, 36, 37] , consistent with the results shown in fig. 5 . a prominent downstream effect of these signaling cascades is the activation of p21 associated kinase 1 (pak1) which in turn can activate a number of different pathways leading to actin network rearrangements that can ultimately lead to the induction of macropinocytosis [38] . fig. 9a -b shows that 20 mm ipa3, an inhibitor of pak1 [39] , specifically inhibits background fluorescence from fdx binding to the outside of cells was determined by performing the same experiment at 4uc (at which no endocytosis takes place) and was subtracted from the mean fluorescence intensity obtained at 37uc to determine the amount of fluorescent fitc-dextran that was internalized at 37uc. data were plotted relative to fitcdextran uptake in pbs in absence of iav. doi:10.1371/journal.ppat.1001329.g008 dyna-ind entry of iav. activation of pak1 in response to growth factor stimulation often involves upstream signal transduction by members of the rho sub-family of small gtpases like cdc42 and/or rac1 [34, 40, 41] . alternatively, activated cdc42 and rac1 can induce actin rearrangements independently of pak1 [34, [40] [41] [42] [43] by direct interaction with wasp or wave family proteins, respectively [44, 45] . however, inhibitors of cdc42 (pirl1 [46] ), rac1 (nsc23766 [47] ) or n-wasp (wiskostatin [48] ) did not display inhibitory effects on dyna-ind or dyna-dep entry of iav (fig. 9c-d) . instead, pirl1 and wiskostatin induced a significant, concentration dependent increase of entry. this stimulatory effect was not observed for the control vaccinia virus strain wr, which enters cells via a rac1dependent, macropinocytotic pathway [43] (fig. 9e) , indicating that this effect is specific for iav. the results suggest a requirement for pak1 in dyna-ind entry of iav that does not require activation by either cdc42 or rac1. growth factor inducible activation of the tyrosine kinase src has also been linked to the induction of macropinocytosis [49] [50] [51] ; consistent with this observation the src inhibitor pp2 [52] specifically inhibited dyna-ind entry of iav (fig. 9a-b) . remarkably, 17-aageldanamycin, a specific inhibitor of the chaperone protein hsp90 [53] , also caused specific inhibition of dyna-ind entry (fig. 9a-b) . hsp90 affects the folding and activity of many proteins but the recent demonstration of direct activation of the catalytic activity of src by hsp90 [54] provides another indication of the involvement of src in dyna-ind endocytosis of iav. in conclusion, like for other viruses utilizing a macropinocytic entry pathway, pak1 seems to play a crucial role in dyna-ind entry by iav. however, this pathway is independent of rac1 or cdc42 but may require src, either upstream and/or downstream of pak1. the data presented in this study demonstrate for the first time that iav can enter cells via dyna-ind macropinocytosis in addition to the previously described dyna-dep classical cme pathway [1, 2] . several lines of evidence indicate that the dyna-ind entry route of iav that we identified corresponds with macropinocytosis. first of all, the entry pathway is dependent on the presence of serum, a well-known inducer of macropinocytosis. second, iav colocalized in vesicles with soluble fitc-dextran, a marker for macropinocytosis. third, dyna-ind iav entry was sensitive to the amiloride-derivative eipa, the hallmark inhibitor of macropinocytosis [14, [55] [56] [57] [58] . fourth, this iav entry pathway is sensitive to inhibitors or dominant-negative mutants affecting actomyosin dynamics. fifth, the specific inhibition of dyna-ind iav entry by a number of inhibitors of growth factor receptor tyrosine kinases as well as downstream effectors thereof also points at the involvement of macropinocytosis. finally, macropinocytosis is independent of dynamin [12, 14, 19] . despite this extensive list of arguments, viral entry by macropinocytosis needs to be considered with caution. the characteristics of the dyna-ind route of cell entry by iav are similar, but not identical to the macropinocytic entry routes taken by other viruses, like two different strains of vaccinia virus and by coxsackie virus b [34, 35] . as is shown in table 1 and discussed in more detail below, the macropinocytic pathways used by each of these viruses have a few unique characteristics. this may very well reflect the growing notion that macropinocytosis represents a number of differentially induced and regulated processes, rather than being a single endocytic pathway [13, 14] . macropinocytosis has collectively been described as an inducible form of endocytosis by which fluid-phase cargo travels via non-coated, relatively large and heterogeneous organelles that have emanated from extensive protrusions (e.g lamellar ruffles, circular ruffles or retracting blebs) of the plasma membrane [13] . in the case of dyna-ind iav entry more extensive studies using electron microscopy will be required to study the morphology of membrane protrusions with which iav may associate. in addition, live cell imaging microscopy will be required to characterize the exact itinerary that is taken by iav virions traveling via a macropinocytic process. this is especially important as different routes of iav entry are likely to converge at some point in the endocytic pathway. although unlikely, co-localization of iav particles with fluid-phase dextran as shown in fig. 8b may thus represent a situation occurring after convergence of several different routes. the use of microscopy to study macropinocytosis is however complicated by the lack of specific membrane-associated markers for any early step of this endocytic process. a model (fig. 10 ) based on our results explains the key steps involved in the macropinocytic entry pathway of iav, which are described in more detail below. by manipulating the inoculation conditions we were able to experimentally dissect iav entry into a dyna-dep and dyna-ind route. the dyna-ind route required the presence of 10% fcs in the entry assay medium. previously, a strict dependency on a dyna-dep entry route for iav was concluded from experiments with a cell line expressing an inducible dominantnegative mutant of dynamin 2 [59] . in that study, as well as in other entry studies of iav, entry was performed in dmem containing 2% serum or bsa. also in our hands 2.5% serum ( fig. 2a) or 0.2% bsa (result not shown) was not sufficient to allow dyna-ind entry. we are currently investigating which serum component is responsible for the observed effects on iav entry. dialysis of fcs (mw cut off .10 kda) did not affect its capacity to induce dyna-ind endocytosis (result not shown), indicating that low molecular weight solutes are not responsible for the observed effect. our evidence for a dyna-dep and a serum inducible dyna-ind entry route is based on the use of pharmacological (dynasore, a highly specific inhibitor of dynamin) as well as genetic (sirna directed against dynamin 2) tools, ruling out the possibility that the inhibitory effect of dynasore was due for instance to absorption of the inhibitor by serum components. whereas dynasore resulted in near 100% inhibition of dyna-dep entry, only 65% inhibition was observed upon sirna induced silencing of dynamin 2 indicating that the residual levels of dynamin 2 that remain after 48 hrs of silencing still support a low level of dyna-dep entry (fig. 4h) . reversible inhibitors like dynasore [60] offer a major advantage for characterization of iav entry pathways. they can be applied for a limited period thus preventing the secondary adaptive effects of cells that may occur in response to long-term down regulation of a gene product by genetic methods like sirna interference. both entry routes were consistently identified by a viral entry assay quantified by virus induced expression of a luciferase reporter as well as by a vlp entry assay allowing direct analysis of the membrane fusion mediated entry step. the consistent performance of an ha with a strict preference for binding to a2-3 linked sialic acids (from iav-wsn; our unpublished data) and an ha also binding to a2-6 linked sialic acids (from 1918 iav [61] ) in the vlp entry assay indicates that both pathways can be utilized by has of different specificity and may therefore be relevant to avian as well as human iav infections. consistently, serum-inducible dyna-ind entry was observed both in avian df1 cells and in a human lung epithelial carcinoma cell line a549 (fig. 3) . the dyna-dep and dyna-ind iav entry pathways were found by our quantitative assays to be fully redundant. in the presence of serum, the combination of dynasore (inhibiting dyna-dep entry) and eipa (inhibiting dyna-ind entry) completely abolished entry whereas either drug alone had no effect. eipa, an inhibitor of plasma membrane na+/h+ exchangers, has been shown to invariably inhibit macropinocytosis [14, [55] [56] [57] [58] . as other routes of endocytosis are generally not affected, eipa is considered as a hallmark inhibitor of macropinocytosis [14] , although results obtained with eipa should be considered with care as long as a mechanistic explanation for its effect on macropinocytosis is not yet fully clear [62] . occasionally, a moderate two-to three-fold inhibition by dynasore alone was observed (result not shown) indicating that the capacity of the serum-inducible entry pathway is somewhat variable, possibly depending on slight variations in serum quality and factors like cell distribution in the wells that have been reported to influence viral infection [63] . a redundancy in the utilization of cme as well as a clathrin-independent route for entry of iav has been visualized previously by quantitative live cell imaging [4] . both routes were operative simultaneously in the same sample and the specific down-regulation of cme did not affect the total number of entry events. in response to specific extra-cellular signals (e.g. serum induction), changes in the actomyosin network occur that give rise to membrane protrusions required for macropinosome formation [13] . compounds inhibiting actin polymerization (cytochalasin b and d), depolymerization (jasplakinolide) or sequestering soluble actin (latrunculin a) all specifically inhibited dyna-ind iav entry. in addition, the requirement for myosinii activity was established by a specific inhibitor (blebbistatin) of myosin ii atpase activity and by the expression of a dominant negative mutant of myosiniia heavy chain. also, the regulation of myosinii activity by phosphorylation of myosin light chain through the action of mlck is suggested by the inhibitory effect of mlck inhibitors ml-7 and ml-9 as well as by the similar effect of an expressed mlck dominant negative mutant. recently, a function for the actin cytoskeleton in iav entry was reported to be required for the entry into polarized epithelial cells but not for entry into non-polarized cells [64] . when using the low-serum conditions used in that paper (2% fcs), we only observed dyna-dep entry that was not affected by actin dynamics inhibitors. perhaps, the polarized cells permit dyna-ind entry at lower serum concentrations. the changes in actin network dynamics that can lead to the formation of macropinosomes can be triggered by a number of signaling cascades. actin dynamics are induced by the activation of growth factor receptor tyrosine kinases by their respective figure 10 . a model for iav entry by macropinocytosis. the model summarizes the inhibitory (red boxes) or stimulatory (blue boxes) effects of compounds on dynamin-independent iav entry. the effect of over-expression of dominant-negative mutants is indicated by red-lined boxes. the pathway requires the presence of serum factors in the entry medium and results in the enhanced uptake of dextran and its co-localization with iav in large vesicles (green boxes). we hypothesize that the interaction of serum factors and/or iav with receptor tyrosine kinases (rtks) is the primary signal for the induction of macropinocytosis. a number of rtks have been shown to be involved in this process in different cell lines. remarkably, a recently published genome-wide sirna screen of iav infection identified the fgf receptor as a host factor required for influenza virus replication [22] . activation of rho family gtpases cdc42 and/or rac1 has been shown to be essential for signal transduction leading to macropinocytosis in many cases [13, 14] but inhibitors are without effect or are stimulatory in the case of iav entry. downstream effectors of rho family gtpases include scaffold proteins like n-wasp and wave and protein kinases like pak1. macropinocytic entry of iav however seems to require a rho family gtpaseindependent pak1 activation mechanism. in addition, src family kinases, which can be directly activated by rtks, play a role. pak1 and src have previously been linked to the activation of macropinocytosis via their effect on changes in actomyosin dynamics, a process which is crucial to any form of macropinosome formation [13, 14] . apart from n-wasp-or wave-containing macromolecular assemblies other actin binding proteins can induce such changes (e.g. cortactin, which can be activated by src [81] ) and thereby induce the formation of one of the different plasma membrane protrusions that can result in the formation of macropinosomes. in addition to an effect on the formation of plasma membrane protrusions and subsequent macropinosome formation, inhibitors can also affect downstream trafficking and maturation of macropinosomes which might be actindependent, but this is not depicted in the scheme. doi:10.1371/journal.ppat.1001329.g010 growth factor ligands that are normally present in serum [12] [13] [14] 17, 36, 37] the signal transduction cascades that link activation of growth factor receptor tyrosine kinases to actin remodeling and macropinocytosis are only beginning to be revealed. the specific inhibition of dyna-ind entry of iav by ipa3, an inhibitor of pak1, provides proof for the involvement of these cascades. pak1 is a key serine/threonine kinase regulating actin network dynamics but its crucial function in several pathways of endocytosis as well as numerous other cellular processes does not make it a very specific marker [65] . even so, macropinocytosis has consistently been demonstrated to require pak1 activation, both in the induction of the process and/or in further downstream trafficking events of macropinosomes [13, 14] . growth factor dependent activation of pak1 has most often been demonstrated to depend on upstream activation of small gtpases rac1 or cdc42 [34, 40, 41] . different strains of vaccinia virus were recently shown to induce their uptake by macropinocytosis via activation of either rac1 or cdc42 [34] . activation of rac1 has been linked to the induction of macropinocytosis via actin network-mediated formation of lamellipodia and/or circular ruffles whereas cdc42 has most often been implied in the formation of filopodia [44] . an inhibitory effect of the rac1 inhibitor nsc23766 or the cdc42 inhibitor pirl1 on iav entry, however, could not be demonstrated. remarkably, cdc42 inhibitor pirl1 enhanced iav entry and a similar effect was observed by wiskostatin, an inhibitor of n-wasp which functions directly downstream of cdc42 as a scaffolding complex required for the activation of actin polymerization leading to filopodia formation. similarly, the macropinocytosis-like entry pathway taken by coxsackie b virus was also shown to require pak1 activity that was independent of rac1 activation [35] . direct examination of the magnitude and timing of the activation of pak1 will be required to obtain more insight in the involvement of this complex pathway. the induction of macropinocytosis by a pak1dependent mechanism has been associated with ruffling at the cell membrane [12, 14, 15, 37] . the identification of sub-membranous regions with increased actin staining by phalloidin has been interpreted as evidence for ruffling. this was not unambiguously identified by confocal microscopy in the experiments presented in fig. 8 and fig. s2 and needs to be investigated in depth by life cell imaging techniques. in agreement with our observation that the dyna-ind entry of iav was inhibited by pp2, an inhibitor of src family kinases, the non-receptor tyrosine kinase c-src has been shown to function as a key signaling intermediate in the induction of macropinocytosis via a mechanism independent of rac1 or cdc42 [49] [50] [51] . downstream effects of c-src on actin networks proceed, amongst others, via phosphorylation of cortactin by c-src resulting in accelerated macropinosome formation [50] . c-src has been shown to associate with macropinosomes [49, 51] , both during their formation and their trafficking, while c-src kinase activity is required for macropinocytosis following egf stimulation of hela cells [49] . interaction of hsp90 with c-src was recently shown to induce c-src kinase activity [54] . also hsp90 has been demonstrated to associate with macropinosomes, while its specific inhibitor geldanamycin reduced the membrane ruffling that preceded macropinocytosis [66] . thus, the inhibition of iav entry via macropinocytosis by aa-geldanamcyin may very well involve the effects of hsp90 on c-src. as detailed above, the dyna-ind entry pathway of iav shares many characteristics with the endocytic pathway macropinocytosis. this is corroborated by the observation that iav particles and dextran colocalize in large vesicles in the presence of fcs. several viruses have recently been reported to enter cells via macropinocytosis [12, 14] . apart from common factors like the requirement for pak1 activation, actin dynamics and independence of dynamin, virus specific details have been described [34, 35] (table 1 ). in part these might be contributed to differences in experimental conditions (e.g. cell types tested) but diversity in the molecular mechanisms by which macropinocytosis can be induced and executed is likely to exist and to be exploited by viruses. whereas vaccinia virus is able to trigger its own macropinocytic uptake [34, 43] , we have described a macropinocytosis pathway that is operational under conditions that are activated by components in serum. still, this does not exclude signaling induced by virus-host cell interactions, which are for instance suggested by the significant increase of fitc-dextran uptake in the presence of iav. the possible requirement for costimulatory signals from serum components and virus imposes an additional layer of complexity on the analysis of iav entry via dyna-ind pathways. influenza viruses cause respiratory infections by targeting the epithelial cells lining the respiratory tract. these surfaces are covered by a mucous layer composed of a variety of small solutes and glycoproteins derived among others from goblet cells [67] . this semi-fluid layer in turn conditions the underlying cells and determines their physiological state, including the activities of their uptake and secretion pathways. it will be important to determine to what extent the dyna-dep and dyna-ind iav entry pathways are operational under the conditions prevailing along the respiratory tract. current knowledge on the protein composition of the fluids covering the respiratory epithelium is rapidly expanding by the application of proteomic methods to determine the protein composition of bronchial alveolar lavage fluids (balf). these studies have extended the previous notion that balf is highly similar in composition to serum. for example, just as for the serum proteome more than 85% of the total protein mass of the balf proteome is accounted for by albumin, immunoglobulins, transferring, a1-antitrypsin and haptoglobin. in addition, many other proteins have been identified both in serum and in balf including growth factors that can bind to growth factor receptor tyrosine kinases [68] [69] [70] . thus, balf is likely to harbor, just as serum, the protein factors that can activate signaling pathways that are crucial for the induction of dyna-ind entry of iav. in agreement herewith, macropinocytosis has been described as a functional entry pathway of haemophilus influenzae into primary human bronchial epithelial cells [71] although the factors involved in signaling the process have not been identified yet. in addition to infecting the respiratory tract, iav has been shown to be able to cause systemic infections involving multiple organs. this has mainly been studied in avian infections [72, 73] or by infection of mice with human-derived h1n1 or h3n2 iavs [74] but is poorly documented for human infections and may have been underestimated thus far. obviously, during potential systemic spreading of iav, the serum-rich conditions that we have demonstrated here to enable the use of alternative entry pathways will be encountered and may contribute to such spreading. mdck, a549, df-1 and hela cells were maintained in complete dulbecco's modified eagle's medium (dmem) (lonza, biowittaker) containing 10% (v/v) fetal calf serum (fcs; bodinco b.v.), 100 u/ml penicillin, and 100 mg/ml streptomycin. chinese hamster-e36 cells were maintained at 37uc in a-minimal essential medium (gibco) supplemented with 10% (v/v) fcs, 100 u/ml penicillin, and 100 mg/ml streptomycin. cells were passaged twice weekly. influenza a/wsn/33 (h1n1) (iav-wsn) was grown in mdck cells. briefly, ,70% confluent mdck cells were infected with iav-wsn at a moi of 0.02. supernatant was harvested after 48 hr of incubation at 37uc and cell debris was removed by centrifigutation (10 min at 2000 rpm). virus was stored at 280uc and virus titers were determined by measuring the tcid 50 on hela cells. the iav-wsn luciferase pseudovirus (wsn-ren) system has previously been described [27] . briefly, wsn-ren pseudovirus harbors a ha segment in which the ha coding region is replaced by renilla luciferase. the pseudovirus is produced in a mdck cell line that stably expresses the ha of iav-wsn. wr-luc, a firefly luciferase encoding vaccinia virus (strain wr) was previously described [75] . vsv-fl, a firefly luciferase encoding vsv virus was also previously described [76] . stocks of bafilomycin a1 (bafa1), dynasore, cytochalasin d, cytochalasin b, blebbistatin, 17-aa-geldanamycin, ml-7, ml-9, pp-2, 5-(n-ethyl-n-isopropyl)amiloride (eipa), ipa-3 (all obtained from sigma-aldrich), latrunculin a (enzo), jasplakinolide, wiskostatin, nsc23766 (all obtained from calbiochem) and pirl1 (chembridge) were prepared in dimethylsulfoxide (dmso). all stocks were stored at 220uc. a kinase inhibitor library composed of 80 kinase inhibitors was obtained from biomol (2832a[v2.2]). hela cells (10,000 cells/well in 96-well plates) were treated with 2 munits of vibrio cholerae neuraminidase (roche) in 50 ml phosphate-buffered saline (pbs) for 2 hr. after washing with pbs cells were infected with iav as described. virus-like particles (vlps) were produced as described [26] . briefly, 293t cells were transfected using lipofectamine 2000 (invitrogen) with pcaggs-blam1 (encoding a beta-lactamase reporter protein fused to the influenza matrix protein-1), pcaggs-ha (encoding ha derived from either a/newyork/ 1/1918 or iav-wsn) and pcaggs-na (encoding iav neuraminidase [na] derived from either a/brevigmission/1/18 or iav-wsn) and maintained in optimem. supernatants were harvested 72 h after transfection and centrifuged to remove debris. vlps were used for inoculation of cells without further concentration. vlps were incubated for 30 min at 37uc with trypsin/tpck for activation of ha. mdck or hela cells grown to near confluency in 24-well plates were inoculated with 250 ul of vlps after pre-treatment of the cells with inhibitors as indicated. infection was synchronized by centrifugation at 1500 rpm for 90 min at 4uc and was performed by further incubation at 37uc for 2 h in the absence or presence of 10% fcs and inhibitors as indicated. detection of beta-lactamase activity was performed as described [25] by loading cells with ccf2-am substrate (invitrogen) and subsequent analysis by flow cytometry on a lsrii flow cytometer (becton dickinson). typically 10,000 events were collected and analyzed using flowjo 8.5.2 software. the reporter construct phh-gluc was derived from plasmid phh-fluc [22] by replacing the firefly luciferase coding region with the gaussia luciferase coding region of pgluc-basic (new england biolabs). unique spei and xbai restriction sites were introduced into phh-fluc using the quikchange xl site-directed mutagenesis kit (stratagene) and oligonucleotides spe4262 (5-9gcctttctttatgtttttggcactagtcattttaccg-atgtcactcag), spe4263 (59-ctgagtgacatcggtaa-aatgactagtgccaaaaacataaagaaaggc), xba4260 (59-gtatttttctttacaatctagactttccgcccttc-ttgg) and xba4261 (ccaagaagggcggaaagtctag-attgtaaagaaaaatac). a spei site was introduced by sitedirected mutagenesis in pgluc-basic directly following the start codon of the gaussia luciferase coding sequence. the unique spei -xbai fragment of pgluc-basic was subsequently cloned into the spei-xbai site of phh-fluc resulting in plasmid phh-gluc. cells were seeded in 96-well plates at a density of 10,000 cells/ well and transfected the next day with 10 ng phh-gluc using lipofectamine 2000 (invitrogen) according to the manufacturer's protocol. after 24 hrs the transfected cells were treated with inhibitors and infected as indicated. at 16 hr p.i. samples from the supernatant were assayed for luciferase activity using the renilla luciferase assay system (promega) according to the manufacturer's instructions, and the relative light units (rlu) were determined with a berthold centro lb 960 plate luminometer. wr-luc and vsv-fl were used to inoculate hela cells (10,000 cells/well) at an moi of 2, in complete dulbecco's modified eagle's medium (dmem) (lonza, biowittaker). after 7 hr the luciferase activity was detected using the steadyglo assay kit (promega). the addition of 10% (v/v) fcs did not change infection levels for both viruses. cells were fixed with 3.7% paraformaldehyde (pfa) in pbs and subsequently permeabilized with 0.1% triton-x-100 in pbs. after blocking with normal goat serum iav-infected cells were incubated for 1 h with a monoclonal antibody directed against the nucleoprotein (np) (hb-65; kindly provided by dr. ben peeters). after washing, the cells were incubated with a 1:400 dilution of alexa fluor 488-or 568-labeled goat anti-mouse igg (molecular probes) secondary antibody for 1 h. nuclei were subsequently stained with topro-3 and after three washing steps, the coverslips were mounted in fluorsave (calbiochem). actin was stained using phalloidin labeled with alexa fluor 633. the immunofluorescence staining was analyzed using a confocal laser-scanning microscope (leica tcs sp2). fitc, gfp or alexa fluor 488 were excited at 488 nm, alexa fluor 568 at 568 nm, and topro-3 at 633 nm. hela cells were grown in 24-well plates on glass coverslips (50,000 cells/well). prior to fitc-dextran uptake cells were serum-starved for 2 hr in pbs. fitc-dextran (mw70,000, sigma-aldrich) was incubated with hela cells (final concentration of 0.5 mg/ml) in 500 ml pbs or in pbs containing 10% fcs in the absence or presence of iav (strain wsn; moi 10; concentrated and purified by centrifugation through a 15 to 30% sucrose gradient with a 50% sucrose cushion at the bottom) at 37uc. after 15 min cells were washed 4 times with pbs at 4uc, fixed with 3.7% pfa in pbs and subsequently permeabilized with 0.1% triton-x-100 in pbs. slides were stained for examination by confocal microscopy as described above. for quantification of fitc-dextran uptake 1.5610 5 hela cells were infected with iav-wsn (moi 10) in suspension in a volume of 1 ml in the presence of fitc-dextran (1 mg/ml). infections were performed for 15 min in pbs (containing 2% bsa to reduce unspecific binding of fitc-dextran) or in pbs containing 10% fcs at 37uc or at 4uc (control for binding of fitc-dextran to cells in the absence of endocytosis). mock-infected samples were analysed in parallel. infection was terminated by addition of 3 ml ice-cold pbs followed by three washes with cold pbs and fixation with 3.7% pfa. 20,000 cells were analyzed by facs and results were represented as the mean fluorescence which was plotted relative to the uptake in the mock-infection in pbs (after subtraction of background fluorescence obtained at 4uc). the effect of dynasore and eipa on dextran and transferrin uptake hela cells (grown on glass cover slips) were incubated at 4uc for 1 hr with 50 mg/ml alexa633-labeled transferin (invitrogen) in pbs. after 1 hr the medium was replaced by pbs or pbs supplemented with 10% fcs containing iav (strain wsn; moi 10) and 0.5 mg/ml fitc-dextran (sigma; 70 kda) and cells were transferred to 37uc for 15 min. after 15 min cells were fixed and stained as described above and examined by confocal microscopy. cells were fixed with 3.7% pfa in pbs and subsequently permeabilized with 0.1% triton-x-100 in pbs. peroxidase was visualized using an aec substrate kit from vector laboratories. iav-positive cells were detected using bright-field light microscopy. two sirna duplexes targeting different sites within the coding sequences of dynamin 2 were obtained from ambion inc (15581 (dynamin 2 sirna 1) and 146559 (dynamin 2 sirna2)). a scrambled sirna (ambion inc.) was taken along as a control for non-specific effects of the transfection procedure and was used for normalization. one day after seeding in 96-well plates (6,000 cells/well), the hela cells were transfected with a final concentration of 10 nm sirna using oligofectamine (invitrogen). 48 h after transfection, the cells were inoculated with the wsn-ren pseudovirus (moi 0.5) in pbs or in pbs containing 10% fcs. after 2 h of infection the entry medium was replaced by complete growth medium containing 10 nm bafa1 to prevent further entry. at 16 h post infection intracellular renilla luciferase expression was determined as described above. each sirna experiment was performed in triplicate. cell viability was not affected as determined by performing a wst-1 cell-viability assay (roche). functional knockdown of dynamin 2 mrna levels was performed by quantitative rt-pcr. using a taqman gene expression assay for dnm2 (hs00191900_m1, ambion) and using 18s rna (hs03928985_g1, ambion) as a control for normalization. the comparative ct-method was used for quantification of the results [77] . reduction of dynamin 2 protein levels was determined by western blotting using polyclonal goat-anti-dynamin 2 c18 (santa-cruz sc-6400). a monoclonal against alpha-tubulin (dm1a, sigma t9026) was used to detect tubulin for normalization. results were quantified by densitometric scanning of the dynamin 2 and tubulin signals displayed in fig. 4h . hela cells were grown in 24-well plates on glass coverslips (50,000 cells/well) for 24 hrs. cells were then transfected (1 mg of dna with lipofectamine 2000 as described above) with plasmids encoding wild-type or dominant-negative (dn) human mlck fused to gfp [78] , wild-type or dn rab5 fused to gfp [79] , or myoii-tail or myoii-head domain fused to gfp [80] . 24 hr after transfection cells were inoculated with iav-wsn (moi 1) in pbs or in pbs containing 10% fcs and 80 mm dynasore. 4 hr after infection cells were fixed and stained for examination by confocal microscopy as described above. an unpaired student's t-test was used for detemination of statistically significant differences. the use of the term significant in text refers to a comparison of values for which p,0.05. studies on the mechanism of influenza-virus entry into cells infectious entry pathway of influenza virus in a canine kidney cell line assembly of endocytotic machinery around individual influenza viruses during viral entry epsin 1 is a cargo-specific adaptor for the clathrinmediated endocytosis of influenza virus influenza virus entry and infection require host cell n-linked glycoprotein influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis endocytosis of influenza viruses mechanisms of endocytosis molecular mechanisms of clathrin-independent endocytosis endocytosis unplugged: multiple ways to enter the cell virus entry: open sesame virus entry by endocytosis defining macropinocytosis virus entry by macropinocytosis ruffles induced by salmonella and other stimuli direct macropinocytosis of bacteria phosphatidylserine (ps) induces ps receptor-mediated macropinocytosis and promotes clearance of apoptotic cells origin, originality, functions, subversions and molecular signaling of macropinocytosis clathrin-independent endocytosis: a unique platform for cell signaling and pm remodeling pathways of clathrin-independent endocytosis dynamin, endocytosis and intracellular signaling ctbp3/ bars drives membrane fission in dynamin-independent transport pathways virus-inducible reporter genes as a tool for detecting and quantifying iav replication involvement of the vacuolar h(+)-atpase in animal virus entry role of clathrin-mediated endocytosis during vesicular stomatitis virus into different host cells vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization an enzymatic virus-like particle assay for sensitive detection of virus entry human host factors required for influenza virus replication virucidal activity of hypericin against enveloped and non-enveloped dna and rna viruses modulation of influenza virus replication by alteration of sodium ion transport and protein kinase c activity the specificities of protein kinase inhibitors: an update rab proteins as membrane organizers differential requirements of rab5 and rab7 for endocytosis of influenza and other enveloped viruses vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry virus-induced abl and fyn kinase signals permit coxsackievirus entry through the epithelial tight junctions phosphoinositide metabolism during membrane ruffling and macropinosome formation in egf-stimulated a431 cells membrane ruffling and signal transduction regulation of macropinocytosis by p21-activated kinase-1 an isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase the small gtp-binding protein rac regulates growth factor-induced membrane ruffling activation of rho gtpases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells ras-related gtpases and the cytoskeleton vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells rho gtpases and actin dynamics in membrane protrusions and vesicle trafficking regulation of actin dynamics by wasp family proteins the cdc42 inhibitor secramine b prevents camp-induced k+ conductance in intestinal epithelial cells rational design and characterization of a rac gtpase-specific small molecule inhibitor chemical inhibition of n-wasp by stabilization of a native autoinhibited conformation role of src-family kinases in formation and trafficking of macropinosomes src triggers circular ruffling and macropinocytosis at the apical surface of polarized mdck cells ) c-src trafficking and co-localization with the egf receptor promotes egf ligand-independent egf receptor activation and signaling modulation of the fcepsilon receptor i signaling by tyrosine kinase inhibitors: search for therapeutic targets of inflammatory and allergy diseases ansamycin inhibitors of hsp90: nature's prototype for anti-chaperone therapy geldanamycin inhibits tyrosine phosphorylation-dependent nf-kappab activation distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma a431 cells adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake a clathrin independent macropinocytosis-like entry mechanism used by bluetongue virus-1 during infection of bhk cells kaposi's sarcoma-associated herpesvirus utilizes an actin polymerizationdependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells early stages of influenza virus entry in mv-1 lung cells: involvement of dynamin dynasore, a cell-permeable inhibitor of dynamin a single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity amiloride inhibits macropinocytosis by lowering submembranous ph and preventing rac1 and cdc42 signaling population context determines cell-to-cell variability in endocytosis and virus infection role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells an emerging role for p21-activated kinases (paks) in viral infections histone deacetylase 6 regulates growth factor-induced actin remodeling and endocytosis mucus and mucins mapping the lung proteome in cystic fibrosis proteomics and the lung: analysis of bronchoalveolar lavage fluid proteome analysis of bronchoalveolar lavage in lung diseases infection of primary human bronchial epithelial cells by haemophilus influenza: macropinocytosis as a mechanism of airway epithelial cell entry a mouse model for the evaluation of pathogenesis and immunity to influenza a (h5n1) viruses isolated from humans biological heterogeneity, including systemic replication in mice, of h5n1 influenza a virus isolates from humans in hong kong multiorgan distribution of human influenza a virus strains observed in a mouse model expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals carrier cell-based delivery of an oncolytic virus circumvents antiviral immunity mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1 dynamin and rab5 regulate grk2-dependent internalization of dopamine d2 receptors myosin iia is involved in the endocytosis of cxcr4 induced by sdf-1a cortactin signalling and dynamic actin networks the following persons are gratefully acknowledged for providing us with plasmids. dr. a. pekosz for plasmid phh-fluc; dr. m yanez-mo for miia-egfp ''head'' and ''tail'' constructs; dr. p. gallagher for dn-mlck-egfp; dr. p. van der sluijs for dn-s43n-rab5a-egfp. we thank dr. m. esteban for providing us with vaccinia wr-luc virus and dr. j. bell for providing vsv-fl virus. dr. b peeters is acknowledged for a gift of monoclonal antibody against iav np (hb-65). confocal images were acquired at the center for cellular imaging (cci) in the faculty of veterinary medicine, utrecht university and we thank dr. r. wubbolts for help and technical advice. key: cord-255980-u0c6pg8n authors: du, lanying; zhao, guangyu; zhang, xiujuan; liu, zhonghua; yu, hong; zheng, bo-jian; zhou, yusen; jiang, shibo title: development of a safe and convenient neutralization assay for rapid screening of influenza ha-specific neutralizing monoclonal antibodies date: 2010-07-02 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2010.05.161 sha: doc_id: 255980 cord_uid: u0c6pg8n abstract the worldwide outbreak of the swine-origin 2009 h1n1 influenza a virus (iav) and an increasing number of influenza cases caused by a highly pathogenic avian influenza (hpai) h5n1 have accelerated the need to develop vaccines and antiviral agents against iavs. among various antivirals, neutralizing monoclonal antibodies (mabs) are considered important passive therapeutics having an immediate effect against viral pathogens. here we report a pseudovirus neutralization assay for rapid screening of neutralizing mabs targeting hemagglutinin (ha) of h5n1 and h1n1 iav. in this study, we generated six pseudoviruses with an hiv-1 backbone, respectively, expressing ha of four clades of h5n1 iav and the 2009 epidemic h1n1 iav. the resulting pseudoviruses were able to infect a variety of human and non-human cells, with 293t cells from human kidney as the most susceptible target cells. using the established pseudovirus neutralization assay, we showed that three of ten selected mabs specific to ha could potently neutralize infection of a pseudovirus bearing ha from the homologous iav a/vietnam/1194/2004(h5n1) strain. this was highly consistent with the result of a microneutralization assay testing the same strain of a live iav. since the pseudovirus neutralization assay does not involve an infectious virus and can be performed without the requirement of a biosafety-3 laboratory, it may be applied for safe and rapid screening of neutralizing mabs and antiviral agents targeting ha of iavs. the worldwide outbreak of the swine-origin h1n1 influenza a virus (iav) poses an increasing threat of a future influenza epidemic potentially induced by the highly pathogenic avian influenza (hpai) h5n1 virus. since its re-emergence in 2003, the h5n1 iav has caused 294 deaths among a total of 498 confirmed cases as of may 06, 2010 (http://www.who.int/csr/disease/ avian_influenza/country/cases_table_2010_05_06/en/index.html) [1, 2] , highlighting the significance of developing vaccines and antiviral agents against divergent iavs. among all structural proteins of influenza viruses, hemagglutinin (ha) plays important roles in viral binding to receptors, virus entry and membrane fusion. during virus infection, ha first mediates virus attachment and subsequent entry into target cells [3] [4] [5] . thus, ha protein is considered the primary target for inducing protective immunity against iavs [6] [7] [8] . indeed, antibodies specific for ha have been shown to inhibit and/or neutralize virus infection in vaccinated hosts and anti-ha monoclonal antibodies (mabs) are regarded as one of the important passive therapeutics for treatment of influenza [9] [10] [11] [12] . it has also been shown that mabs targeting ha glycoprotein may broadly neutralize homologous and heterologous strains with different clades of iav infection and prevent formation of escape mutants [13] [14] [15] [16] . to identify more potent and specific neutralizing mabs as immunotherapeutics for treatment of influenza, it is essential to develop an assay for rapid screening of neutralizing mabs against ha of iavs. currently, microneutralization and plaque reduction assays are mainly used to evaluate the neutralizing activity of antibodies [16, 17] ; however, the infectious, replication-competent live iavs and biosafety level 3 (bsl-3) facilities are generally required for performing these assays. here we report the development of a safe and convenient neutralization assay for rapid screening neutralizing mabs targeting ha of h5n1 and h1n1 iav. we first generated a series of pseudoviruses bearing has of four clades of h5n1 iav isolated from avians and humans, respectively, and the 2009 epidemic h1n1 iav, and an hiv-1 backbone (pnl4-3.luc.re). these pseudoviruses are single-cycle and replication-deficient viruses [18] [19] [20] . then we used these pseudoviruses to establish a neutralizing assay in a bsl-2 laboratory, based on which we screened a panel of mabs specific for the ha of an h5n1 virus and successfully identified three specific neutralizing mabs, suggesting that this established assay is safe and efficient for screening neutralizing mabs targeting ha of iavs. table 1) were synthesized by integrated dna technologies (coralville, ia) and inserted into pcdna3.1 vector as above. the constructed recombinant plasmids containing ha were, respectively, designated as qh-ha-, xj-ha-, ah-ha-, hk97-ha-, 1194-ha-and h1n1-ha-pcdna3.1 and confirmed for correct insertion by sequencing analysis. generation of influenza pseudovirus was done as previously described with some modifications [21, 22] . briefly, 293t cells (atcc, manassas, va) were co-transfected with 10 lg plasmid encoding env-defective, luciferase-expressing hiv-1 (pnl4-3.luc.re) and 10 lg each of the 6 plasmids, including qh-ha-, xj-ha-, ah-ha-, hk97-ha-, 1194-ha-and h1n1-ha-pcdna3.1, respectively, into 100-mm culture dishes using calcium phosphate method. cells were changed into fresh dmem 8 h later, and exogenous bacterial neuraminidase (na) from vibrio cholerae (sigma, st. louis, mo) was added at the concentration of 4.8 lg/ml 24 and 48 h post-transfection. the transfection without adding na was used as parallel controls. supernatants containing ha pseudovirus with or without na were harvested 72 h post-transfection, and used for single-cycle infection. the viral particles with corresponding ha were named as qh-ha, xj-ha, ah-ha, hk-ha, 1194-ha, and h1n1-ha pseudoviruses, respectively ( table 1 ). the plasmids encoding vesicular stomatitis virus g protein (vsv-g), vsv-g-pcdna3.1, and the pcdna3.1 vector only were used to co-transfect with pnl4-3.luc.re plasmid to generate vsv-g pseudovirus and pseudovirus without env (envà) as controls. for titration of the pseudovirus, 293t cells were infected with 100 ll of each pseudovirus at 2-fold dilution in 96-well culture plates, followed by detection of luciferase activity 72 h later. the titer of pseudovirus was expressed as relative luciferase unit (rlu). the p24 in produced pseudovirus was measured by elisa. briefly, 96-well elisa plates were pre-coated with anti-hiv-1 p24 (clone nih 183-h12, 5 lg/ml) overnight at 4°c and blocked with 2% non-fat milk at 37°c for 2 h. lysed pseudoviruses were added to the plates and incubated at 37°c for 1 h. after washes, hiv-igg sera were added (1 lg/ml) and incubated at 37°c for 1 h, followed by sequentially incubated with biotin-labeled goat anti-human igg (1:10,000) and streptavidin-conjugated horseradish peroxidase (hrp) (1:10,000) at 37°c for 1 h. the substrate 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb) (zymed, carlsbad, ca) was added to the plates, and the reaction was stopped by 1 n h 2 so 4 . the absorbance at 450 nm (a450) was measured by elisa plate reader (tecan, san jose, ca). hiv-1 p24 in the viral core and ha on the surface of the generated pseudoviruses were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), followed by western blot according to our previous protocols with some modifications [23] . briefly, lysed pseudoviruses were resolved by 10-20% tricine gel (invitrogen), which were then transferred to nitrocellulose membranes (bio-rad laboratories, hercules, ca), and blocked in 5% non-fat milk overnight at 4°c. the blots were, respectively, incubated with anti-hiv-1 p24 (clone nih 183-h12) and anti-ha #8 mab (produced below) at 1:1000 dilution. after three washes, the blots were incubated with hrp-conjugated goat anti-mouse igg (1:5000, zymed) for 1 h at room temperature. signals were visualized with ecl western blot substrate reagents and amersham hyperfilm (ge healthcare, piscataway, nj). to detect cell tropism, 10 4 cells/well of 293t, madin-darby canine kidney (mdck), a549, chinese hamster ovary (cho-k1), and vero cells (atcc) were, respectively, transduced with produced pseudoviruses. fresh dmem was added 24 h later, and luciferase activity was detected 72 h post-transduction and expressed as rlu. the genes encoding full-length ha of iav (a/vietnam/1194/ 2004(h5n1)) plus 6â his tag were amplified by pcr and inserted into the pacgp67a baculovirus transfer vector (bd biosciences, san jose, ca) to construct rha-bacul plasmid. the expression of rha protein was conducted according to the manufacturer's protocols and our previously described method [23] . briefly, 2 lg rha-bacul plasmid was mixed with 0.5 lg bd baculogold linearized baculovirus dna and sit at room temperature for 5 min before addition of 1 ml bd baculogold transfection buffer b. the mixture was added to sf9 insect cells (atcc) pre-covered with 1 ml transfection buffer a, followed by incubation of the cells at 28°c for 4 h and further culture of the cells in fresh sf-900 ii sfm (invitrogen) for 4 days. supernatants were collected and infected cells with three more cycles for virus amplification. cell culture supernatant from the 4th cycle was collected for purification of rha protein using his columns (promega, madison, wi). five female balb/c mice aged 4-6 weeks were subcutaneously vaccinated with purified rha protein (20 lg/mouse) in the presence of freund's complete adjuvant (fca, sigma) and boosted twice with the same immunogen (10 lg/mouse) containing freund's incomplete adjuvant (fia, sigma) at 21-day intervals. the hybridoma technique for generation and screening of specific mabs against ha protein was performed according to the standard protocol [22] . briefly, vaccinated mice were sacrificed 10 days after the last boost, and the splenocytes were fused with mouse myeloma cells (sp2/0). the ha-specific mab-screening positive hybridomas were screened by elisa using rha protein as the coating antigen. positive cells were expanded, retested and subcloned to generate stable hybridoma cell lines. the mabs were purified from culture supernatant using nprotein a sepharose 4 fast flow (ge healthcare). neutralizing activity of mabs against infection of ha pseudovirus was performed as follows. briefly, pseudovirus-containing supernatants were incubated with serially diluted h5n1 ha mabs at 37°c for 1 h before adding to 293t cells pre-plated in 96-well culture plates (10 4 /well). mab 33g4 from severe acute respiratory syndrome coronavirus (sars) [22] was used as the negative control. twenty-four hours later, cells were re-fed with fresh medium, which was followed by lysing cells 72 h later using cell lysis buffer (promega) and transferring the lysates into 96-well luminometer plates. luciferase substrate (promega) was added to the plates, and relative luciferase activity was determined in ultra 384 luminometer (tecan). the neutralization of ha pseudovirus was calculated [24] and presented as 50% neutralizing antibody titer (nt 50 ). the titer of anti-influenza ha neutralizing activity of mabs was determined by microneutralization assay as follows. briefly, serial 2-fold dilution of mabs was mixed with 100tcid 50 of a/vietnam/ 1194/04(h5n1) and incubated at 37°c for 2 h before adding to mdck cells in quadruplicate. medium was replaced with fresh dmem 2 h later, and cell culture was continued for 72 h at 37°c. viral cytopathic effect (cpe) was observed daily and recorded on day 3 post-infection. the neutralizing antibody titer (nt 50 ) was determined by the ha test and defined as the lowest dilution that was negative for ha when 50 ll of cell culture supernatant was incubated with equal volume of 0.5% turkey red blood cells (lampire biological laboratories, pipersville, pa) at room temperature for 30 min. values were presented as mean with standard deviation (sd). statistical significance among different groups was calculated by student's t test using stata statistical software. p values less than 0.05 were considered significant. to verify whether these ha pseudoviruses contain the hiv-1 p24, we first used elisa to detect hiv-1 p24 in the pseudoviruscontaining supernatants. all six ha pseudoviruses, one vsv-g pseudovirus and one envà pseudovirus contained about 50 ng/ ml of hiv p24. then, we did western blot to further detect the reactivity of ha pseudovirus with mabs specific for hiv-1 p24 and iav ha, respectively. as shown in fig. 1 , clear bands, respectively, corresponding to p24 and ha were observed in all six ha pseudoviruses, suggesting that the ha of influenza pseudovirus was incorporated into the hiv-1 viral particles. exogenous na was added to 293t cells 24 and 48 h post-transfection to further compare the transduction activity of pseudovirus in the absence and presence of na. fig. 2a indicates that ha pseudovirus containing na induced a significantly higher level of infection, with an average 50-fold increase of rlu in contrast to pseudovirus without na in transduced 293t cells. the highest infectivity was reached by qh-ha, a pseudovirus expressing the ha of a highly pathogenic h5n1 iav in goose (table 1 ). since ha pseudoviruses containing na may be able to improve the infection rate significantly, they were used for subsequent experiments to detect pseudovirus cell tropism and test neutralizating activity for ha mabs. to detect cell tropism of the produced ha pseudovirus, cells from various tissues and different hosts, including mdck (dog kidney), a549 (human lung), cho-k1 (chinese hamster ovary), vero (african green monkey kidney) and 293t (human kidney) cells were, respectively, transduced with six pseudoviruses containing hiv-1 p24 (50 ng/ml), and luciferase activity was detected 72 h post-infection. vsv-g pseudovirus, which has a broad host range capable of infecting multiple tissues in various hosts, and envà pseudovirus were used as the positive and negative controls. as shown in fig. 2b with the infectivity from a549 and mdck cells being relatively higher than that from vero and cho-k1 cells. particularly, these pseudoviruses demonstrated a significantly higher level of infective ability in 293t cells than other cells, suggesting that the cells derived from human kidney may be more susceptible than those derived from other human tissues or non-human hosts to the ha pseudovirus infection. in addition, avian (goose)-origin pseudovirus (qh-ha) showed a higher affinity and infectivity than human-hosted pseudoviruses, such as xj-ha, ah-ha, hk-ha, 1194-ha and h1n1-ha. as expected, the positive control, vsv-g pseudovirus, was able to infect all tested cell lines, while negative control, envà pseudovirus was unable to infect any of these cell lines. elisa was used to initially screen mabs generated by immunization of mice with rha of a/vietnam/1194/2004(h5n1). as shown in fig. 3a , 20 mabs were initially screened for ha-specific antibody responses, 10 of which with the highest antibody titer were selected to detect neutralizing activity against five pseudoviruses expressing ha of h5n1 iavs (qh-ha, xj-ha, ah-ha, hk-ha, 1194-ha), one pseudovirus bearing ha of 2009 epidemic h1n1 iav (h1n1-ha) ( table 1) , and the vsv-g pseudovirus in 293t cells since this cell line demonstrated the highest ability to support virus infection. as shown in fig. 3b , among selected 10 mabs specific to ha of a/vietnam/1194/2004(h5n1), 3 mabs (#8, #9 and #17), particularly #8 mab, neutralized the homologous strain of pseudotyped h5n1 (1194-ha) virus infection, while other haspecific mabs showed non-neutralizing activity against homologous and heterologous h5n1 (qh-ha, xj-ha, ah-ha, hk-ha, 1194-ha) and 2009 epidemic h1n1 (h1n1-ha) pseudoviruses, as well as vsv-g pseudovirus. in addition, the unrelated control mab 33g4, which is specific for sars-cov spike protein, could not neutralize any of the above pseudoviruses. these results indicate that the established pseudovirus neutralization assay is specific to ha, suggesting its potential application for quick and effective screening of mabs targeting ha of iavs. the selected 10 mabs were further tested for the neutralizing activity against a wild-type h5n1 virus (a/vietnam/1194/2004) using microneutralization assay, and the results were compared with those from pseudovirus neutralization assay based on the homologous h5n1 virus expressing ha of h5n1 (1194-ha). table 2 shows that #8, #9 and #17 mabs, especially #8 mab, could inhibit infection of live h5n1 virus (a/vietnam/1194/2004), while other mabs had no neutralizing activity against this virus in tested mdck cells. since the results correspond to those of the pseudovirus neutralizing assay based on the ha of the same virus type, the established pseudovirus neutralization assay was confirmed as a reliable method for testing the neutralizing activity of mabs against ha of iavs. in this study, six pseudoviruses were produced in the supernatant of 293t cells in the presence or absence of exogenous na, respectively, expressing ha of a variety of four clades (clade 0, 1, 2.2, and 2.3) of hpai h5n1 virus strains and the epidemic strain of 2009 h1n1 virus isolated from avian goose and humans (table 1) . although packaged ha pseudoviruses without na may infect target cells, the addition of exogenous na to 293t cells co-transfected by hiv-1 (pnl4-3.luc.re) and ha plasmid was shown to significantly increase the transduction efficiency ( fig. 2a) , possibly because na protein may help to remove sialic acid from the host cells to facilitate viral release [25] , thus making it an indispensable component of ha-based iav infection. these results are consistent with previous reports by rong and colleagues [21] . the produced ha pseudoviruses were demonstrated to infect a number of nonhuman (mdck, vero and cho-k1) and human (293t and a549) cells. because the human 293t and a549 cells maintained higher transduction efficacy (fig. 2b) , it can be reasonably inferred that both the avian-origin h5n1 and the swine-origin h1n1 viruses isolated from avian (goose) and humans have a higher tendency to infect humans than other hosts, such as chinese hamster and african green monkey. these results further highlight the importance of developing anti-iav agents and vaccines for treatment and prevention of influenza. a pseudovirus neutralization assay was further developed based on the produced ha pseudoviruses and used to screen neutralizing mabs against ha of h5n1 virus (a/vietnam/1194/2004). three of ten selected mabs were able to neutralize infection by the pseudovirus carrying ha from the homologous h5n1 iav, while none of them neutralized pseudoviruses expressing ha from heterologous iavs and the unrelated vsv-g pseudovirus in the tested cell lines (fig. 3b) , the result of which is highly consistent with that from microneutralization assay ( table 2 ). these findings demonstrated that the established pseudovirus neutralization assay is highly specific and sensitive, thus making it especially suited to the quick screening of a large amount of mabs in a short period of time. compared with microneutralization assay and plaque reduction neutralization assay that require the handling of a live virus and the use of approved bsl-3 laboratory facilities, the established non-infectious virus-based pseudovirus neutralization assay is relatively safer and particularly useful when access to a bsl-3 laboratory is restricted. neutralizing antibodies targeting ha of iavs play crucial roles in neutralizing virus infection, clearing virus and suppressing virus spread [26] [27] [28] . thus, application of the pseudovirus neutralization assay may be extended to evaluate the efficacy of ha-based influenza vaccine candidates and antiviral agents targeting ha of iavs. this neutralization method could also be optimized for detecting specific neutralizing activity of clinical patient samples, which may be useful for predicting prognosis of the disease. to conclude, the ha pseudovirus-based neutralizing assay can be applied to screen neutralizing mabs and therapeutics targeting iav ha, and to evaluate the ha-based iav vaccines, as well as to detect neutralizing activity of clinical samples, making it possible to assess virus-neutralizing activities safely and rapidly without using live infectious influenza viruses. the detection limit is 1:100. a using pseudovirus neutralization assay. b using microneutralization assay. origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic h5n1 influenza viruses: outbreaks and biological properties structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion length requirements for membrane fusion of influenza virus hemagglutinin peptide linkers to transmembrane or fusion peptide domains receptor binding and membrane fusion in virus entry: the influenza hemagglutinin recombinant parainfluenza virus 5 (piv5) expressing the influenza a virus hemagglutinin provides immunity in mice to influenza a virus challenge dna vaccine encoding hemagglutinin provides protective immunity against h5n1 influenza virus infection in mice safety and immunogenicity of a baculovirus-expressed hemagglutinin influenza vaccine: a randomized controlled trial neutralizing human monoclonal antibody against h5n1 influenza ha selected from a fab-phage display library passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza a h5 hemagglutinin in mice new class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets neutralizing anti-influenza virus monoclonal antibodies: therapeutics and tools for discovery prophylactic and therapeutic efficacy of human monoclonal antibodies against h5n1 influenza combination therapy using chimeric monoclonal antibodies protects mice from lethal h5n1 infection and prevents formation of escape mutants human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza a virus subtype h5n1 cross-protective potential of a novel monoclonal antibody directed against antigenic site b of the hemagglutinin of influenza a viruses naturally occurring human monoclonal antibodies neutralize both 1918 and 2009 pandemic influenza a (h1n1) viruses development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein generation and characterization of an h5n1 avian influenza virus hemagglutinin glycoprotein pseudotyped lentivirus pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to h5n1 influenza viruses analysis of hemagglutininmediated entry tropism of h5n1 avian influenza receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies recombinant receptor-binding domain of sars-cov spike protein expressed in mammalian, insect and e. coli cells elicits potent neutralizing antibody and protective immunity theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies emergence and pandemic potential of swine-origin h1n1 influenza virus contributions of the avian influenza virus ha, na, and m2 surface proteins to the induction of neutralizing antibodies and protective immunity heterosubtype neutralizing responses to influenza a (h5n1) viruses are mediated by antibodies to virus haemagglutinin generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza a h5n1 viruses this study was supported by the national high technology key: cord-002407-25cawzi0 authors: nogales, aitor; martínez-sobrido, luis title: reverse genetics approaches for the development of influenza vaccines date: 2016-12-22 journal: int j mol sci doi: 10.3390/ijms18010020 sha: doc_id: 2407 cord_uid: 25cawzi0 influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. however, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. the segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. with the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary dna copies of the viral genome by transfection of susceptible cells. these reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. in addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. in this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines. influenza a (iav) and b (ibv) viruses belong to the orthomyxoviridae family of enveloped viruses [1] . iav is able to infect several species and mostly exists in the wild aquatic fowl reservoir [2] [3] [4] . on the other hand, ibv is mainly restricted and adapted to humans, although sporadic infections of seals have been documented [5, 6] . iav and ibv genomes contain eight negative sense, single-stranded viral (v)rna segments [1] (figure 1 ). iav and ibv vrnas contain a central coding region that is flanked at both terminal ends by non-coding regions (ncrs), which serve as promoters to initiate genome replication and gene transcription by the viral polymerase complex [1, 7] . influenza vrnas in the virion are found as viral ribonucleoprotein (vrnp) complexes encapsidated by the viral nucleoprotein (np) and a single copy of the viral polymerase complex. influenza virus-encoded rna-dependent rna polymerase (rdrp) [8] is a trimeric complex consisting of the polymerase basic 1 (pb1) and 2 (pb2) and acidic (pa) proteins and, together with the viral np, are the minimal components involved in viral replication and transcription [9] . iav and ibv share many features, but they differ in their host range, virion structure, genomic organization and glycan binding specificities [1, 10] . despite having similar genomes encoding homologous proteins, iav and ibv are distinguished by the different lengths of proteins and noncoding regions (ncrs) that serve as promoters for genome replication and gene transcription [5, 11, 12] (figure 1 ). likewise, they can also be distinguished by the presence of accessory proteins encoded from overlapping open reading frames (orfs) and by the antigenic differences of internal proteins [13] (figure 1a ,b). for instance, iav and ibv both encode ion channel proteins from the gene m segment 7, m2 and bm2, respectively. the m2 and bm2 proteins of iav or ibv are encoded together with the matrix protein 1 (m1) and both are incorporated into virions and expressed on the surface of virus-infected cells [1] . however, the m2 protein of iav is translated from a spliced mrna [14] , while the ibv bm2 protein is translated using a different strategy, where the initiation codon of bm2 protein overlaps the termination codon of m1 protein (uaaug, a stop-start pentanucleotide) [15] . in addition, ibv expresses the nb ion channel, which is absent in type a influenza virus [1] ( figure 1b) . however, both influenza viruses encode two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) ( figure 1a ,b). iav and ibv ha proteins are involved in binding to cellular receptors and responsible for the fusion of the viral and endosomal membranes [16] . infection with iav or ibv induces a protective immunity mediated, at least partially, by antibodies directed against the viral ha, which is the main immunogenic target in both natural infections and vaccine approaches. influenza na glycoprotein is responsible for the cleavage of sialic acid moieties from iav and ibv share many features, but they differ in their host range, virion structure, genomic organization and glycan binding specificities [1, 10] . despite having similar genomes encoding homologous proteins, iav and ibv are distinguished by the different lengths of proteins and non-coding regions (ncrs) that serve as promoters for genome replication and gene transcription [5, 11, 12] (figure 1 ). likewise, they can also be distinguished by the presence of accessory proteins encoded from overlapping open reading frames (orfs) and by the antigenic differences of internal proteins [13] (figure 1a ,b). for instance, iav and ibv both encode ion channel proteins from the gene m segment 7, m2 and bm2, respectively. the m2 and bm2 proteins of iav or ibv are encoded together with the matrix protein 1 (m1) and both are incorporated into virions and expressed on the surface of virus-infected cells [1] . however, the m2 protein of iav is translated from a spliced mrna [14] , while the ibv bm2 protein is translated using a different strategy, where the initiation codon of bm2 protein overlaps the termination codon of m1 protein (uaaug, a stop-start pentanucleotide) [15] . in addition, ibv expresses the nb ion channel, which is absent in type a influenza virus [1] ( figure 1b) . however, both influenza viruses encode two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) ( figure 1a ,b). iav and ibv ha proteins are involved in binding to cellular receptors and responsible for the fusion of the viral and endosomal membranes [16] . infection with iav or ibv induces a protective immunity mediated, at least partially, by antibodies directed against the viral ha, which is the main immunogenic target in both natural infections and vaccine approaches. influenza na glycoprotein is responsible for the cleavage of sialic acid moieties from sialyloligosaccharides and facilitates the release of newly produced virions from infected cells [17, 18] . iavs are classified on the basis of the antigenic properties of ha and na into 18 ha (h1-h18) and 11 na (n1-n11) subtypes [1, 19, 20] . however, only iav h1n1 and h3n2 subtypes are currently circulating in humans. on the other hand, two major lineages of ibv are circulating in humans, the victoria-like and yamagata-like subtypes that are divergent from the ancestral ibv (b/lee/1940) and have been co-circulating in humans since the 1980s [5, 21, 22] . these two subtypes are the predominant circulating virus strains about once every three years [23] [24] [25] . infection with influenza viruses begins when the viral ha protein binds to its cellular receptor, a sialylated glycoprotein containing α-2,3 or α-2,6 linkages [16] . upon the binding to the receptor, the uptake of the virus by receptor-mediated endocytosis is initiated, and the cell membrane engulfs the virus particles in an endosome. after endocytosis and upon acidification of the endosome, influenza viral ha undergoes a conformational change responsible for the fusion of the viral and the endosome membrane [16] . then, the m2 (iav) or bm2 (ibv) ion channel proteins promote the release of the vrnp complexes from the virion core into the cytoplasm of infected cells [14, 26] . the vrnps are translocated from the cytoplasm to the nucleus of infected cells to initiate viral genome replication and gene transcription [27] . the nuclear export (nep) and matrix 1 (m1) proteins are responsible for the nuclear export of newly-synthesized vrnps into the cytoplasm of infected cells. notably, a single copy of each of the eight vrnas is packaged into new virions [28, 29] . selective package of influenza vrnas into nascent virions is mediated by rna-rna interactions of vrna packaging signals present at the terminal ends of each of the vrna segments [30] . finally, the receptor-destroying enzymatic activity of na is responsible for the release of newly-synthesized viral particles from the surface of infected cells [17] . among the first battles between the host and the virus, cellular type i interferon (ifn-i) plays an important role in controlling viral infection [31] . therefore, viruses have developed multiple strategies to hijack the host cellular immune response. influenza vrna segment 8, or the nonstructural (ns) gene, encodes two distinct proteins through a direct or alternative splicing mechanism [1] . influenza virus segment 8 produces the nonstructural protein 1 (ns1) as a primary transcript, whereas nep is produced by alternative splicing of the ns mrna [32] . ns1 has multiple functions during the replication cycle of influenza virus, but most notably inhibits induction of ifn-i response and innate immune activation [31] . influenza viruses pose a threat to human health and are responsible for global epidemics every year [33] [34] [35] [36] [37] . in fact, influenza virus is one of the most significant causes of morbidity and mortality yearly, leading to a significant economic impact [38] . despite the implementation of effective and comprehensive vaccination programs, the world health organization (who) estimates that seasonal influenza virus infections results in about one billion infections, 3-5 million cases of severe disease and between 300,000 and 500,000 deaths around the world annually [39] . moreover, just in the united states (u.s.), influenza viral infections result in an average of 87 billion dollars of cost due to prophylactic, therapeutic and hospitalization costs, as well as missed school or work days [38, [40] [41] [42] . in addition to seasonal influenza, iav can cause sporadic pandemics of great consequences when novel viruses are introduced into humans [43] . the mechanisms responsible for the emergence of seasonal and pandemic iavs are antigenic drift and antigenic shift, respectively [12, [44] [45] [46] . in the case of seasonal influenza, mutations in the viral genome result in the selection of antigenic variants with changes in viral tropism, increased levels of viral fitness or in the ability to escape neutralizing antibody (nab) responses induced upon previous natural infections or vaccinations [44, 45] . moreover, antigenic drift can lead to variant viruses resistant to antivirals [47] . on the contrary, antigenic shift results from co-infection of a host cell or organism with two or more iavs where vrnas in viral progenies are reassorted [12, 46] . by obtaining viral segment constellations that confer influenza virus optimal replication, transmissibility and immunologic escape, reassortant iavs can lead to a pandemic in immunologically-naive populations. iavs and ibvs can reassort intratypically between subtypes or lineages, but intertypic reassortment, or genetic swapping of segments between iav and ibv, has not been reported [21, 48, 49] . the absence of intertypic reassortment is mediated by the lack of compatible packaging signals between iav and ibv [12] . in the last century, three iav pandemics have occurred: the h1n1 spanish flu of 1918, the h2n2 asian flu of 1957 and the h3n2 hong kong flu of 1968 [44, 50] . of these three, the 1918 h1n1 spanish flu was particularly fatal and responsible for approximately 50 million deaths around the world [51] . although not classified as true pandemics, three epidemics of influenza h1n1 viruses in 1947, 1976 and 1977 were feared to have pandemic potential. in 2009, a swine-origin h1n1 iav was responsible for the first influenza pandemic of the 21st century and infected, in less than one year, more than 600,000 individuals around the world [52, 53] . although influenza virus infections are primarily spread by person-to-person transmission via aerosolized droplets, infection with new avian-or swine-origin iav can take place and may have great risk for pandemic potential if the virus acquires the ability to be transmitted between humans [54, 55] . ibvs usually contribute less to seasonal epidemics than iavs of the h3n2 subtype, but they contribute more than type a h1n1 influenza strains and are the predominant circulating virus strains once every three years [23] [24] [25] . moreover, during the last decade, ibv has been the cause of several acute respiratory illness outbreaks [56] [57] [58] [59] [60] . to date, no influenza pandemics have been linked to ibvs. public health concerns posed by influenza virus infections are aggravated by the ability of influenza viruses to efficiently transmit and the limited therapeutic options to treat viral infections [37] . thus, vaccination remains our best medical intervention to protect humans against seasonal influenza virus. however, the efficiency of current influenza vaccines is suboptimal [61] . the segmented genome of influenza viruses provides an evolutionary advantage of reassortment, or the exchange of viral genome segments between different viral strains within the same type. in addition to this exchange of genome material (or antigenic shift), influenza viruses can introduce mutations in their genomes (or antigenic drift), leading to viral mutants with resistance against current antivirals or nabs [62] [63] [64] . because of the antigenic drift, influenza vaccines need to be reformulated yearly to ensure that the ha and na present in the vaccine match those present in circulating seasonal viruses. to date, three types of influenza virus vaccines are approved by the food and drug administration (fda) for human use: recombinant viral ha, inactivated virus and live-attenuated viruses [65] [66] [67] [68] [69] . regardless of the type of vaccine, seasonal influenza vaccines contain antigens from the three circulating influenza virus strains: iav subtypes h1n1 and h3n2; and ibv (victoria-like or yamagata-like lineage) [67, 70] . recently, to improve the efficacy of seasonal influenza vaccines, a quadrivalent influenza vaccine formulation that includes both ibv lineages components (victoria-like and yamagata-like lineage) has been approved by the fda [14] . the most common influenza vaccine is the inactivated influenza vaccine (iiv). iiv, which is administered intramuscularly, has been shown to elicit protective humoral immunity by producing nabs that target epitopes on ha [61, 71] . in contrast to iiv, the live-attenuated influenza vaccine (laiv) and its administration mimic the natural route of virus infection, which consequently having risks and benefits [72] . an advantage is that laiv elicits both more rapid and efficient innate and adaptive immune responses [65] and can provide more efficient cross-reactive t-cell-mediated protection against heterologous influenza viruses [72, 73] . although both iiv and laiv have been shown to be efficient for the treatment of influenza viral infections, there is an unmet need to increase the effectiveness of seasonal influenza vaccines. likewise, there is an urgent need to develop effective vaccines for the treatment of potential pandemic influenza viruses. although vaccination is the main method to prevent influenza infections in humans, antivirals offer an additional countermeasure against new rapidly-spreading and/or potentially pandemic influenza viruses [74] [75] [76] . therapeutic choices to control influenza infection are currently limited to two classes of fda-approved antivirals targeting either the viral m2 ion channel (amantadine, rimantadine) [77] [78] [79] or the sialidase active site of na (oseltamivir, zanamivir) [80] . the first inhibits the initial steps of the virus life cycle, while the second inhibits virus release. however, na inhibitors are the only type of antivirals approved for the prophylaxis and treatment of ibv infections [81] , although data from clinical trials have shown that oseltamivir is less effective against ibvs than against iavs [82] . importantly, mutations in the viral genome can lead to influenza antivirals being ineffective, like in the case of m2 blockers, which are no longer recommended against circulating seasonal influenza viral strains [83] . therefore, the emergence of drug-resistant influenza variants is an increasing concern for controlling influenza infections [76, 84] , and there is a significant need for the identification of novel compounds with antivirals properties. influenza viruses evade human pre-existing immunity by accumulating mutations (antigenic drift), allowing reinfection of individuals previously exposed to natural infections or vaccinated. thus, vaccine companies have to reformulate the composition of influenza vaccines yearly to ensure a good match between the viruses present in the vaccine and those seasonally circulating in humans [33, 34, 85] . the who global influenza surveillance network (gisn) [35] , which includes more than 120 national influenza centers in over 90 countries [86] , tracks the evolution and epidemiology of influenza viruses and uses the collected data for the vaccine strain selection process. these studies also help to understand virus evolution and epidemiology in different geographic areas [19] . (4) the national institute of infectious disease in tokyo, japan. basically, the degree of immunity induced by one influenza strain that is effective against another strain is mainly dependent on the antigenic difference between both viral strains [87, 88] . the influenza viral glycoprotein ha, the primary target of the protective neutralizing immune responses [88, 89] , is the focus of influenza virus surveillance and the primary component targeted by currently licensed influenza vaccines [72, 89] . influenza vaccine production is challenging because of the wait time that is needed to identify the predominant circulating virus and the production time that is needed to manufacture the vaccine. the national influenza centers perform virus isolation on certain samples obtained from patients to identify circulating viruses and to determine if the viruses grow efficiently in culture. normally, influenza viruses are isolated using madin darby canine kidney (mdck) cells instead of chicken embryonated eggs because higher isolation rates, especially for h3n2 iav strains, have been reported using mdck cells [90] [91] [92] . although most influenza viruses are isolated from mammalian cells, viruses must be generated in embryonated eggs for vaccine manufacturing due to regulations [93] . nowadays, the vaccine strains must be selected almost 7-9 months ahead of the influenza season in which they will be used. the recommendation for the strains included in the vaccine composition for the northern hemisphere is made in february to allow time for the~300 million doses of vaccine to be produced in time for vaccinating people in october/november. this allows for influenza season preparation, which typically peaks sometime between december and march [86] . on the other hand, for the southern hemisphere, recommendations are provided in september, and vaccination takes place in march/april of the following year [86] . seasonal influenza vaccines must protect against h1n1, h3n2 and b viral strains currently circulating in humans globally [34, 86] . the main goals of influenza vaccines are the protection against infection and disease caused by influenza infections and to restrict virus transmission within the population [67, 94] . nab responses, commonly assessed by measuring hemagglutination inhibition (hai) titers, are currently used as a serological marker of the efficient immunological response to the vaccine. the effectiveness of influenza vaccines is variable and usually higher in children and in healthy adults under the age of 65. in individuals above 65 years of age, lower effectiveness has been observed [61, 71, 95] . recommendations for influenza vaccination differ between countries, but since influenza vaccines do not induce long-lasting antibody protection, annual influenza vaccinations are recommended. nowadays, the most used influenza vaccines can be divided into iiv and laiv [65, 66, 72, 73, 96 ]. killed virus vaccines or iiv are generally administered intramuscularly and can be classified as whole virus vaccines or split vaccines [66, 71, 97, 98] . whole virus vaccines were the first to be developed. the influenza virus is grown in embryonated chicken eggs, subsequently purified, concentrated and chemically inactivated with formaldehyde [98] (figure 2 ). whole virus vaccines are safe and well tolerated, with an efficacy of 60%-90% in children and adults. on the other hand, the split-virus vaccine exposes all viral proteins and subviral elements upon dissociation of the virions by a nonionic detergent treatment step [97, 99] . most influenza vaccines in the u.s. and europe are egg-produced, formaldehyde-inactivated, then chemically disrupted with nonionic detergents after purification. unlike virus-based vaccines, subunit influenza vaccines consist of purified viral ha or ha/na proteins without the other viral components [68] . these subunit vaccines can be produced in eggs if the viral proteins are prepared from viruses where ha/na have been purified by removal of other viral component. in addition, the subunit vaccines can be generated using novel manufacturing technologies [98, 100] , which allows for the production of large quantities of the viral ha using baculovirus expression systems and recombinant dna technologies [100] . the iav strains that are used in vaccine manufacture are high-growth 6 + 2 reassortants containing the ha and na gene segments from the target strains in the backbone of influenza a/puerto rico/8/1934 h1n1 (pr8) or other high growth virus. influenza pr8 replicates at high titers in eggs and cells and also has a favorable safety profile in humans [98, 101] (figure 2a ). to generate the reassortant viruses, eggs are co-infected with pr8 and seasonal strains (figure 2a ). selection of appropriated seed vaccine viruses is made by amplification in the presence of pr8 ha and na nabs [98] (figure 2a) . then, selected viruses are cloned and sequenced for confirmation. the ibv vaccines are typically wild-type (wt) viruses. however, ibv reassortants are used if wt ibv growth properties are not optimal for efficient growth and vaccine production [98] . the remaining class of vaccines consists of live-attenuated influenza viruses. attenuated human laivs were developed in the 1960s by serial passage of the virus in eggs using suboptimal conditions of temperature. the resulting attenuated viruses displayed a temperature-sensitive (ts) cold-adapted (ca) attenuated (att) phenotype that grew at 25 • c, but not at temperatures found in the lower respiratory tract (>35 • c) [102] [103] [104] [105] . because this ts, ca, att phenotype restricts virus replication to the upper respiratory track, these viruses could induce local protective immunological responses [65, 95, 96, 98] . laiv have been available in the u.s. since 2003 and are administrated intranasally. the advantages of a live virus vaccine, as compared to the inactivated virus vaccine, is that it is applied to the nasal mucosa where the vaccine can induce local immunity (including nabs), generate a cellmediated immune response and provide a cross-reactive and longer lasting immune response [106] . laiv have been available in the u.s. since 2003 and are administrated intranasally. the advantages of a live virus vaccine, as compared to the inactivated virus vaccine, is that it is applied to the nasal mucosa where the vaccine can induce local immunity (including nabs), generate a cell-mediated immune response and provide a cross-reactive and longer lasting immune response [106] . the current laivs consist of the internal viral segments (pb2, pb1, pa, np, m and ns) of an attenuated master donor virus (mdv) and the ha and na viral segments from the selected seasonal virus strain ( figure 2b ). the mdvs used are a/ann arbor/6/60 (h2n2) and b/ann arbor/1/66 for iav and ibv, respectively [103, 104, [107] [108] [109] [110] [111] [112] [113] . similar to mdv iav, mdv ibv was originally derived by serial passage of the parental wt virus and isolated at successively reduced temperatures in primary chicken kidney (pck) cells [114] . the resulting ibv mdv grows efficiently at 25 • c (ca), but its growth is restricted at 37 • c (ts). the genetic changes in the mdv strains have been recently characterized. the mdv iav includes five mutations in two of the viral polymerases (pb2 n265s; and pb1 k391e, d581g and a661t) and np (d34g) [103, 104, 115] . the mdv ibv has been reported to contain two mutant amino acids in np (a114 and h410) and one in pa (m431) that are responsible for the ts, ca signature [108] . two additional residues in m1 (q159 and v183) provide the mdv ibv an attenuated (att) phenotype [108] . although laivs have been approved for clinical use, to date, their mechanism of attenuation has not been completely understood. however, the tolerability of laivs in specific populations is an important concern because of the inherent risk of immunizing with live viruses. thus, laivs are not recommended for immunocompromised patients or asthmatics [116] and are not approved for use in children under two years of age [72, 117] . moreover, laivs harboring different ha and na viral segments can be unequally safe or immunogenic from year to year as the viral ha and na are different. similar to u.s. laivs, in russia, two ts, ca mdv iavs have been obtained by using a similar temperature adaptation approach. the two russian mdvs were originated from the same parental a/leningrad/134/57 h2n2 (len/57) influenza strain [118, 119] . similarly to the u.s. mdv, the russian mdvs have been selected by growing len/57 in embryonated chicken eggs at lower (25 • c) temperatures. the mdv len/17 was obtained after 17 passages of len/57 at 25 • c and has been used in the preparation of the russian laiv for use in adults. the genetic changes in the mdvs len/17 have been identified, and these include four mutations in three viral proteins (pb2 v478l; pb1 k265n and v591i; nep m100i) [120] . the second donor strain (len/47) was obtained after a total of 47 passages and has been used for vaccinating children less than 16 years of age, although the genetic changes responsible for the further ts, ca phenotype have not been well characterized [121] . in all cases, like for iiv, the mdvs contain six internal genes to generate vaccine strains in combination with the seasonal recommended ha and na genes from the circulating strains ( figure 2b ). laivs are generated either by classical reassortment in eggs (as previously described for the iiv) ( figure 2b ) or by reverse genetics, as indicated below [98] . most of the influenza virus vaccines have been traditionally produced in eggs, but with the progress made in mammalian cell culture technologies, influenza vaccine manufacturers have invested in these novel cell culture systems for the mass production of influenza vaccines without the need of eggs. the majority of the currently licensed influenza vaccines that are made by biotechnology companies use fertilized chicken egg-based production technology, but this process has multiple drawbacks. this form of manufacturing depends on the access to embryonated eggs, relies on the ability of influenza viruses to efficiently grow in eggs and is a resource-and time-intensive process [91, [122] [123] [124] . moreover, the risk of egg contamination by avian pathogens or microbes represents a risk for the production of influenza vaccines [125, 126] . importantly, in the case of an iav pandemic, the egg supplies can be compromised. for iiv, one dose for adults contains approximately 45 µg of ha (15 µg of viral ha for each of the three antigenic h1n1, h3n2 and ibv components), meaning one egg = 7-10 dose of vaccine. laivs share similar egg-based production process steps. the laiv is recovered from infected eggs and then purified and concentrated [90] . importantly, new vaccine production approaches that do not depend on the propagation of influenza viruses in eggs (e.g., cell cultures) represent an excellent option to increase influenza vaccine production. mammalian cell cultures have been used in the biopharmaceutical industry for the production of therapeutic proteins and/or vaccines [127, 128] . in 2012, the fda approved a cell-based production process for influenza vaccines, but the manufacturing process begins with egg-grown vaccine viruses per fda regulations. influenza vaccine production using fda-approved mdck or vero (african green monkey kidney) cells may eventually and completely replace the use of eggs for the production of influenza vaccines in the future. influenza vaccine production in mammalian cell lines offers several advantages over egg-based production: it allows faster and greater production capacity, improved availability of substrate for virus growth [122, 127] and eliminates reliance on the supply of embryonated chicken eggs [129, 130] . in addition, cell cultures can be cryopreserved and scaled up in bioreactors at any time. adjuvants have been shown to enhance the immune response elicited by an antigen and could be used to improve the immunogenicity of iiv [131, 132] . the use of adjuvants could also reduce vaccine dose, stretching antigen and vaccine supplies. currently, fda-licensed adjuvants for influenza vaccine usage include aluminum salt (alum) and the squalene oil-in-water emulsion systems mf59 (wadman 2005 (novartis)) [133] and as03 (glaxosmithkline) [134] . however, most of the current iivs do not contain any type of adjuvant, but many are under investigation. genetics techniques to generate recombinant viruses were first developed for dna viruses and based on the transfection of cells with plasmids encoding the viral genome or by heterologous recombination between plasmids bearing viral sequences with the virus genome and the helper virus [135] . initial genetics approaches for dna viruses were followed by manipulations of positive-sense rna viral genomes [136, 137] . transfection of plasmid dna, or rna transcribed directly in vitro from plasmids, containing the genome of poliovirus into susceptible cells led to the generation of recombinant infectious poliovirus [138] . however, the genomes of negative-sense rna viruses, including influenza, were less suitable to molecular biology manipulations in comparison with dna or positive-sense rna viruses since their genomes are complementary to mrna in their orientation and, therefore, not infectious by themselves [1, 7] . they require the presence of vrna(s) and the viral rdrps to initiate the replication cycle of the virus [33, 44] . the advent of reverse genetics and molecular engineering has transformed the influenza field, allowing multiple questions to be answered using genetically-engineered recombinant influenza viruses [139] . such studies include mechanisms of viral genome replication and gene transcription, pathogenicity and virulence, virus-host interactions or host range and transmissibility [12, 13, 139, 140] . moreover, these technologies have been implemented to develop influenza vaccines [141] and to generate recombinant influenza viruses expressing foreign proteins as vaccine vectors [142] [143] [144] or harboring reporter genes to easily track viral infections [142, [145] [146] [147] . plasmid-based reverse genetics for influenza virus allows for the simultaneous expression of the viral components involved in viral genome replication and gene transcription (pb2, pb1, pa and np) and the eight negative-stranded vrnas in transfected susceptible cells, which together generate de novo, recombinant iavs or ibvs (figure 3 ) [48] . the goal to generate vrna in vivo from cloned complementary (c)dnas was achieved when the rna polymerase i (pol i) system for influenza vrna synthesis was established [148, 149] . pol i is a nuclear enzyme that transcribes ribosomal (r)rna, which like influenza vrna does not contain a cap structure on the 5 or poly (a) structures on the 3 ends [150, 151] . importantly, pol i initiates and terminates transcription at defined promoter and terminator sequences allowing the generation of vrnas without additional nucleotides at their 5 or 3 ends, which is required for efficient generation of recombinant viruses using reverse genetics. nevertheless, the pol i promoter is species specific [152, 153] and was originally established for influenza rescue in human cells [135, 140, 149, 154] . currently, the pol i promoters of different species have been identified, allowing the generation of recombinant influenza viruses using reverse genetics techniques in avian, canine, equine or murine cells lines [155] [156] [157] [158] . nevertheless, the pol i promoter is species specific [152, 153] and was originally established for influenza rescue in human cells [135, 140, 149, 154] . currently, the pol i promoters of different species have been identified, allowing the generation of recombinant influenza viruses using reverse genetics techniques in avian, canine, equine or murine cells lines [155] [156] [157] [158] . influenza viruses require the presence of eight vrna segments for efficient virus fitness and successful production of virion progeny. the initial description of influenza reverse genetics, originally established in 1999 for iav [154, 159] , required the use of 12 plasmids to generate recombinant influenza viruses: four polymerase ii (pol ii) protein expression plasmids, encoding the viral rdrp complex (pb2, pb1 and pa) and np for vrnp reconstitution; and eight pol i-driven plasmids for expression of the eight vrna segments [154, 159] . however, it was later described that influenza viruses require the presence of eight vrna segments for efficient virus fitness and successful production of virion progeny. the initial description of influenza reverse genetics, originally established in 1999 for iav [154, 159] , required the use of 12 plasmids to generate recombinant influenza viruses: four polymerase ii (pol ii) protein expression plasmids, encoding the viral rdrp complex (pb2, pb1 and pa) and np for vrnp reconstitution; and eight pol i-driven plasmids for expression of the eight vrna segments [154, 159] . however, it was later described that only eight ambisense and/or bidirectional plasmids were needed for complete reconstitution of influenza viruses ( figure 3a ) [150, 151] . the eight plasmid-based rescue system is now the most common method for the generation of recombinant influenza viruses. because fewer plasmids are required, the eight-plasmid approach is more successful than the initial twelve-plasmid reverse genetic technique. the core of the eight-plasmid rescue system is that each plasmid contains an "ambisense cassette" that includes rna pol i and/or ii sequences, which drives the transcription of vrnas (pol i) and protein (pol ii) expression from the same viral cdnas ( figure 3b ). using a similar technology in 2002, this reverse genetics technique allowed the recovery of ibv entirely from ambisense plasmids [13, [160] [161] [162] . now, iav and ibv reverse genetics techniques are well established and commonly used in multiple research laboratories for different research purposes. influenza reverse genetics techniques have had an important effect on expanding our knowledge of the molecular biology and pathogenesis of influenza viruses, allowing researchers to answer important questions in the biology of iav and ibv that were not possible using conventional virological or biochemical procedures [12, 144, 147] . scientists can now mutate specific nucleotides in the influenza viral genome to elucidate the nature of regulatory sequences or the contribution of specific amino acids to the function of influenza viral proteins. for instance, reverse genetics technologies have enabled the identification and characterization of the cis-acting elements required for virus cell entry, uncoating, genome replication and gene transcription, encapsidation, packaging and viral release [13, 135, [163] [164] [165] . moreover, by engineering viral vectors suitable for the expression of foreign proteins in infected cells, multiple recombinant influenza viruses harboring reporter fluorescent and/or luminescent genes have been generated and used to identify antivirals or nabs in vitro and/or in vivo [142, 146, 147] . finally, reverse genetics have allowed the creation of single-cycle infectious iavs (sciiavs) that are restricted to one cycle of replication in parental cell lines. however, in complementing cell lines, sciiavs can replicate efficiently and to levels comparable to wt forms of influenza viruses [144] . highly virulent iav have the potential to pose a greater human threat than many other biosafety level (bsl)-3 and bsl-4 pathogens because of their efficient transmission and limited therapeutic options [47] . traditional immunological approaches (e.g., hai or microneutralization assays) to identify the presence of iav nabs rely on the manipulation of live forms of viruses and need the use of special bsl conditions. thus, novel approaches that allow the detection of viral nabs without the use of live forms of iavs and highly contained bsl laboratories would facilitate these serological assays. in this regard and because of their safety profile, sciiavs represent an excellent alternative to identify new antivirals and/or nabs [144] . moreover, several sciiavs have been shown to represent an excellent option for their implementation as safe, immunogenic and protective vaccines and/or vaccine vectors [69, 144] . influenza plasmid-based reverse genetics represent a better alternative to circumvent the process of generating reassortant virus by co-infection of chicken embryonated eggs for the generation of influenza vaccines (figure 2 ) [135] . moreover, the generation of recombinant influenza viruses using plasmid-based reverse genetics approaches is simple, well established and currently in use in several laboratories around the world [139, 166] (figure 4) . briefly, the eight-ambisense plasmids are co-transfected into susceptible fda-approved cells, and viable virus can be recovered from the tissue culture supernatants [139, 166] , then amplified in either embryonic eggs or fda-approved for vaccine production cells (figure 4) . for many years, iivs have been produced by reassortment in eggs ( figure 2) [90, 98, 122] . however, strains with the desired genotype (six internal genes of a high-growth virus and the ha and na glycoproteins of the seasonal influenza virus) could be produced easily and more quickly by implementing reverse genetics approaches. this process would overcome the need of chicken embryonated eggs to generate the desired reassortant virus and, therefore, minimizing the time associated with the selection process of the reassortant iiv ( figure 4a) . moreover, the improvements in surveillance, as well as the de novo gene synthesis for the production of the ha and na viral segments from the selected strains could reduce considerably the time of vaccine production. although influenza reverse genetics could be useful for the production of vaccine seed strains, to date, it has not been possible to predict which gene segment(s) constellations are required for efficient growth of different vaccine viruses. moreover, the time to produce vaccine viruses using reverse genetics versus the time needed to generate them using the classical reassortment approach could be a major consideration for the cost effectiveness in vaccine production and manufacturing. thus, the traditional viral reassortment has remained preferred because it allows the generation of a number of diverse gene(s) combinations in order to select recombinant viruses with better fitness [98] . during the last few decades, considerable improvements have been accomplished in the development of influenza vaccines. however, novel approaches to increase the effectiveness of seasonal influenza vaccines are needed. reverse genetics technologies have proven a valuable tool to develop reassortant strains for the production of laiv candidates ( figure 4b ). currently, seed viruses containing six gene segments from the mdv a/ann arbor/6/60 (h2n2) and the ha and na from the selected seasonal virus can be quickly generated using reverse genetics systems [98, 131] ( figure 4b ). the ha and na from selected seasonal influenza viruses can be amplified by rt-pcr or quickly chemically synthetized and then cloned in ambisense plasmids used for virus rescue. this technology could speed up the development of new laivs, bypassing the need to isolate the exact virus reassortment in eggs ( figure 2 ). the best way to combat influenza virus infection is to prevent it. thus, an urgent need exists to develop novel and more effective influenza vaccines. plasmid-based reverse genetic technologies have allowed the engineering of recombinant influenza viruses that contain single or multiple mutations in the viral genome, which can be potentially implemented as novel or improved vaccine approaches. in fact, several novel vaccine candidates have been developed with promising results in animal models of experimentation. here, we review and discuss some of them. because of ns1's ability to hijack the host innate immune ifn-i response, a variety of potential vaccine strategies have been developed, which are based on the use of modified ns1 proteins as a means for virus attenuation [167] [168] [169] [170] [171] [172] . equine [170] , swine [169, 173, 174] , avian [167, 175, 176] , canine [177] and human [178, 179] iavs with partial truncations in or deletions of the viral ns1 protein are all attenuated in vitro and in vivo [167, 169, 170, 179] . importantly, these recombinant iavs can induce a protective immune response upon a single intranasal vaccination in mice [177, [179] [180] [181] , horses [170] , pigs [169, 173, 174] , birds [167, 175, 176] and macaques [178] ; therefore, they represent excellent laiv candidates to prevent iav infections. in addition, a similar approach has been employed to develop attenuated ibvs with similar results [180] . mice inoculated with the ns1-truncated or -deleted mutants elicited an antibody response and showed protection against wt virus challenge [180] . thus, these ns1-truncated influenza a and b viruses represent excellent candidates as safe, immunogenic and protective laivs for multiple virus strains in different animal models. the genetic code in animals encodes for 20 different amino acids (aa) using 61 codons. this degeneracy of the genetic code allows amino acids, except tryptophan (w) and methionine (m), to be encoded by more than one synonymous codon [46] . viruses, including influenza, rely on the host cell translation machinery to synthesize their viral proteins for the formation of infectious virus progeny. as an evolutionary consequence, viruses have modified their codon usage according to the host they infect [182] . experimentally, protein synthesis can be downregulated by synthetically deoptimizing the codon usage of a gene [141, 182] . the generation of recombinant viruses containing genes with deoptimized codons is now feasible [141, 182] , and their level of attenuation depends on the viral gene targeted and the number of codon changes made during the codon deoptimization process [182] . for influenza viruses, many regions in the viral genome cannot be altered because of their important role in viral replication and transcription (e.g., ncrs), packaging (e.g., packaging signals), the presence of multiple overlapping orfs (e.g., segments 7 and 8), etc. [48] . to date, recombinant iavs that have been attenuated using a codon-pair [183, 184] or a codon bias [182] deoptimization approach to decrease expression levels of the viral pb1, ha and np [183] , na and ha [184] or ns1 and nep [182] have been generated. importantly, influenza viruses generated by codon deoptimization showed similar viral replication kinetics to wt virus in mdck cells, which is important for their effective use for vaccine production. however, to date, the ability of these recombinant iavs containing codon-pair or codon bias deoptimized viral segments to replicate in eggs has not yet been evaluated. importantly, all of the codon deoptimized iavs were attenuated in mice and able to provide, upon a single immunization dose, protection against a lethal challenge with a wt form of the virus, showing that laivs were safe, immunogenic and protective. however, mice do not accurately reflect virus pathogenesis and immunological responses seen in humans [81] and do not have the same codon usage bias as humans. therefore, studies aimed to demonstrate the safety, immunogenicity and protection efficacy of codon deoptimized recombinant iavs in other well-established animal models of influenza (e.g., guinea pigs, ferrets or nonhuman primates) could lead to their implementation as laivs in future vaccinations [141] . other promising approaches for the development of laivs relate to the use of sciiavs [144] . sciiavs based on their safety profile, ability to elicit protective humoral and cellular responses and protective effectiveness represent a feasible alternative to current influenza vaccines for the treatment of influenza viral infections [69, 144] . however, to date, no single-cycle infectious ibvs (sciibv) have been reported. when delivered intranasally, sciiav has been shown to be safe in a mouse model of influenza infection, without signs of illness or mortality [69, [185] [186] [187] [188] [189] . moreover, and similar to the current laivs, intranasal immunization with a single dose of sciiavs elicited localized mucosal immune responses and recruitment of influenza-specific cd8 t-cells into the lungs of vaccinated animals [145, 185, 186, 190] , the latest being the main contributor of immunity against challenge with heterologous influenza viruses [69, 190] . importantly, sciiavs protected mice against lethal influenza virus challenges [69, 144] . moreover, similar safety, immunogenicity and protection efficacy of sciiavs were observed in ferrets [69] . vaccination of pigs with sciiav has also been shown to be immunogenic and protective against challenge with swine influenza viruses [191] . moreover, sciiavs expressing foreign genes represent an excellent option for their implementation as bivalent vaccines. for instance, sciiavs expressing the surface protein a (pspa) of streptococcus pneumoniae, the hemagglutinin-neuraminidase (hn) protein of hpiv-3 or the fusion (f) protein of respiratory syncytial virus (rsv) [192] [193] [194] , were able to induced abs against the foreign polypeptides and reduced the viral load of the heterologous pathogen while retaining their ability to protect against challenge with iav. it is worth indicating that, for vaccine purposes, it is important to consider what influenza viral gene is replaced, since it was shown that sciiav replication is required for protection [69, 185, 189] . although substitution of the polymerase (pb2, pb1 and pa) segments may allow insertion of larger foreign genes, removing the viral polymerase from sciiavs limits their ability to express more polymerase during the single-cycle round of infection, decreasing total viral antigens and, thus, limiting their protective efficacy. thus, sciiavs where the viral ha or na has been removed were able to confer, upon a single immunization, protection against a lethal challenge with influenza. [190] . on the contrary, a single dose of a sciiav where the viral pb2 has been removed was only as efficacious as an inactivated virus [189] . it is worth noting that sciiavs combine the advantages (better immunogenic properties of the laiv and the safety profile of the iiv) and circumvent the disadvantages (safety of the laiv and poor immunogenicity of the iiv) of current influenza vaccine approaches [144] . while sciiavs have been shown to be safe, immunogenic and protective against lethal challenges with wild-type forms of iavs in animal studies, no human trials have yet been performed. more recently, the rearrangement of the influenza virus genome has been shown to have great potential for the development of improved laivs against influenza virus, as well as vaccine vectors against other pathogens [195, 196] . avian influenza virus subtypes h5n1 and h9n2 have pandemic potential [197, 198] . however, h5n1 iiv induces limited adaptive immune responses, and in the case of laiv, there are safety concerns about the possibility of reassortment between the viral segments in the laiv and circulating h5n1 strains. to overcome these concerns, a bivalent laiv against influenza a/vietnam/120320/04 h5n1 and a/guinea fowl/hong kong/wf10/1999 h9n2 was generated using viral genome rearrangement [195] . to that end, the nep was removed from the ns viral segment of the h9n2 virus and substituted by the ha of the h5n1 virus. h9n2 ns1 and h5n1 ha were separated by the foot-and-mouth disease virus (fmdv) 2a autocleavage site to allow co-linear expression of both viral proteins. then, nep was cloned down-strain of the h9n2 pb1 segment separated by another fmdv 2a autocleavage site. the rearranged h9n2 virus expressing the h5n1 ha was able to provide complete protection against challenge with a/vietnam/1203/2004 h5n1 and also against a potential pandemic h9:ph1n1 iav reassortant virus in both mice and ferrets [195] . segments 7 (m) and 8 (ns) of iavs use an alternative splicing mechanism to express two different viral proteins from the same viral segment. recently, we have generated recombinant influenza a/puerto rico/8/34 h1n1 viruses containing modified m and/or ns segments, in which the overlapping orfs of the m1 and m2 viral proteins (m segment) and/or the ns1 and nep proteins (ns segment) were separated with the porcine teschovirus 1 (ptv-1) 2a autocleavage site [196] . recombinant viruses with a rearranged m segment were affected or impaired in replication in vitro at nonpermissive temperatures (37 and 39 • c, respectively), whereas high viral titers were obtained at permissive low temperatures (33 • c) [196] . notably, viruses containing the m split segment were highly attenuated in vivo, but able to confer, upon a single immunization dose, complete protection against a lethal homologous challenge with wild-type pr8 [196] . importantly, viruses with a reorganized m segment were able to confer better protection than a temperature-sensitive, laiv pr8 virus [103, 104, 115, 196] . these studies demonstrate that the rearrangement of the influenza viral genome can be used for the generation of safe, immunogenic and protective laivs. current influenza vaccines induce immunity to the influenza virus strain-specific ha antigen and are not very effective against new pandemic viruses, given that ha is highly susceptible to frequent changes by antigenic drift and shift [12, [44] [45] [46] [74] [75] [76] . to overcome these drawbacks, different approaches aimed to develop a universal influenza vaccine able to induce cross-protective broadly neutralizing immunity against conserved viral antigens, such as the ectodomain of m2 (m2e) [199] , the ha stalk domain [200] or na [201] , have been explored. the m2e antigen is a linear peptide that is very well conserved across iav strains. although the mechanism of m2e-specific immunity is unclear, protective anti-m2 antibodies have been observed in multiple animal models, including mice, ferrets and primates [36] . however, these conserved antigenic targets need to be presented in a carrier system or conjugated to adjuvant molecules. promising results have been obtained with virus-like particles (vlps), which are morphologically similar to the virus and present surface proteins in a highly immunogenic form. because vlps do not contain the viral genome, they are considered safer than viral vaccines, yet still induce strong humoral and cellular immune responses [202] . vlps are most commonly made by expression of ha, na and m1 [203, 204] , although ha and na alone may be sufficient for vlp production [205] . reverse genetics approaches to generate recombinant viruses, including influenza, have been described for representative family members of negative-sense, single-stranded rna viruses. these plasmid-based reverse genetics methods have provided scientists with a unique opportunity to study different aspects of the biology and pathogenesis of these viruses, both in vitro and in vivo, as well as to generate attenuated forms to be used as vaccines [135] . in this review, we discussed the use of reverse genetics for the generation of influenza vaccines with a special focus on laivs. in general, laivs are highly immunogenic, and immunization usually induces faster and substantially higher levels of both systemic and local mucosal antibody and t-cell responses, providing better protection than their inactivated counterparts. prevention of influenza virus infection requires seasonal vaccinations, and identification of the correct virus subtype to include in the vaccine leaves little time for vaccine development, scale up and distribution. although influenza vaccines work well most of the years, if a new viral variant emerges after the strain to be included in the vaccine has been selected, the efficacy of the vaccine would be sub-optimal for this new variant strain. this usually results in decreased efficacy of the vaccine. reverse genetics approaches have also 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authors: lauterbach, sarah e.; nelson, sarah w.; robinson, meghann e.; lorbach, josh n.; nolting, jacqueline m.; bowman, andrew s. title: assessing exhibition swine as potential disseminators of infectious disease through the detection of five respiratory pathogens at agricultural exhibitions date: 2019-09-18 journal: vet res doi: 10.1186/s13567-019-0684-5 sha: doc_id: 3888 cord_uid: lgutt1r9 widespread geographic movement and extensive comingling of exhibition swine facilitates the spread and transmission of infectious pathogens. nasal samples were collected from 2862 pigs at 102 exhibitions and tested for five pathogens. at least one pathogen was molecularly detected in pigs at 63 (61.8%) exhibitions. influenza a virus was most prevalent and was detected in 498 (17.4%) samples. influenza d virus was detected in two (0.07%) samples. more than one pathogen was detected in 165 (5.8%) samples. influenza a virus remains a top threat to animal and human health, but other pathogens may be disseminated through the exhibition swine population. agricultural swine exhibitions showcase a variety of swine related educational programs, livestock auctions, and competitive shows for youth throughout the united states. swine exhibitions are unique agricultural settings where large numbers of pigs from various geographic locales that would otherwise not come in contact with one another are co-housed and comingled for the length of the exhibition. long exhibition durations (3-7 days), direct contact of swine, and relaxed biosecurity strategies have all been recognized as risk factors for the rapid transmission of infectious pathogens infecting swine during exhibitions [1, 2] . influenza a virus (iav) has been routinely detected in swine at agricultural exhibitions, where subclinical infection is common and can make recognition difficult [3] [4] [5] [6] . swine are important mixing vessels of iav, where genetic reassortment between multiple iav strains may produce novel, potentially more virulent strains [7] . the comingling of large numbers of iav infected swine at exhibitions may increase the chance for the emergence of these reassortant viruses. in addition to the extensive interaction of swine, exhibitions also create a unique human-animal interface, where exhibiting families and the general public are permitted to interact with swine. this unique swine-human interface facilitates zoonotic transmission of iavs during swine exhibitions [8] . continued detection of variant iavs, iavs infecting humans that normally circulate in swine, in individuals who have association with swine exhibitions has begun to heighten disease awareness and surveillance in exhibition swine [9, 10] . combined with the ability of iavs to reassort in swine, the increased swine-human interface of exhibitions creates opportunities for novel iav strains to enter the human population, potentially leading to an influenza pandemic. continued disease surveillance is vital to understanding the epidemiology of iav and other pathogens infecting swine at agricultural exhibitions in order to protect animal and public health. commonly raised on small farms with varying management practices, the exhibition swine population is unlike commercial swine, which are managed in large herds under strict biosecurity protocols [11, 12] . coupled with extensive movement over a large geographic area in order to attend multiple exhibitions, exhibition swine facilitate the widespread dissemination of infectious pathogens throughout the country [11, 13] . therefore, exhibition swine represent a unique niche in the total swine population that may play an important role in the ecology and epidemiology of all swine infectious diseases. with continued detection of iav and increasing awareness of disease in exhibition swine, the current study aims to identify pathogens in addition to iav that may be circulating in this swine population. other swine respiratory viruses such as porcine hemagglutinating encephalomyelitis virus (phev), porcine reproductive and respiratory syndrome virus (prrsv), porcine parainfluenza virus 1 (ppiv1), and influenza d virus (idv) have only briefly been described in exhibition swine and their prevalence and overall impact in exhibition swine remains unknown [12, [14] [15] [16] . with varying degrees of severity and impact on swine and human health, understanding the overall disease ecology and epidemiology of the exhibition swine population is vital to the development and implementation of disease mitigation strategies designed to protect animal and public health. here, we describe the overall estimated prevalence of five infectious respiratory viruses in swine at agricultural exhibitions and assess the potential epidemiological role of exhibition swine. in 2016, as part of an ongoing iav surveillance program, 2862 pigs at 102 exhibitions across six states were sampled by nasal swab or nasal wipe [17, 18] . all samples underwent nucleic acid extraction and were tested individually for iav using real-time reverse transcription polymerase chain reaction (rrt-pcr) as previously described [19] . five microliters of extracted nucleic acid of no more than seven individual pigs from the same exhibition were pooled and screened for ppiv1, phev, and idv using rrt-pcr as previously described [14, 16, 20] and for prrsv using the vetmax prrsv reagents and manufacturer's protocol (thermofisher scientific, waltham, ma, usa). nucleic acid of samples within positive pools was subsequently tested individually using the same methods. during field sample collection, exhibitions with ≥ 1 pig showing clinical signs consistent with influenza like illness (ili) were recorded as having pigs with ili [1] . five of the exhibitions were excluded from statistical analysis due to lack of clinical sign data. logistic regression (stata special edition 14.2, college station, tx, usa) was performed to determine any association between ili noted at the exhibition and the detection of a pathogen in the pigs. at least one pathogen was detected in the pigs at 63 (61.8%) of the 102 swine exhibitions tested. influenza a virus was detected among the pigs at 37 (36.3%) and was detected at more exhibitions than any of the other viruses. in contrast, influenza d virus was detected in the pigs at the fewest exhibitions; only pigs at two (2.0%) of the exhibitions tested positive for idv (table 1 ). more than one pathogen was detected among the pigs at 31 (30.4%) exhibitions; four pathogens were detected at three (2.9%), representing the highest number of pathogens detected among the pigs at any of the exhibitions. exhibitions with pigs testing positive for at least one pathogen had 2.4 times the odds of having noted ili compared to exhibitions where no pathogens were detected (p < 0.05). detection of iav in swine at exhibitions was the only pathogen with a significant positive association to noted ili at exhibitions (or = 1.3, p < 0.05). however, with fewer positives, this association may be hidden for the other pathogens. out of the 2862 pigs sampled, 785 (27.4%) tested positive for at least one pathogen. the most commonly detected pathogen was iav which was detected in 498 (17.4%) pigs. influenza a virus was detected in more pigs than the next two most prevalent pathogens combined, phev and ppiv1, which were detected in 251 (8.8%) and 201 (7.0%) pigs, respectively. influenza d virus was the least prevalent and was only detected in two (0.07%) pigs (table 2) . co-infections were common with 165 (5.8%) pigs testing positive for more than one pathogen, including four (0.14%) pigs testing positive for three pathogens, which were the most pathogens found in co-infected, individual pigs. due to widespread movement and prolonged intermingling, exhibition swine and the variety of pathogens they carry should be considered significant for both swine and human health. there has been increased attention on iav in swine at exhibitions from veterinarians, health officials, and exhibition attendees due to recent iav zoonotic transmission events [9, 21, 22] . previous active surveillance has described the epidemiology of iav in swine at exhibitions in the u.s.; at exhibitions where ≥ 1 pig tests positive for iav, over 60% of the pigs will have active iav infection by the conclusion of the exhibition [3] . there were 18 cases of variant influenza infections associated with the iavs detected in this population of exhibition swine, and iav should remain a priority for infectious disease surveillance and mitigation in exhibition swine [22] . several mitigation strategies have been suggested and implemented at exhibitions as the concern for animal and public health rises. many of these strategies are aimed at reducing the risk of iav transmission between pigs and humans before, during, and after agricultural swine exhibitions [1, 2] . still, iav continues to impact exhibition swine and was detected at almost double the prevalence of phev, the next most prevalent virus of those included in this study. despite the persistent spread of iav through exhibition swine and its implications for both animal and human health, other infectious pathogens can be detected in exhibition swine and should not be ignored. identification of active infections in exhibition swine is vital to protecting animal and human health during agricultural exhibitions. pigs at exhibitions with at least one pathogen detected in the pigs were more likely to exhibit ili. however, subclinical infections are common and there is overlap in clinical signs associated with various respiratory pathogens in swine; because of this, clinical signs should not be solely relied upon for the detection and subsequent diagnosis of infectious pathogens in swine at exhibitions [3, 6] . proper biosecurity should be used at all times, even in the absence of clinical signs, in order to reduce the risk of intra-and interspecies transmission of all pathogens during exhibitions. focus on animal and public health at swine exhibitions is a priority, but the potential for introduction of infectious pathogens from exhibition swine into commercial swine facilities should also be considered. an estimated 39% of exhibition pigs are raised near commercial swine, with many of those returning home after attending an exhibition [12] . detection of pathogens such as phev and prrsv in exhibition swine raises concern for commercial swine due to the potential economic implications should they enter the production system. prrsv alone causes an estimated $600 million in economic loses annually in the united states swine production system [23] . detection of ppiv1 and idv also raises concern, though they are more recently described in swine and their overall consequences to swine health are still unknown [15, 16] . in addition to the five pathogens assessed in the present study, there is potential for other pathogens to travel with and spread through exhibition swine that may also present a threat to commercial swine, including foreign animal-diseases. exhibition swine should not be disregarded as potential hosts and disseminators of diseases such as african swine fever virus (asfv); movement and comingling of pigs have been recognized as risk factors for the spread of asfv in endemic regions [24] . if overlooked, exhibition pigs may create a niche for devastating pathogens, with extensive animal movement resulting in rapid spread across the country. the findings of this study highlight the potential role of exhibition swine in the disease ecology and epidemiology of the united states swine population. lack of sustained disease surveillance and application of mitigation strategies for swine at agricultural exhibitions will allow for continued variant iav infections that pose considerable risk to public health and could ultimately lead to a devastating disease epizootic within the u.s. swine herd. exploration of risk factors 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attendees of an agricultural fair movement patterns of exhibition swine and associations of influenza a virus infection with swine management practices potential role of noncommercial swine populations in the epidemiology and control of porcine reproductive and respiratory syndrome virus introduction, evolution, and dissemination of influenza a viruses in exhibition swine in the united states during 2009 to 2013 porcine hemagglutinating encephalomyelitis virus and respiratory disease in exhibition swine complete genome sequence of an influenza d virus strain identified in a pig with subclinical infection in the united states widespread detection and characterization of porcine parainfluenza virus 1 in pigs in the usa nasal wipes for influenza a virus detection and isolation from swine utility of snout wipe samples for influenza a virus surveillance in exhibition swine populations prevalence of influenza a virus in exhibition swine during arrival at agricultural fairs isolation of a novel swine influenza virus from oklahoma in 2011 which is distantly related to human influenza c viruses two cases of flu linked to contact with swine at fowlerville fair. the detroit news influenza a(h3n2) virus in swine at lauterbach et al. vet res (2019) 50:63 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year • at bmc, research is always in progress. learn more biomedcentral.com/submissions ready to submit your research ? choose bmc and benefit from: agricultural fairs and transmission to humans assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers small-scale pig farmers' behavior, silent release of african swine fever virus and consequences for disease spread publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank field staff michele zentkovich, alison martin, courtney wright, lauren smith, and alex tweedy for collection of the samples as well as research assistants rachel patton, libby mcgarry and dillon mcbride for help in the laboratory. we also thank the exhibition officials and participants for partaking in the study. funding was provided by the centers of excellence for influenza research and surveillance, national institute of allergy and infectious diseases, national institutes of health, department of health and human services, under contract nos. hhsn266200700007c and hhsn272201400006c. iav: influenza a virus; phev: porcine hemagglutinating encephalomyelitis virus; ppiv1: porcine parainfluenza virus 1; prrsv: porcine reproductive and respiratory syndrome virus; idv: influenza d virus; rrt-pcr: real-time reverse transcription polymerase chain reaction; ili: influenza like illness.authors' contributions sel: data analysis, data interpretation, manuscript preparation. swn: laboratory sample testing, data analysis, manuscript preparation. mer: study design, laboratory sample testing, data analysis, manuscript revision. jnl: study design, data interpretation, manuscript revision. jmn: study design, data interpretation, manuscript revision. asb: study conception and design, data interpretation, manuscript revision. all authors read and approved the final manuscript. funding was provided by the centers of excellence for influenza research and surveillance, national institute of allergy and infectious diseases, national institutes of health, department of health and human services, under contract nos. hhsn266200700007c and hhsn272201400006c. all data generated or analyzed during this study are included in this published article. ethics approval for animal use in this research was provided by the institutional animal care and use committee at the ohio state university under approval number 2009a0134-r2. key: cord-003169-bdw5ke4i authors: guo, hongbo; rabouw, huib; slomp, anne; dai, meiling; van der vegt, floor; van lent, jan w. m.; mcbride, ryan; paulson, james c.; de groot, raoul j.; van kuppeveld, frank j. m.; de vries, erik; de haan, cornelis a. m. title: kinetic analysis of the influenza a virus ha/na balance reveals contribution of na to virus-receptor binding and na-dependent rolling on receptor-containing surfaces date: 2018-08-13 journal: plos pathog doi: 10.1371/journal.ppat.1007233 sha: doc_id: 3169 cord_uid: bdw5ke4i interactions of influenza a virus (iav) with sialic acid (sia) receptors determine viral fitness and host tropism. binding to mucus decoy receptors and receptors on epithelial host cells is determined by a receptor-binding hemagglutinin (ha), a receptor-destroying neuraminidase (na) and a complex in vivo receptor-repertoire. the crucial but poorly understood dynamics of these multivalent virus-receptor interactions cannot be properly analyzed using equilibrium binding models and endpoint binding assays. in this study, the use of biolayer interferometric analysis revealed the virtually irreversible nature of iav binding to surfaces coated with synthetic sialosides or engineered sialoglycoproteins in the absence of na activity. in addition to ha, na was shown to be able to contribute to the initial binding rate while catalytically active. virus-receptor binding in turn contributed to receptor cleavage by na. multiple low-affinity ha-sia interactions resulted in overall extremely high avidity but also permitted a dynamic binding mode, in which na activity was driving rolling of virus particles over the receptor-surface. virus dissociation only took place after receptor density of the complete receptor-surface was sufficiently decreased due to na activity of rolling iav particles. the results indicate that in vivo iav particles, after landing on the mucus layer, reside continuously in a receptor-bound state while rolling through the mucus layer and over epithelial cell surfaces driven by the ha-na-receptor balance. quantitative bli analysis enabled functional examination of this balance which governs this dynamic and motile interaction that is expected to be crucial for penetration of the mucus layer and subsequent infection of cells by iav but likely also by other enveloped viruses carrying a receptor-destroying enzyme in addition to a receptor-binding protein. introduction specificity, avidity and dynamics of influenza a virus (iav)-receptor interactions are determining factors in host tropism and pathogenesis. virus attachment to sialic acid (sia) receptors on host cell surfaces and decoy mucins is mediated by hemagglutinin (ha) [1] [2] [3] , while neuraminidase (na) removes receptors by cleaving sias [4] [5] [6] . a precisely tuned functional ha/na balance [7] [8] [9] [10] [11] [12] [13] [14] [15] is required for efficient infection of, and replication in, a specific host. ha and na properties affect host-and cell-specificity but have been studied in much more detail for ha because of the relative lack of accurate na assays [16] . iavs that infect humans bind preferentially to α2,6 sialosides, having an α2,6-linkage between sia moieties and the penultimate residue, whereas avian iavs prefer binding to α2,3-linked sias [17] [18] [19] . cell surface glycan composition, as well as branching, modification and linkage-type of the internal carbohydrate chain residues, is variable between host and cell type and also strongly affects binding affinity (reviewed in [20] ). nas of avian iavs are highly active and prefer cleavage of α2,3-linked sialoglycans, while human virus nas cleave α2,3-and α2,6-linked sias with lower activity [21] [22] [23] . the ha/na balance is important for initiating iav infection. abundantly sialylated mucins in the mucus layer covering the airway epithelial cells bind ha and may trap iavs before they reach the epithelial cells [24, 25] . this needs to be counteracted by na activity sufficiently matching ha binding specificity. this is also required to prevent the virus-trapping at sites on the cell surface that do not support efficient endocytosis [26, 27] . na activity-dependent de-sialylation of the surface of the infected cell and the virus envelope glycoproteins facilitates budding and release of new virus particles and prevents virus aggregation [28] . as a consequence, viral fitness depends on continuous fine-tuning of the ha/na balance during virus evolution. replication of viruses, harboring mismatched pairs of ha and na or propagated in the presence of inhibitory decoy receptors or na inhibitors, could be rescued by adaptive mutations in the ha, na or both proteins [7, 11, 13, 29] . cross-species transmission events, leading to human pandemics in the past, required adaptions in both ha and na [10, 30] . when infecting a novel host species, a non-optimal ha/na balance is for instance adapted by evolving increased na activity as well as decreased binding by ha to the decoy glycan-receptor repertoire on mucins present in the mucus, and which differs between different hosts. [29, [31] [32] [33] . there is no straightforward method to determine the ha/na balance of iavs. hemagglutination of erythrocytes, which harbor poorly defined receptor repertoires, does not well reflect binding avidity and dynamics. current setups of solid phase binding assays (e.g. elisas, glycan arrays) as endpoint binding assays do not handle well the polyvalent aspects of virus binding by masking the dynamics of virus binding, even more so as precise variation of receptor density is difficult. moreover, besides a wide range of synthetic glycans, only very few glycoproteins (mostly fetuin) are being employed in current binding studies, thus limiting studies on the effects of diversity and clustering of glycans as naturally presented on glycoproteins. na activity assays often use soluble substrates poorly reflecting natural sialosides [5, 34] . the soluble substrate 2 0 -(4-methylumbelliferyl)-α-d-n-acetylneuraminic acid (munana) can be used to determine the na activity on a soluble substrate but ignores the effects of the virion context on na activity on a receptor-coated surface (e.g. binding of the virion to the polyvalent substrate via its ha). thus, methods that determine the dynamic effects of ha and na activity urgently need improvement to allow integration into a model that accurately provides a quantitative description of the dynamic interaction between iav particles and (decoy) receptors. biolayer interferometry (bli) is increasingly being used for analyzing virus-receptor interactions [35] [36] [37] [38] , but standard methods for quantifying kinetic parameters describing iavreceptor interactions are lacking. here we have used this label-free real-time binding analysis method to study the dynamics of iav-receptor interactions. our results indicate that the initial virus binding rate is the prime, and physiologically most important kinetic parameter of ha-dependent iav particle binding to synthetic as well as natural glycoprotein receptors. na was shown to contribute to virus binding and to be absolutely required for virus dissociation. in turn, na activity is critically dependent on the ability of virions to bind to a receptor-coated surface. the ha/na balance of different virus-receptor combinations was determined by measuring empirical virus binding and elution parameters. prior to virus elution, na-dependent morphological changes in receptor-associated virus particles were observed as well as rolling of virus particles over a receptor-coated surface in a na activity-dependent manner. bli is a potentially versatile tool to obtain mechanistic and quantitative insight into the dynamics of virus-receptor interaction by real-time recording of virion binding to, and release from, receptor-coated surfaces. these interactions are highly polyvalent by nature and expected to be poorly described by equilibrium binding models. in addition, receptor density and identity are key determinants of the interaction. we therefore established a flexible experimental set-up, using synthetic sialoside receptors as well as n-linked or o-linked sialoglycoproteins which carry sialoglycan receptors attached in the natural linkage-conformation that is encountered in vivo. we first evaluated the applicability of the set-up to a quantitative description of iav-receptor binding kinetics. to assist interpretation of the results, the approximate geometric properties of a streptavidin (sa) bli sensor surface, to which biotinylated glycans or glycoproteins can be tightly attached (k d = 10 −14 ), and of iav virions are depicted in s1 fig (fig 1) . virus association was performed in the presence of the na inhibitor oseltamivir carboxylate (oc) and the lack of virus dissociation after transfer of sensors to buffer containing oc ( fig 1a and fig 1b) showed that virus binding is virtually irreversible (off-rate constant k off % 0) due to highly polyvalent interactions between virus particles and the receptor-coated bli sensor surface. pr8 mtsin is a faster binder than pr8 cam2,3 (fig 1a and fig 1b) , but reached a maximum binding signal of~9 nm at the highest concentration only after 4 hours (s4a fig and s4b fig) . a fully occupied sensor surface theoretically accommodates 3.3e+07 spherical virus particles (100 nm diameter; s3 fig and [39] [40] [41] [42] ). this fitted well with the experimentally determined number of virus particles bound to a maximally loaded sensor as determined by quantification of na activity (s4c fig). importantly, sensor-regeneration followed by re-binding of virus from the same well could be consecutively repeated at least 6 times yielding identical curves (s5 fig) . thus, substantial virus decay during the assay or binding of a limited subset of virus particles with particular properties did not occur. analysis of iav-receptor interactions has predominantly been focused on quantification of an apparent dissociation constant (k d = k off /k on ) by measuring polyvalent binding of virus particles or artificial ha polymers (e.g. by antibody complexation) after a fixed binding time to geometrically poorly characterized receptor-coated surfaces with often unknown receptor density and orientation. however, as virus binding is virtually irreversible, equilibrium binding models do not apply and the binding rate equation ( ) and virus concentration was observed for the fast (pr8 mtsin ; r = 0.997, p = 0.0002) and the slow binding (pr8 cam2,3 ; r = 0.9915, p = 0.0009) virus ( fig 1c) . the effect of receptor density on iav association and dissociation rates can be precisely determined as bli provides quantitative recording of receptor loading levels. lowering the receptor density, resulting in less potential ha-receptor contacts between virus and sensor surface, gradually reduced maximum binding levels ( fig 1d and s4d and s4e fig) as well as the v obs ( fig 1e) . remarkably, irreversible binding was observed at any receptor density that supported binding (fig 1d) . a~2-fold higher fractional receptor loading was required for the weaker binder pr8 cam2,3 (0.593±0.01752) than for pr8 mtsin (0.332±0.00364) to reach 50% of the maximum fractional initial binding rate (p<0.001) ( fig 1e) . pr8 mtsin reached a maximum binding rate at~55% receptor loading whereas the weaker binder pr8 cam2-3 reached its maximum binding rate close to maximum receptor density. a~3-fold reduction in receptor loading was already sufficient to reduce the v obs from maximal to zero for both viruses. this suggests a narrow margin between the number of interacting ha-receptor pairs, below which binding is undetectable due to high reversibility (high k d ) and above which binding is irreversible (see fig 1d, no dissociation at any receptor density). we conclude that v obs , which is directly related to virus concentration and receptor density, is the most suitable parameter for quantification of virus binding strength over a range of receptor densities and is likely to reflect in vivo virus binding, which probably occurs to levels very far from saturation. as the initial binding rate is linear with virus concentration, the relative specificity for two receptors (v rel = v obs1 / v obs2 ) is independent of virus concentration and the relative-binding specificity of viruses can therefore be quantitatively compared without the need for elaborate virus quantitation procedures. as an example, we quantified the effect on receptor-specificity of amino acid substitution e190d in ha of pr8 which is predicted to cause a shift in binding from α2,3-to α2,6-linked sia receptors in h1 [43] . indeed glycan array analysis of the ha proteins of lab strains pr8 cam2,3 and pr8 cam2,6 , which only differ by substitution d190e, showed an absolute specificity shift (fig 2a) . in contrast, virus binding quantified by bli showed a relative specificity shift (v rel = v 3'slnln /v 6'slnln ) of 19.7±0.11 for pr8 cam2,3 to 1.2 ±0.18 for pr8 cam2,6 ( fig 2b and fig 2c) . remarkably, strain wsn ha, which carries 190e plus some additional mutations in comparison to pr8 cam2,3 , displayed efficient binding to α2,3-as well as α2,6-linked synthetic glycans on the microarray, but wsn wt virus did not bind to synthetic glycans in the bli assay. possibly, soluble ha proteins can have access to short glycans on glycan arrays whereas virus envelope-embedded has cannot, depending on virus strain, bind to the same glycans on bli sensors. to test this, we examined binding to glycoproteins carrying α2,3-or α2,6-linked sias on n-linked sialoglycans (3'n fetuin or 6'n transferrin bt; fig 2d and fig 2e) . pr8 cam2,3 virus displayed absolute specificity for 3'n fetuin. however, an absolute α2,3-to α2,6-linked sia specificity shift by mutation e190d, as observed above by glycan array analysis, did not occur 3 , pr8 cam2,6 or wsn wt was determined by glycan array analysis. binding to mono-, bi-and tri-antennary glycans carrying one, two or three lacnac repeats terminated with a α2,3-or α2,6-linked sia is indicated. means of 6 independent replicates are graphed, standard errors of the means are indicated. biotinylated 3'slnln or 6'slnln for virus binding to glycoproteins (v rel = v 3'n fetuin /v 6'n transferrin = 1.4±0.21 for pr8 cam2,6 ), similar to what was observed for the synthetic glycans ( fig 2b and fig 2c) . glycoproteins, in contrast to synthetic glycans, supported wsn wt virus binding to bli sensors which displayed dual binding specificity (v rel = 4.9±0.15) in agreement with the glycan array results for wsn ha proteins. in conclusion, determination of v obs allows quantification of relative receptor specificity differences for virus particles, which is independent of virus concentration (s4g fig, s4h fig and s4i fig) . the application of (tailor-made) glycoproteins enables to elucidate quantitative differences in iav receptor-binding fine specificity, which is a step forward to mimicking virus-receptor interactions that occur in vivo. the versatility of the latter is further illustrated by showing the relative specificity for n-linked versus o-linked glycans, using engineered fetuin constructs (s2 fig). o-linked glycans are abundantly present on soluble mucins present in respiratory mucus. as an initial experiment to explore potential differences in binding dynamics to o-or n-linked glycans we expressed fetuin variants containing three n-linked and/or three olinked glycans and compared the binding of pr8 cam2,3 and pr8 mtsin to these receptors (s6a na activity contributes to the dynamic interaction of virions with receptor-coated surfaces. we first determined whether non-bound virions are able to cleave receptors loaded on bli sensors. we used viruses carrying the same na but a different ha (pr8 mtsin (fig 3b) . in the presence of na activity, the binding rate of tx77 namtsin became increasingly lower in time, presumably reflecting virion release due to ongoing receptor depletion. regeneration of sensors, which removes all virus particles but leaves the biotinylated glycans on the sensors, was followed by a second round of tx77 namtsin binding in order to determine the extent of de-sialylation that occurred in the first round of binding ( fig 3c) . only tx77 namtsin binding in the first round in absence of oc led to receptor desialylation as detected by inefficient binding of tx77 namtsin in the second round. in contrast, α2,3 specific pr8 mtsin did not bind to the sensor and thereby could not remove sias, as shown by efficient re-binding of tx77 namtsin upon regeneration. we conclude that non-bound virions do not contribute to sialidase activity and do not have to be taken into account when studying the effect of virion-associated na activity on iav receptor binding dynamics by bli. we determined the quantitative effect of na activity on the dynamics of iav-receptor interactions of two viruses carrying the same ha, but different na proteins (wsn hamtsin and pr8 mtsin ; s2e fig) . whereas the viruses displayed similar binding to both 3'n fetuin and 3'sln when na was inhibited (fig 4a and 4d ), only pr8 mtsin binding was strongly reduced by its associated na activity (fig 4b and 4e ). this observation corresponded well to thẽ 30-fold lower na activity of wsn hamtsin per virus particle (s7i fig and s7j fig) . we conclude that, in addition to ha binding properties, na activity drastically affects virus binding dynamics in the absence of na inhibitors. the binding curves reflect the ha/na balance which is quantifiable by empirical parameters like initial binding rate, the area under the curve (unit is min nm) and x,y coordinates of the peak (time and binding level). na activity-dependent self-elution of viruses bound in presence of oc provides quantification of other descriptive parameters that characterize the balance between ha, na and receptor. of note, the concentration of released particles is too low for their re-association to take place. as expected, pr8 mtsin eluted much faster from 3'n fetuin or 3'sln than wsn hamt-sin (which does not elute from 3'sln). for both viruses the self-elution rate from 3'n fetuin was higher than from 3'sln ( fig 4c and fig 4f) . this likely results from differences in specific na-dependent influenza a virus rolling on receptor surfaces activity of the nas for these receptors but, as ha and na are in competition for binding/cleavage of the same receptor, might also be affected by the k d of monovalent ha-receptor interactions and thus be a reflection of ha/na balance on a specific receptor. this possibility is further corroborated by an experiment where we compared the elution rates from 3'n fetuin and 6'n transferrin bt for two viruses (pr8 cam2,6 and wsn wt ) carrying the same na (from wsn wt ) but a different ha (s8 fig). both viruses bind at similar, but relatively low, rate to 6'n transferrin bt ( fig 2e) whereas wsn wt displays a~3-fold faster binding rate to 3'n fetuin than pr8 cam2,6 ( fig 2d) . ha clearly affects the self-elution rate as, in combination with the same na, self-elution from 3'n fetuin is much more efficient in companion of the weaker binding ha of pr8 cam2,6 (s8b fig thus, whereas α2,3 sia versus α2,6 sia specificity of na is seemingly opposite for pr8 cam2, 6 and wsn wt , this is not caused by the na itself (which is identical for both viruses) but by differences in their has. also a change of receptor (in this case to fucosylated 3'sln, giving sialyl-lewis x ; sle x ) was shown to have differential effect on the observed binding rate ( fig 4g) and na-driven self-elution ( fig 4h and 4i ). while v obs is similar for sle x and 3'sln in the absence of na activity ( fig 4g) , an active na resulted in a lower v obs and maximum binding level and a smaller area under the curve for 3'sln ( fig 4h) and in a faster virion self-elution from 3'sln ( fig 4i) . thus, receptor binding dynamics on 3'sln and sle x differ due to a lower specific na activity towards sle x which shifts the relative ha/na balance on these receptors. in conclusion, bli can be used to quantify changes in the dynamics of receptor binding due to an altered ha/ na/receptor balance. in a small increase in reflection resulting in negative virus dissociation values, which cannot be explained by additional virus particle binding, was consistently observed during the initial phase of self-elution (e.g. fig 4c, 4f and 4i). the effect was most prominent when na activity was low. the possible role of na activity herein was further examined by performing self-elution of pr8 mtsin from 3'sln and wsn hamtsin from 3'n fetuin in the presence of an oseltamivir concentration range (fig 5a and 5b ). duration and magnitude (maximally~0.12nm) of the increase in the apparent binding level, which was not observed when na was fully inhibited, depended on the degree of na activity. the effect must reflect a neuraminidase activitydependent change in the receptor-bound virus particles as free virus particles that can bind are absent. cleavage of sias by na is expected to gradually decrease the number of ha-sia contacts between receptor-coated surface and virus until the particle dissociates. relaxation of virus particle binding (by less contacts) could result in an altered binding conformation of a particle that, at the maximal number of contacts, might be tightly squeezed against the receptor surface. such a morphological change likely explains the observed, na-activity dependent, changes in reflection. remarkably, the initial binding rate of wsn hamtsin to 3'n fetuin was higher in absence than in presence of a high concentration of oc (compare fig 4a and 4b ). several avian na genotypes have been shown to possess a 2 nd sia-binding site, alternatively referred to as hemadsorption site [44, 45] , but such a site is not conserved in wsn na and oc binding to this site has never been demonstrated. however, active site mutations that abolish catalytic na activity have resulted in na-dependent hemagglutination, which could be inhibited by oc [46, 47] . we therefore examined the effect of oc on binding of wsn hamtsin (low na activity) and pr8 mtsin (high na activity) virions to 3'sln and 3'n fetuin. complete na inhibition gave binding curves displaying a continuous increase of virus binding (fig 5c-5f , blue lines). however, the v obs of wsn hamtsin increased at lower oc concentrations in a concentrationdependent way (fig 5d and fig 5f) . in time, the curves bent down by depletion of sia receptors due to na activity. in contrast, the v obs of pr8 mtsin , which has a highly active na in comparison to wsn hamtsin , directly decreased strongly at lower concentrations of oc ( fig 5c and fig 5e) . the results imply that nas with a relatively low cleavage rate (low k cat ) contribute to the v obs by binding with their active site to the sia receptor. to strengthen this conclusion, we quantified the enhancement of the initial binding rate by wsn wt viruses carrying the same na but four different has ( fig 5g and s9 fig) . whereas all four viruses displayed an effect of na on v obs , the virus with the lowest ha-dependent initial binding rate (pr8 cam2,6 ), was relatively most enhanced in initial binding rate by na-dependent binding (s9e fig and s9f fig) . this demonstrates that the degree of the contribution of na to v obs is influenced by its ha partner. in conclusion, the na protein contributes to the initial binding rate balance by binding with its active site to sia receptors and the magnitude of the effect is probably influenced by the ha/na balance. all four viruses showed faster dissociation when more virus was loaded, indicating that there is positive cooperativity between viruses in respect to self-elution rate. the only plausible mechanism by which viruses can assist each other in self-elution is by exerting na activity on a larger surface area than their own contact area, implicating that virus particles move over the receptor-coated surface. to test this hypothesis a concentration series (12.5 pm to 100 pm) of pr8 mtsin, harboring a highly active na, was bound to 3'n fetuin in presence of oc resulting in virus saturation levels on the sensor surface ranging from~8% to~60% (fig 6e) , based on a maximal binding level of 9 nm after prolonged binding (s4a fig). subsequent na-dependent self-elution ( fig 6f) confirmed that higher virus loading levels result in faster self-elution as shown by plotting fractional virus dissociation against time (fig 6g) . we next determined whether virus particles are ) for 30 minutes in presence of 10 μm oc after which the sensors were washed in pbs and dissociation was examined at a range of oc concentrations as indicated in the panels. (c-f) pr8 mtsin (c, e) and wsn hamtsin (d, f), carrying the same ha mtsin but either na mtsin (high na activity) or na wsn (low na activity), respectively, were bound at 100 pm concentration to biotinylated 3'sln (c, d) or fc-tagged 3'n fetuin (e, f) loaded to maximum level. binding was performed in absence (red lines) or in the presence of a range of oc concentrations as indicated in the panels. significant differences between initial binding curves shown in panels c-f were analyzed by ibm spss statistic 24. in panel c, there was no significant difference between curves with 10μm oc and 625nm oc (p>0.1), whereas the curves of 10μm oc and 625nm oc were significantly different from 39nm, 2.45nm and 0nm (p<0.001). in panel d, the curves of 10μm oc and 625nm were significantly different from those of 39nm, 2.45nm and 0nm. there also was a significant difference between 39nm and 2.45nm. in panel e, the 100nm curves significantly differed from the other three oc concentrations. in panel f, there were significant differences between the 100nm (and 25nm) and 6.25nm and 0nm curves. (g) the ratio (initial virus binding rate v obs in absence of oc)/(initial virus binding rate v obs in presence of 10 μm oc) of four viruses carrying the wsn wt na in the background four different has was plotted (the individual binding curves are shown in s9 fig) . standard deviations and significant differences between the mean initial binding rate ratios are indicated ( ã indicates p<0.05). indeed able to clear sias from a larger surface area than their own contact area during self-elution. after self-elution, sensors were regenerated and re-bound with a high concentration (100 pm) of virus ( fig 6h) . clearly, even at a level where only~8% of the surface was bound with virus, self-elution created a surface to which only very limited re-binding of virus particles could take place indicating extensive removal of sias from the complete surface. as the concentration of virus released from the surface is too low for re-association and non-bound virus does not contribute to receptor cleavage, we conclude that virus particles exert na activity while moving over the receptor-coated surface. migration of attached iav particles over a receptor surface necessarily depends on the very high k d of monovalent ha-sia interactions resulting in their rapid formation and dissociation. we hypothesize that na, in combination with the highly dynamic formation and release of individual ha-sia interactions, drives virus rolling by the generation of a receptor gradient due to its receptor destroying activity. rolling of virus particles is predicted to be faster for virus particles with higher na activity. this was tested by comparing the binding and dissociation dynamics of pr8 mtsin and wsn hamtsin , which have the same ha but have high and low na activity, respectively. the experimental set-up shown in fig 7a links sensors to bli graphs by color coding. first (fig 7b) , biotinylated 3'n+o fetuin -coated sensors were loaded with wsn hamtsin to a~15% saturation level in the presence of oc (blue and red) or incubated in buffer (green). in the next step (fig 7c) , these sensors were incubated with pr8 mtsin in the presence or in the absence of oc. in presence of oc (blue) efficient binding of pr8 mtsin to the large virus-free area takes place. in the absence of oc, high na activity prevents long na-dependent influenza a virus rolling on receptor surfaces lasting binding of pr8 mtsin to an empty sensor (green). the slightly negative slope of the wsn hamtsin loaded sensor (red) represents minor dissociation of wsn hamtsin in absence of oc, as expected on basis of its low na activity. this also implicates that wsn hamtsin elution is not appreciably assisted by pr8 mtsin . in the last step of the experiment (fig 7d) , the sensors were regenerated to remove all bound viruses followed by probing the residual sia content by its association capacity with wsn hamtsin in the presence of oc. as expected, the blue sensor, to which both viruses were bound in presence of oc, was as efficiently bound by wsn hamtsin as in the first round. also as expected, the green sensor to which no virus was bound in the first step and pr8 mtsin in absence of oc in the second step was efficiently cleared from sias, as demonstrated by its poor capacity to re-bind to wsn hamtsin . in contrast, binding of wsn hamtsin to~15% of sensor surface in the first step (red) prevented complete removal of sias by pr8 mtsin in the second step as demonstrated by considerable re-binding of wsn hamt-sin in the third step (red). this implies that wsn hamtsin , by marginal rolling over the surface because of its low na activity, protects its contact surface with the sensor against the na activity of pr8 mtsin , which cleaves sias of all the non-protected surface area. we conclude that na activity is the driver of virus rolling on a receptor-coated surface. the ménage a trois between iav ha, na and (decoy) receptors largely determines viral fitness and host cell tropism. here, by studying their highly dynamic but poorly understood interplay using bli, we obtained novel mechanistic and quantitative insights into iav-host interactions. we combined the key findings into a schematic model (fig 8) . the initial monovalent ha-sia interaction is virus concentration-dependent and governed by a binding rate constant k on (m −1 s −1 ) and a dissociation constant k off (s −1 ). its high k d (k on /k off % 0.3 to~3mm [48] [49] [50] cannot be easily determined and makes monovalent virus-receptor interactions undetectable by bli too. in combination with picomolar virus concentrations, this high k d inevitably results in the observed low virus binding rate. subsequent ha binding steps are intramolecular (determined by a different k on and k off with the unit s −1 ), resulting in multivalent binding. remarkably, and counterintuitive to its receptor destroying activity, na can contribute to the initial binding rate. receptor residence time in the na substrate binding site is determined by the binding constant (k d = k on /k off , range is 30μm~600μm [51] and catalytic rate constant (k cat ). obviously, a low k cat promotes the chance on secondary binding events (mostly of ha), which will also be affected by the na/ha virus incorporation ratio (s7k fig) . bli-detectable virus binding is, due to multivalency, virtually irreversible but highly dynamic as individual ha-and na-sia interactions are rapidly formed and broken in a virus concentration independent mode. the number of simultaneous interactions required for virtually irreversible (k off % 0) binding is low and logically depends on receptor density and the k off and k on values of a virus (fig 1e) . the rapid interconversion of binding states via the association/dissociation events shown in fig 8 enables a virus to roll over the surface. na activity (curled arrows) is required to drive rolling, most likely by creating a receptor gradient that forces a virus to roll away from the empty receptor positions. receptor cleavage eventually results in virus dissociation when receptor density becomes too low to support tight multivalent binding. this model clearly sketches important biological as well experimental consequences. quantification of iav-receptor binding usually focuses on determination of the dissociation constant k d . however, equilibrium binding models did not apply, even at low receptor densities, and binding curves were dominated by concentration-independent avidity effects resulting in virtually irreversible binding. the binding curves are in agreement with a random sequential adsorption model [52, 53] . in such models irreversible particle binding proceeds to a plateau at~55% occupation of the binding surface (the jamming limit). however, iav particle binding could slowly proceed to complete saturation of the surface (fig 1 and s4a fig). we attribute this to virus rolling over the surface, a mechanism by which at an eventually very low rate sufficient space for new binding events is created. current, inherently complex, models for polyvalent binding lack general applicability [54, 55] and cannot be used to determine the kinetic constants of the different binding events shown in part of the viral envelope, containing ha (red symbol) and na (blue symbol), and the sensor surface, coated with sia (purple diamond)-containing receptors (r), is shown. kinetically different steps lead to multivalent interaction between iav and a receptor-coated surface. the initial ha-dependent binding event (step 1 in red) is a virus concentration-dependent intermolecular process governed by a binding rate constant k on (with the unit m −1 s −1 ) and a dissociation constant k off (with the unit s −1 ). subsequent ha binding steps (e.g. steps 2 and 4) are intramolecular with a k on (not necessarily the same as in the first step) and k off (both with the unit s −1 ). for iav, having a k d (k off /k on ) of~0.3 to~3mm for a monovalent ha-receptor interaction, binding at pm concentrations inevitably results in a low binding rate that is mostly determined by the first binding event. na can also contribute to the initial binding rate. this contributory effect can be inhibited by oc and is therefore attributed to binding of receptor to a na catalytic site. the contribution of na to receptor binding is determined by a dissociation constant (k d = k off /k off ) for the substrate (step i in blue) and a catalytic rate constant (k cat ; bold blue arrow) determining the receptor cleavage rate. a lower k cat will result in prolonged receptor binding before cleavage or dissociation takes place. this will enhance the chance for additional binding events (mostly by ha, which is present at higher density than na), thereby promoting the cascade of multivalent interactions responsible for tight virus binding. given the lower k d (30μm~600μm) [51] of na, in comparison to ha, for interaction with sialosides, a considerable contribution to the initial binding rate by na is expected even whereas the na/ha ratio of a virus particle is generally quite low. longer lasting, bli-detectable, binding requires the formation of additional ha-and/or na-sia bonds, which is indicated by the grey shaded area. initial binding events will be hardly detected due to the low levels of equilibrium binding in step 1 and i. during the bli-detectable phase of binding, ha-and na-sia interactions are formed and broken in a virus concentration independent mode with the result that all binding states can rapidly interconvert via binding/dissociation events 2 to 11 and ii to iv. the number of simultaneous interactions that can occur is logically dependent on receptor density and k off /k on ratios but how many simultaneous interactions suffice to keep a virus particle bound to the surface remains unknown. the experiments shown in fig 1 suggest the number of interactions required is very low. theoretically, the dynamics of ha-sia interactions allow a virus to roll over the surface but experiments shown in fig 7 show that na activity strongly stimulates rolling (and eventually leads to virus dissociation). this is schematically indicated by the curled arrows where na cleavage activity creates receptor-free positions on the surface. the receptor gradient caused in this way is probably the driving force for virus rolling but the direction in which the virus rolls (away from the empty position or "reaching over" the empty position) still needs further research. an endpoint binding assay (shown for our data in s4f fig) falsely assumes an equilibrium binding model and further errors into apparent k d determinations are introduced by low binding rates (as observed for weak binders or at low virus concentration, s4a fig and s4b fig) that prevent reaching a binding plateau within 60 min. iav binding to cells is poorly reversible [56] and adherence of only a few particles is already sufficient for productive cell infection. thus, where equilibrium binding levels or binding to saturation are not an issue, the initial binding rate v obs is a physiologically relevant parameter for iav binding. it can be determined by bli, but not easily by endpoint assays like conventional glycan arrays, more recently developed shotgun glycan arrays [57] or receptor-coated 96-well plate based assays. direct proportionality between virus concentration and v obs during the initial binding phase allows quantitative comparison of viruses of unknown concentration by determination of the relative binding rate constants for different receptor pairs. coating of sensors with recombinant glycoproteins, in a homogeneous orientation by virtue of their nterminal tag (e.g. fc-tag, biotinylated bap-tag), provides analysis of receptor surfaces on which the sialoglycans receptors are attached in protein-linked conformations as encountered in vivo. genetic engineering enables binding studies to glycoproteins carrying specific glycantypes (n-linked, o-linked) or glycan-density whereas further tuning of glycan structure can be accomplished by glycoprotein expression in cell lines with specific differences in their glycosyltransferase expression patterns. this will enable a much more refined analysis than the often used biotinylated polyacrylamide molecules carrying randomly distributed glycans and supposed to adopt a spherical configuration with a~15nm diameter [58] . the recent emergence of h3n2 strains displaying na-dependent hemagglutination hint at a role for na in determining the changes in receptor binding that accompany and/or drive virus evolution. this phenomenon is thought to reflect the gained capacity of na to bind receptors that are refractory to cleavage by na [46, 59, 60] . by using highly sensitive bli assays we showed that the na of strain wsn wt contributes to the initial binding rate even though it is capable of receptor cleavage leading to virus elution. this effect is exerted by binding of the receptor to the na catalytic site, as demonstrated by the inhibitory effect of oc, and is negatively correlated with the specific activity of the na. changes in ha-receptor binding specificity and avidity are thought to be prime factors in causing, or responding to, antigenic change [61, 62] or infection of an altered host cell range. secondary changes in na have been proposed to restore a critical ha/na functional balance. now, considering the evidence for a role of na in receptor binding, more complex scenarios should be considered. for instance, changes in na might facilitate (transient) functional changes in ha by (temporarily) contributing to the initial virus binding rate. bli allows quantification of such effects by determination of the relative increase by na of ha-dependent virion binding (fig 5 and s9 fig). a functional ha/na balance has primarily been described by weighing separately determined ha binding and na activity properties, using isolated proteins or virions. bli enables quantification of the separate contributions of ha and na to virus binding rates. the strength of bli lays in studying the simultaneous effect of na and ha, and thereby their balance, on the dynamics of virus-receptor interactions. virtually irreversible binding is the result of multiple ha-sia interactions that rapidly associate and dissociate, thereby providing access for na to temporarily unbound sias. sia cleavage by na creates a receptor density gradient that drives virus rolling, most likely away from the receptor-free spot in the direction of higher receptor density, or alternatively, by taking "a large step" to a site beyond the de-sialylated receptor. ultimately, when receptor density becomes too low, virus dissociation will occur. the resulting binding/dissociation profiles can be recorded by bli and are a quantitative reflection of the ha/na balance. yet lacking a mathematical model, these profiles can be described by empirical parameters (initial binding rate, area under the curve and x,y coordinates of the peak). kinetic binding rate constants for ha and na (k on and k off ) and the na catalytic rate constant (k cat ) will determine virion rolling characteristics (fig 8) . in addition, the ha/na ratio and distribution pattern in the virus envelope will determine the frequency at which na will be present in the contact area between virus and receptor surface where receptor cleavage can take place. as such, ha/na ratio and distribution pattern are additional variables that can be involved in functional evolution of the ha/na balance and its effect on virus rolling. whereas mostly a random distribution of ha over the viral envelope has been observed, the less abundant na has been shown to occur as singular tetramers as well as small clusters thereof. the latter has mostly been observed for iavs with a filamentous morphology [63] [64] [65] . whereas iavs harvested from cell culture usually display a spherical (~100 to 120 nm diameter) or slightly pleomorphic shape, filamentous morphology is frequently, but not always, observed in clinical samples (reviewed in [65, 66] ) although the loss of a filamentous phenotype is not absolutely required for adaptation to growth in cell culture. filamentous iavs usually have a diameter of 80 to 100 nm and a length up to 20 μm. patches of na clusters at the tip and the base of long filaments have been described [63, 64] but other reports suggest the presence of na along the length of the filament [67] . several functions for a filamentous morphology have been proposed (reviewed in [65, 66] ) including a role in clearance of sias from mucus by long budding filaments that have not pinched of from the cell surface [68] . results obtained in this study are confined to the behavior of spherical iavs and additional studies are required to see if filamentous iavs can move over a surface by lateral rolling, crawling or a caterpillar-like motion. interestingly, unidirectional motility of a filamentous influenza c virus over a receptor-coated glass-slide was recently demonstrated by microscopy [69] where as a more random movement of spherical iav particles was observed on fetuin-coated glass slides by total internal reflection microscopy. directional movement of virus particles at two different velocities, both dependent on na activity, was reported [69] . earlier, the combination of a receptor-binding and a receptor-destroying enzyme has been proposed to enable iav movement over a cell surface by a mechanism of repeated cycles of receptor binding, receptor release and receptor cleavage [70] . spherical and filamentous iav particles probably both occur in vivo and may very well have different functions. whereas na-dependent motility of filamentous particles through a mucus layer might be difficult, they might be better suited for spreading the infection to neighboring cells or clearing the mucus layer from their sia content. spherical iav particles on the other hand, are likely obtained during in vivo infection of humans by inhalation of aerosols containing a relatively low number of virions that somewhere get stranded on the mucus layer covering epithelial cells of the respiratory tract. thus, whereas quantitative measurements of spherical iav binding kinetics by bli should yield valuable data for modeling and testing the movement of such particles through a mucus layer and over epithelial cell surfaces, additional experiments are required to determine whether this can be extended to filamentous iav particles. iav was shown to require na activity for penetrating through a mucus layer in vitro [24] . the irreversible but highly dynamic binding mode that leads to virus rolling results in virus self-elution from a receptor-coated surface only upon efficient clearance of receptors from the complete surface. considering the low amount of virions confronted by a large amount of mucus and epithelial cells, we propose that virions, once attached to the mucus, remain receptor bound for their entire extracellular life. soluble, densely sialylated mucins in the mucus layer (2-20 μm thickness) form, by polymerization, a mesh-like structure with an average pore-size of~500nm [71] through which the virions need to move to reach the pericilliary layer. in this layer, the cilia of columnar epithelial cells (0.2 to 0.3 μm in diameter; 6 to 7 μm in length) are covered by membrane-spanning mucins and tethered muco-polysaccharides that protrude into the narrow (~200nm) space between cilia from which soluble mucins or 40 nm beads were shown to be excluded [72] . we consider it likely that rolling is necessary for gaining the directionality and even traction required for efficiently penetrating this heavily sialylated maze to reach sites on the epithelial cell membrane that permit virus entry. whether, and to which extent, rolling also takes place over the epithelial cell surface in order to arrive at a site that permits entry by endocytosis remains an open question. the overall density of sias on the cell surface is estimated to be high [73, 74] , but also very heterogeneous. iav entry by clathrin-mediated endocytosis was shown to take place by de novo formation of clathrincoated pits at sites where a virus particle was bound [75] . thus, it seems likely that in case of rolling the virus becomes arrested at a specific place in time. tight binding to specific receptors might be required for this but these have so far not been identified. note that the virus may also surf together with a tightly-bound glycoprotein over the cell surface. rolling might also be important for cell-to-cell virus spread. virions budding from de-sialylated cells, resulting from na activity, may even utilize sialylated mucins covering the epithelial cell layer as a rolling track to neighboring cells. in this perspective, an optimal ha/na balance should be considered as the combination of ha and na kinetic parameters, including factors like ha/na ratio and distribution, that optimally supports virion rolling over distinct surfaces coated with a diversity of receptors. also protective antibodies targeting iav receptor binding sites will function within this context with an important role for the ha/na/receptor balance as they will compete with receptors for interaction with iav [61, 62] . the bli-based methodology that we have established here is well-suited to quantify such processes using a highly versatile, modifiable and controllable receptor repertoire. [77] as well as all other viruses were grown in mdck-ii cells as described previously [78] and stored at −80˚c. virus titers were determined by measuring the tcid50 on mdck-ii cells. sequences of the ha and na genes were confirmed by sequence analysis (macrogen). for quantification, virus samples were concentrated by tca precipitation [79] and applied to standard 10% sds-page gels for separation of viral proteins followed by silver staining. silver-stained polymerase bands were quantified by densitometry on silver staining gels as outlined and shown in supplementary s7a fig, s7b fig and s7c fig. ha and na amounts were quantified by western blotting of standard 10% sds/page gels. monoclonal antibodies used for detection and quantification by densitometry were fi6 (for quantification of ha) [80] , n1-7d3 (for quantification of pr8 mtsin n1) [81] and gt288 (for quantification of wsn n1). recombinant purified ha and na proteins (see below) were used for standard curves (s7ds7i fig) . before the application of samples, 400-mesh copper grids with a pure carbon film were exposed to a glow discharge in air for 20 s to make them hydrophilic. ten microliters of the virus preparations was applied to the grids and incubated for 2 min. excess sample was blotted with a filter paper. for negative staining, 10 μl of 2% phosphotungstic acid at ph 6.8 was applied. after 1 min, excess stain was blotted and grids were left to dry. the specimens were examined in a jeol jem1400 transmission electron microscope at 120 kv and images were taken at a magnification of 30.000x with a matataki 2k x 2k camera. human codon-optimized h1-encoding and n1-encoding cdnas of pr8 mtsin and wsn wt (accession no. p03454.1 for wsn ha, acf54601.1 for wsn na, adx99484.1 for pr8 ha, and p03468.2 for pr8 na) were cloned in pcd5 (ha) or pfrt (na) expression vectors flanked by cd5 signal peptide-, gcn4-isoleucine-zipper trimerization (for ha) or tetramerization (for na) domains, and strep-tag ii-encoding sequences similarly as described previously [16, 82] . codon-optimized cdna fragments encoding the variable domains of the heavy and light chains of antibody fi6 [80] were synthesized by genscript usa inc and cloned inframe into pcaggs vectors containing human igg1 heavy and light constant domains, respectively, similarly as described previously [83] . codon-optimized bos taurus fetuin-encoding cdna (accession no. np_776409.1) was cloned in the pcaggs expression vector fused in frame to sequences encoding a human fc-tag with or without a tandem repeat of the 15-amino-acid biotin acceptor peptide (bap)-tag [84] . biotinylated fetuin is referred to as fetuin bt. mutations for knocking out o-linked glycosylation sites were introduced into the fetuin gene by using the q5 site-directed mutagenesis kit (neb) and confirmed by sequencing. human codon-optimized cdna encoding biotin protein ligase (bira) [84] was cloned in pcd5 in frame with sequences encoding a cd5 signal peptide and a 6his tag. expression vectors were transfected into hek293t (atcc crl-3216),cho k1 (atcc ccl-61) and gnti-deficient cho 15b cells [85] (from ineke braakman, utrecht university, the netherlands) using polyethyleneimine i (pei) similarly as described previously [16, 82] . cho cells are deficient in α2,6 sialyltransferases and therefore exclusively synthesize 2,3 sia linkages [86, 87] . tissue culture supernatants were harvested 5-6 days post transfection. recombinant ha and na proteins were purified using strep-tactin sepharose beads according to the manufacturer's instructions (iba, germany). fc tag-containing proteins were purified using protein a sepharose beads (ge healthcare), similarly as described previously [83] . for an overview of the different fetuin constructs see s2c fig. the concentration of purified protein was determined by using a nanodrop 1000 spectrophotometer (isogen life sciences) according to the manufacturer's instructions and analyzed by 10% sds-page followed by visualization of protein bands using a colloidal blue staining kit (invitrogen). biotinylated transferrin (sigma) contains two glycan chains exclusively carrying α2,6 sias [88, 89] (referred to as 6'n transferrin bt). the activity of iav virus particles as well as recombinant soluble nas was determined by using a fluorimetric assay similarly as described previously [16] . in short, viruses and na preparations were subjected to 2-fold serial dilutions in reaction buffer (50 mm tris-hcl, 4 mm cacl2, ph 6.0) in a flat-bottom 96-well black plate (greiner bio-one). subsequently, a similar volume of reaction buffer containing 200 μm 2 0 -(4-methylumbelliferyl)-α-d-n-acetylneuraminic acid (munana; sigma) was added to each well, mixed well, and incubated at 37˚c for 60 min. the reaction was terminated with the stop solution (0.1 m glycine, 25% ethanol, ph 10.7). the fluorescence of the 4-mu reaction product was immediately determined in relative fluorescence units (rfus) using a fluostar optima plate reader (bmg labtech, mornington, australia) with excitation and emission wavelengths at 340 and 490 nm, respectively. microarrays were printed as described previously [90] . the glycan array analysis of the ha proteins was performed as previously described [91] . briefly, 200 μg/ml recombinant ha was precomplexed with a horseradish peroxidase-linked anti-streptavidin tag antibody and an alexa fluor 488 anti-mouse antibody (4:2:1 molar ratio) for 30 min at 0˚c, prior to incubation for 90 min on the microarray slide under a microscope cover glass in a humidified chamber at room temperature. after repeated washes with phosphate-buffered saline (pbs) with 0.05% tween, pbs, and deionized water, the slides were immediately subjected to imaging. bli analysis was performed on an octet qk machine using standard streptavidin (sa) or protein a bio-sensors. pbs with calcium and magnesium (pbs+/+) was used as standard assay buffer. receptor loading was performed by loading biotinylated receptors (synthetic glycans or proteins) to sa sensors or fc-tagged glycoprotein receptors to protein a sensors. unless otherwise specified, sensor were loaded with receptor to maximum levels (no further increase in reflection) using 100 nm synthetic glycan or 4 μg/ml glycoprotein as loading sample concentration. after loading sensors were washed in pbs+/+ until a stable baseline was obtained. virus binding was performed by moving receptor-loaded sensors to wells containing 100 μl virus sample at the indicated concentrations (the use and concentration of oc is indicated where applicable). then virus-loaded sensors are usually moved to pbs+/+ in presence of 10 μm oc to examine virus dissociation (consistently producing a flat line), or washed 3 times for 3 seconds in pbs+/+ to remove oc and next transferred to pbs+/+ without oc to measure na activity-driven self-elution. alternatively, in order to determine simultaneous action of ha and na virus binding was analyzed from the start in pbs+/+ in the absence of oc. regeneration of sensors, preserving the binding of biotinylated receptors but removing all bound virus, was performed by dipping sensors briefly in 10 mm tris/glycine buffer ph 2. pr8 mtsinspecific antibody (03/242 from nibsc) was used for detection of virus binding in presence of oc with or without prior sensor generation. fetuin-coated sensors were also analyzed for their lectin-binding properties. to this end, fetuin-coated sensors were incubated with different lectins (80ng/ul; sna, mal-i, mal-ii, eca, all from vector labs) and lectin-binding curves were obtained. each bli experiment was repeated at least twice. representative experiments were graphed. initial binding rates, corresponding to the sloops of the binding curves during the first few minutes of the virus-binding experiments, were determined by second order polynominal equation (graphpad prism 7.04). the correlation between virus particle numbers and the initial binding rate was determined by linear regression and pearson r analysis using graphpad prism 7.04 software. significant differences between curves were analyzed by univariate analysis of variance model using ibm spss statistic 24. fractional receptor densities correlating with half maximum initial binding rates were determined by non-linear regression analysis using graphpad prism 7.04 software. significance analysis was based on two tailed unpaired t test or one way anova followed by tukey's multiple comparisons test (graphpad prism 7.04). streptavidin-coated (sa) biosensors contain 10 9 biotin binding sites (pall-fortebio). sa tetramers carry four biotin binding sites, ordered two by two at opposing planes of the cubic structure [92] . only two binding sites (spaced at 2.5 nm distance as determined by x-ray crystallography [93] ) on one side of a surface-coated sa tetramer (25 nm center to center distance assuming regular hexagonal packaging) are assumed to have exposed biotin binding sites [93] . ha trimers are closely packaged on the virus surface (s3 fig, in agreement with [94] [95] [96] [97] ) and the center to center distance has been determined at 12nm [94] [95] [96] [97] . a fully loaded streptavidin can, in principle, form a bivalent interaction with an ha-trimer in which the sia-binding sites are spaced at 4nm distance [94, 97] . lowering the receptor-density results in a non-homogenous sensor surface with streptavidins carrying 0, 1 or 2 receptor molecules. as a result, increasing amounts of surfacearea will have a receptor density too low to bind virus at decreasing receptor concentrations thus contributing to the observed decrease in maximum binding levels and initial binding rate when lowering receptor density (fig 1d and fig 1e) . (b) labstrains pr8 and wsn wt are spherical viruses with a diameter of~100 nm (s3 fig) [39] [40] [41] [42] . when virus particles can be flattened for 0.2 times the radius 10% of the virus surface will be in contact with the sensor. (c) when 10% of the virus surface is in contact with the sensor,~7 ha trimers can interact with receptor-loaded sa molecules at the virus-sensor contact interface (inner red circle). in principle two receptor molecules on a sa molecule can interact with an ha trimer but whether this occurs simultaneously will depend on the exact geometry of the specific glycan that was loaded. (d) at saturating levels of virus binding (hexagonal packaging) the majority of sa molecules are not present at the contact interface and therefore cannot be cleaved by na without virus movement. [88, 89] . biotinylated fetuin was made by expressing a construct encoding a bap-tag fused to fetuin that, by co-transfection with a plasmid carrying a biotinylation enzyme, yields c-terminally biotinylated 3'n+o fetuin (3'n+o fetuin bt) upon expression in cho k1 cells. (d) confirmation of sia linkage-type specificity of glycoproteins using lectin binding. the glycoproteins were analyzed for linkage type specificity of their sialic acids using lectins mal i (specific for siaα2,3galα1,4glcnac linkages abundantly present on n-linked glycans), mal ii (specific for siaα2,3galα1,3galnac linkages abundantly present on o-linked glycans), sna (specific for siaα2,6galα1,4glcnac linkages abundantly present on n-linked glycans), and eca (specific for terminal galα1,4glcnac epitopes present on non-sialylated n-linked glycan antennae). (e) viruses used for binding to receptor-loaded sensors are wild type pr8 mtsin , wild type wsn wt and recombinant viruses carrying the ha encoding segments of pr8 mtsin (wsn hamtsin ) or pr8 cam (pr8 cam2,6 ) in the background of seven wsn segments. pr8 cam2,3 is identical to pr8 cam2,6 except for a substitution (d190e) that was introduced in ha to obtain a shift from α2,6 to α2,3 linkage-type binding specificity. tx77 namtsin carries the ha encoding segment of a/bilthoven/1761/76 (h3n2) in the background of seven pr8 mtsin segments [77] . whereas both pr8 mtsin and pr8 cam2,3 bound to fetuin containing n-glycans (a and b), only pr8 cam2,3 was able to bind fetuin containing o-glycans, albeit at a 6-fold lower initial binding rate than to n-glycosylated fetuin (c). when n-and o-linked glycans are both present (a), the initial binding rate seems to be determined by the stronger binding to n-linked glycans as binding of pr8 cam2,3 is not accelerated despite its ability to bind α2,3 sialylated o-linked glycans. (e-g) confirmation of presence of n-and/or o-glycan on recombinant fetuin. the glycoproteins were treated with pngase f (neb) for 4 hours at 37˚c under non-denaturing conditions, which effectively removes almost all n-linked oligosaccharides from glycoproteins. the glycoproteins were analyzed for linkage type specificity using lectins mal i (specific for siaα2,3galα1,4glcnac linkages abundantly present on n-linked glycans), mal ii (specific for siaα2,3galα1,3galnac linkages abundantly present on o-linked glycans), and eca (specific for terminal galα1,4glcnac epitopes present on non-sialylated n-linked glycan antennae). (e) after pngase f treatment, the binding level of mal i to 3' n+o (blue) and 3' n fetuins (red) significantly reduced, whereas the binding level to 3' o fetuin (green) remains the same, indicating the presence of sialylated n-linked oligosaccharides specifically on 3' n+o and 3' n fetuin. (f) after pngase f treatment, the binding level of eca to 3' n+o (blue) and 3' n fetuins (red) dramatically decreased, whereas the binding level to 3' o fetuin (green) remains the same, indicating the presence of non-sialylated n-linked oligosaccharides specifically on 3' n+o and 3' n fetuin. (g) similar binding of mal ii to glycoproteins was observed after treatment with pngase f, indicating that o-linked oligosaccharides were not removed by pngase f treatment. reliable determination of v obs depends on precise determination of virus concentration. hemagglutination titers or infectious titers do not reveal absolute or relative particle of different viruses. quantitative pcr or western blotting (usually targeting np) are sensitive to variation caused by rna or protein contamination of virus preparations and, when comparing different viruses, to differences in specificity of probes or antibodies. therefore, densitometric quantification of the polymerase content of a virus preparation was used, assuming that every particle carries eight c) and (e). the obtained numbers/particle fit well to numbers obtained by different methods by others [98, 99] . (g, h, i) quantification of na by similar procedures as applied for ha in panel d-f. antibodies gt288-gtx629696 (wsn) and n1-7d3 (pr8) were used and quantification was calibrated using a western blot of a standard concentration series of recombinant soluble na of pr8 mtsin and wsn expressed in hek293t cells run in parallel. the na proteins were expressed similarly are described previously [100] . (j) na activity of pr8 mtsin and wsn wt virus particles and expressed recombinant na soluble tetramers was determined using a two-fold dilution series in a soluble substrate na activity assay (munana assay). the activity of recombinant wsn na was 5.4-fold lower than of pr8 mtsin na, whereas the na activity of the cognate virus particles showed a 31.4-fold lower activity for wsn wt particles. this difference is explained by the 6.4-fold higher incorporation level of na in pr8 mtsin particles as determined by western blotting (g-i). the ha content of both viruses was similar (d-f). (k) ha/na ratio was determined from panels f and i. both viruses bind at similar, but relatively low, rate to 6'n transferrin bt (fig 2e) whereas wsn wt displays a~3-fold faster binding rate to 3'n fetuin than pr8 cam2,6 ( fig 2d) . ha clearly affects the self-elution rate as, in combination with the same na, self-elution from 3'n fetuin is much more efficient in companion of the weaker binding ha of pr8 cam2,6 (b) than in companion of the stronger binding ha of wsn wt (a). self-elution rates from 6'n transferrin bt are more similar for both viruses. thus, whereas α2,3 versus α2,6 sia specificity of na is seemingly opposite for pr8 cam2,6 and wsn wt , this is not caused by the na itself (which is identical for both viruses) but by differences in their has. in absence of oc, ongoing receptor cleavage reduces receptor density in time and thus the binding rate of additional virus particles, resulting in bending of the curves. as receptor cleavage by na is in competition with receptor 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productivity cultures comparative-study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture-medium of recombinant chinese-hamster ovary cells screening for n-glycosylated proteins by liquid chromatography mass spectrometry site-specific carbohydrate profiling of human transferrin by nano-flow liquid chromatography/electrospray ionization mass spectrometry arraying the post-translational glycoproteome (ptg) only two residues are responsible for the dramatic difference in receptor binding between swine and new pandemic h1 hemagglutinin structural origins of high-affinity biotin binding to streptavidin streptavidin-biotin binding in the presence of a polymer spacer. a theoretical description kinetic evidence from image analysis of hemagglutinin-reconstituted vesicles zernike phase contrast electron microscopy of ice-embedded influenza a virus electron microscopy of the influenza virus submembranal structure structure of influenza haemagglutinin at neutral and at fusogenic ph by electron cryo-microscopy influenza virus pleiomorphy characterized by cryoelectron tomography polypeptides specified by the influenza virus genome i. evidence for eight distinct gene products specified by fowl plague virus mutation of the second sialic acid-binding site, resulting in reduced neuraminidase activity, preceded the emergence of h7n9 influenza a virus oseltamivir carboxylate was kindly provided roche. recombinant pr8 virus tx77 namtsin was kindly provided by dr. r. fouchier (erasmus medical center, rotterdam, the netherlands). n1-7d3 was generously provided by dr. x. saelens (molecular virology unit, university of ghent). cho 15b cell was kindly provided by ineke braakman (utrecht university, the netherlands). key: cord-004280-c470nlie authors: coleman, kristen k.; sigler, william v. title: airborne influenza a virus exposure in an elementary school date: 2020-02-05 journal: sci rep doi: 10.1038/s41598-020-58588-1 sha: doc_id: 4280 cord_uid: c470nlie influenza contributes significantly to childhood morbidity and mortality. given the magnitude of the school-aged child population, a sizeable proportion of influenza virus transmission events are expected to occur within school settings. however, influenza virus activity in schools is not well-understood, likely due to our limited ability to accurately monitor for respiratory viruses without disrupting the school environment. in this study, we evaluated the use of a bioaerosol sampling method to noninvasively detect and quantify airborne influenza a virus (iav) densities in a public elementary school. air samples were collected from multiple locations in the school, two days per week, throughout an eight-week sampling period during influenza season. real-time rt-pcr targeting the iav m gene revealed detectable iav on five occasions in densities ranging from 2.0 × 10(−1) to 1.9 × 10(4). no significant differences in iav densities were related to student presence/absence. the majority of iav-associated particles were ≤4 μm in diameter, and theoretical calculations indicate infectious thresholds after minutes of exposure. our study represents the first identification and quantification of airborne influenza virus in an elementary school, and the results suggest that airborne iav has the potential to circulate in schools during influenza season, in large enough doses known to cause infection. a total of 128 air samples were collected over an eight-week period (february-march), followed by qrt-pcr targeting the influenza a virus (iav) m gene, which revealed detectable iav in 5% (5/96) of the air samples collected indoors, in densities of 2.0 × 10 −1 , 1.9 × 10 3 , 3.8 × 10 3 , 1.5 × 10 4 and 1.9 × 10 4 m gene copies m −3 air (table 1) . iav was not detected in any of the outdoor reference samples. an r 2 value of 0.97 for each standard curve was achieved for the qrt-pcr assays and the detection limit for iav m gene was 1 rna copy per reaction volume (25 μl) when the cut-off for positive result was set at 40 cycles. because each influenza virus particle packages one copy of the m gene 34 , our results can also directly reflect the number of virus particles m −3 air. however, our results are reported in copy numbers m −3 air to remain consistent with other quantitative studies measuring airborne iav densities. significantly different (p = 0.049) airborne iav densities were detected between all three indoor locations (i.e., gymnasium, classroom, and corridor) and all positive samples were collected during the last two weeks of 66 , and a 20-30% relative humidity level; descriptive of an average elementary school student in the usa weighing ~23-32 kg with an assumed tidal volume (v t ) of 7 ml per kg of body mass. † irrespective of iav target gene; based on the assumption that one tcid 50 is equivalent to ~300 pcr-detectable iav rna copies [47] [48] [49] [50] . the study (month of march, table 1 ). sample location was strongly correlated with iav density (r 2 = 0.95), with the highest density of iav detected in the corridor. two (6%) of the air samples collected from the gymnasium were positive for iav, as well as two (6%) from the corridor, and one (3%) from the classroom. no significant difference in iav densities was observed between samples collected during and after school (p = 0.14). indoor relative humidity (rh) and temperatures remained relatively consistent (19-29% rh and 20-23 °c) throughout the study. no significant difference in iav densities was observed between samples collected at different rh levels (p = 0.74) or temperatures (p = 0.10). significantly different (p = 0.049) airborne iav densities were detected among particle size fractions. particle size was strongly correlated with iav density (r 2 = 0.95), with 91% of virus-laden particles detected in respirable size fractions (≤4 μm in diameter). the largest virus-laden particles (>4 μm, 9% of total particles) were detected only in the gymnasium, while the smallest (<1 μm; 5% of total particles) were detected only in the classroom. no significant difference in virus-laden particle size was observed among samples collected at different rh levels (p = 0.82) or temperatures (p = 0.44), nor during and after school hours (p = 0.44). theoretical exposure threshold calculations accounting for body mass, tidal volume, and breathing rate predict that exposure to an equivalent of 1.1 × 10 4 -2.8 × 10 5 iav rna copies m −3 air for one minute is sufficient to induce infection in a student ( table 2 ). based on the airborne iav densities detected in the school, we estimated that students in the classroom (day 80), gymnasium (day 80), and corridor (day 84 and 87) were at risk of infection following 6, 3 and 1 minute(s) of breathing, respectively (table 1 ). aerosolization is an important mechanism for spread of iav 25 . since children are significantly burdened by influenza and play a key role in transmission 5, 29, 35, 36 , we established a protocol through which iav densities could be monitored in a school setting. viral densities were compared to theoretical iav exposure thresholds, above which students are expected to become infected. student illness and absenteeism caused by respiratory diseases is thought to occur, in part, because of prolonged time spent in school buildings, 50% of which experience impaired indoor air quality 31 . therefore, understanding how the student environment influences disease transmission can not only help to better address student health, but also improve sanitation/cleaning practices and inform predictive efforts. to our knowledge, our work represents the first identification and quantification of airborne iav in an elementary school, showing that schools can not only harbour airborne iav densities similar to those found in clinical settings 37, 38 , but also in infectious doses. furthermore, the detection and quantification of airborne iav in the school airshed warrants future investigations to determine the relationship between iav densities and student illness. three locations in an elementary school were sampled for airborne iav, including the main corridor, gymnasium, and a classroom. because each location featured different iav densities and particle sizes, the exposures of students to iav differed according to sample location. for example, students were likely at an elevated risk of iav infection by breathing in the classroom on day 80, especially since the particles were <1 μm in diameter. infection could have also arisen from breathing for 3 minutes in the gymnasium on day 80, but is less likely, as particles were >4 μm in diameter. while no specific activity can explain elevated iav densities in the classroom, two defined activities in the gymnasium throughout the school day are thought to promote elevated iav densities. first, the gymnasium is used by approximately 25 students during each class period for physical education instruction. physical activity/exercise can result in aerosolized iav through increased respiratory rates, and increased respiratory distress due to bronchoconstriction 39 , which has been demonstrated to affect up to 16% of children 40 . second, the gymnasium hosts the student lunch period, which lasts for 50 minutes, during which two cohorts (approximately 235 students each) eat for 25 minutes. the entire student body is not only represented in the gymnasium during the lunch period, but is also moving, en masse, as each cohort enters and exits the space, theoretically creating turbulence to maintain suspension of iav (if present) throughout the period, and likely into the post-lunch hours. airborne iav densities were highest in the school corridor, which was expected, and can be partially explained by two factors. first, student lockers are situated along the walls of the corridor which encourages air turbulence as students pass through the corridor and open and shut locker doors. second, the corridor represents the only available passage between classrooms and therefore each student passes through the corridor multiple times per day, which dually increases the probability that an infectious student will shed virus in the area, and that other students will be exposed. in a college student office setting, zhang and li (2018) 41 demonstrated that the frequency of close contact (within 1 m) is 9.64 contacts per hour per student, which contributed to 45% of reported iav infections. additionally, previous studies have suggested that influenza illness and death rates could be decreased by as much as 50% by reducing the contact rates of infected persons 42 . given the high airborne iav densities detected in the school corridor, along with elevated student contact rates, it is plausible to conclude that the school corridor is a "hotspot" for influenza virus transmission. studies focusing on clinical environments have demonstrated that a considerable proportion (48-53%) of total airborne iav-laden particles are respirable 37, 38 . in the current study, the majority (91%) of airborne particles associated with iav were respirable (≤4 μm in diameter), representing the most infectious fraction based on size. particles >4 μm in diameter are deposited predominantly in the nasal cavity or trachea, and are subject to mucociliary clearance before initiating infection 43 . in contrast, particles ≤4 μm in diameter can be deposited deep into the lungs, and are more likely to result in lower respiratory tract infections, which disproportionately impact children during influenza pandemics 24 . therefore, identifying the particle size distribution of airborne iav is critical for understanding the potential transmission and infectious impact of the virus. although we successfully detected airborne iav in the school with appropriate sensitivity to accurately quantify iav densities, environmental factors created sample processing challenges and difficulty interpreting the data. airborne pathogen densities in nonclinical environments can be several orders of magnitude lower than (2020) 10:1859 | https://doi.org/10.1038/s41598-020-58588-1 www.nature.com/scientificreports www.nature.com/scientificreports/ those detected in clinical environments 37, 38, 44 . furthermore, desiccation stress is known to limit the stability of airborne iav 45 . therefore, in the school, lower iav densities and high variability were anticipated, and sampling parameters were chosen to maximize detection, including short sampling durations to preserve the integrity of captured iav rna. we chose a flow rate of 3.5 l/min for four hours at a time, which was previously demonstrated to efficiently capture and preserve airborne iav rna for rt-pcr detection 37, 38 . next, iav packages a single copy of our rna target, the m gene 34 , and therefore a 1:1 relationship between the number of gene copies detected and number of virions is a valid assumption. however, brown et al. (2015) indicated that iav densities can be overestimated when quantifying gene copies 46 , and studies have assessed the relationship between rna copy number and the number of viable viruses, suggesting that one tcid 50 is equivalent to ~300 pcr-detectable iav rna copies [47] [48] [49] [50] . while iav viability was not directly assessed in the current study, our quantitative results do provide an estimate of the potential for iav transmission in the school environment, consistent with the prevailing public health proposals for conservative estimates of disease transmission risk 51 . lower densities of airborne iav were expected in the absence of students from the building. however, no significant difference in airborne iav density, as a function of student presence or absence, was observed. this result was unexpected, as children are thought to be key vehicles of iav transmission and are viewed here as the major factor contributing iav to the school airshed. we did not collect individual student health data during the study, and therefore could not definitively link the prevalence of influenza among the student population with virus detections. however, the detection of iav during the absence of all students from the building was nonetheless an important observation, indicating persistence of the virus in the airshed. airborne iav can remain viable for up to 36 hours 52,53 and is likely facilitated by two factors. first, it has been demonstrated that temperature and rh influence the viability and transmission of influenza viruses [54] [55] [56] [57] [58] . however, research has recently demonstrated that rh does not influence influenza virus viability 59 , but rather the rate of aerosol deposition, which influences the concentration of virus particles in the air. in the current study, the environmental conditions during the school day (19-29% rh and 20-23 °c) were optimal for iav persistence. second, children shed iav for a longer duration than adults shed the virus 60 , encouraging prolonged iav aerosolization in a school setting where children predominate. overall, our findings demonstrate that on the short term, iav is not fully cleared from the school environment upon removal of students, and support the assertion that schools should be considered an iav transmission hotspot, even in the absence of students. although airborne influenza virus has been detected in select indoor settings 37, 38, 44, [61] [62] [63] [64] [65] , the ability to consistently detect the virus in the airshed remains limited in environments featuring low iav densities. we now have the first molecular evidence of airborne iav in an elementary school, during a portion of the influenza season when students were exposed for appropriate durations to densities of influenza-laden particles that could facilitate infection. furthermore, given the magnitude of the school-aged population, our data provide justification for considering schools as influenza hotspots, warranting further study to determine the relationship between airborne iav densities and student health to improve influenza management in the greater community. bioaerosol sampling. air samplings were performed four times per week during an eight-week sampling period (february-march) in an elementary school (toledo, oh area) that enrolls approximately 470 students, in grade levels k-6. airborne iav was sampled in the school gymnasium, a classroom, main corridor, and an outdoor reference, using two-stage bioaerosol cyclone samplers provided by the national institute for occupational safety and health (niosh) and chosen for their portability, durability, minimal preparation time, and efficiency that equals that of commercial samplers 14 . each sampler was placed 1.2 m from the ground, simulating the average elementary school student's breathing level, and connected to an skc airchek xr5000 pump (skc, eighty four, pennsylvania) with 6.35-mm tygon tubing, operating at a flow rate of 3.5 l of air min −1 , collecting a total of 840 l of air for each sample. the pump flow rate and sampling duration was based on previous studies that demonstrated efficient capture of airborne influenza virus rna for rt-pcr detection 14, 37, 38 . each sampler collects particles >4 μm in diameter into a 15 ml centrifuge tube, particles 1-4 μm in diameter into a 1.5 ml centrifuge tube, while particles <1 μm in diameter are collected onto a 37-mm diameter, polytetrafluoroethylene filter with 2-μm pores. the influence of student presence/absence on airborne influenza virus detection was determined by collecting samples early in the school day (8:00 am-12:00 pm, students present), and in the afternoon/evening (3:00-7:00 pm, students absent). after sampling, collection tubes and filter cassettes were transported to the laboratory on ice and stored at −80 °c, if not immediately processed. samplers were washed with isopropanol and air dried between sample collections. temperature and rh were continuously recorded inside the school building near each sampler using hobo dataloggers (onset; bourne, ma, usa). sample rna was extracted and purified using the magmax viral rna isolation kit (ambion) following the manufacturer's instructions with slight modifications, including the addition of lysis/binding solution directly to (i) sampler tubes, and (ii) 50-ml falcon tubes containing the ptfe filters. xeno rna control (ambion), a synthetic rna transcript, was added to the sample lysis solution to act as an internal, positive control target for assessing the efficiency of rna recovery. purified rna was eluted in 30 μl of elution buffer. all analysis materials were purchased rnase-and pyrogen-free, if possible, and otherwise depyrogenated by autoclaving at 250 °c for 30 minutes. one-step, real-time, rt-pcr targeting the influenza a virus m gene was performed in an applied biosystems step-one real-time pcr system with commercial taqman aiv-matrix reagents (ambion) in a total reaction volume of 25 μl. reverse transcription was performed at 65 °c for 5 min, 50 °c for 2 min, and 95 °c for 10 min, followed by 65 cycles of qpcr analysis at 95 °c for 15 s, and annealing/elongation at 60 °c for 1 min. three negative control reactions (no template) were included in each qrt-pcr assay. to quantify the viral load present in scientific reports | (2020) 10:1859 | https://doi.org/10.1038/s41598-020-58588-1 www.nature.com/scientificreports www.nature.com/scientificreports/ each sample, the ct value from each reaction was compared to those of a standard curve derived from a dilution series of known quantities of iav m gene copies. iav exposure threshold calculations. based on observed densities of airborne iav, we estimated the breathing time, above which a student could theoretically become infected. to calculate the time, we used a known range of 0.6-3.0 tcid 50 (tcid 50 is the number of iav particles that induce infection in 50% of inoculated tissue culture cells) 66 , which has previously been used to estimate the risk of airborne iav infection after exposures consistent with a one-hour clinical visit, an eight-hour workday, and after 24 hours indoors 44 . since approximately 300 rna copies is equivalent to one tcid 50 47-50 , the resulting threshold iav density capable of initiating an infection is theoretically equivalent to 50-900 rna copies. assuming the average elementary school student weighs 23-32 kg, has a respiratory rate of 20 breaths min −1 , and a tidal volume (air volume displaced in a single breath) of 7 ml per kg of body mass, we calculated an inhalation volume range of 3200-4480 ml air min −1 for a typical student, which was then compared with our estimated iav densities to determine the number of minutes of breathing necessary to cause an infection. statistical analysis. data were imported into stata version 15.0 (statacorp, college station, tx, usa) and a two-sample t-test or one-way analysis of variance was performed to test for significant differences in iav densities and particle sizes between indoor sample locations (gym, classroom, corridor) and environmental conditions (temperature, rh, and student presence). regression analyses were then performed to test for correlations between variables demonstrated to be statistically significant. datasets generated during this study are available from the corresponding author upon reasonable request. outbreak of 2009 pandemic influenza a (h1n1) at a new york city school epidemiological and clinical characteristics of the outbreak of 2009 pandemic influenza a (h1n1) at a middle school in luoyang effectiveness of non-pharmaceutical interventions in controlling an influenza a outbreak in a school pandemic (h1n1) 2009 virus outbreak in a school in london influenza and school-based influenza-like illness surveillance: a pilot initiative in maryland interrupting the transmission of respiratory tract infections: theory and practice using data on social contacts to estimate 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estimates of viable virus loss resulting from aerosolization and air-sampling influenza virus in human exhaled breath: an observational study application of a fluorogenic pcr assay for typing and subtyping of influenza viruses in respiratory samples simultaneous detection of influenza viruses a and b using real-time quantitative pcr biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity the case for 'plausible conservatism'in choosing and altering defaults. science and judgment in risk assessment decay of influenza a viruses of human and avian origin influenza a of human, swine, equine and avian origin: comparison of survival in aerosol form experimental air-borne influenza infection. i. influence of humidity on survival of virus in air influenza virus transmission is dependent on relative humidity and temperature dynamics of airborne influenza a viruses indoors and dependence on humidity indirect health effects of relative humidity in indoor environments the effect of environmental parameters on the survival of airborne infectious agents influenza virus infectivity is retained in aerosols and droplets independent of relative humidity amantadine therapy of epidemic influenza a2-hong kong bioaerosol sampling in clinical settings: a promising, noninvasive approach for detecting respiratory viruses molecular surveillance of respiratory viruses with bioaerosol sampling in an airport. tropical diseases detection of influenza and other respiratory viruses in air sampled from a university campus: a longitudinal study monitoring for airborne respiratory viruses in a general pediatric ward in singapore bioaerosol sampling for respiratory viruses in singapore's mass rapid transit human influenza resulting from aerosol inhalation this study was approved by the university of toledo irb (ref no: 200094). we thank the ottawa hills elementary school principal, kori kawczynski, and superintendent, dr. kevin miller, for graciously allowing us access to their school; dr. bill lindsley (national institute for occupational safety and health, niosh) for loaning the aerosol samplers used in this project and guiding us in their use; drs. april ames, daryl dwyer, sheryl milz, and daryl moorhead for their helpful insight and support throughout the study; and former ms student, marcus keller, and undergraduate student jessica dilworth for volunteering their time in the laboratory and out in the field. the authors declare no competing interests. correspondence and requests for materials should be addressed to k.k.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-004018-33zi29bg authors: coombs, kevin m.; simon, philippe f.; mcleish, nigel j.; zahedi-amiri, ali; kobasa, darwyn title: aptamer profiling of a549 cells infected with low-pathogenicity and high-pathogenicity influenza viruses date: 2019-11-05 journal: viruses doi: 10.3390/v11111028 sha: doc_id: 4018 cord_uid: 33zi29bg influenza a viruses (iavs) are important animal and human emerging and re-emerging pathogens that are responsible for yearly seasonal epidemics and sporadic pandemics. iavs cause a wide range of clinical illnesses, from relatively mild infections by seasonal strains, to acute respiratory distress during infections with highly pathogenic avian iavs (hpai). for this study, we infected a549 human lung cells with lab prototype a/pr/8/34 (h1n1) (pr8), a seasonal h1n1 (rv733), the 2009 pandemic h1n1 (pdm09), or with two avian strains, an h5n1 hpai strain or an h7n9 strain that has low pathogenicity in birds but high pathogenicity in humans. we used a newly-developed aptamer-based multiplexed technique (somascan(®)) to examine >1300 human lung cell proteins affected by the different iav strains, and identified more than 500 significantly dysregulated cellular proteins. our analyses indicated that the avian strains induced more profound changes in the a549 global proteome compared to all tested low-pathogenicity h1n1 strains. the pr8 strain induced a general activation, primarily by upregulating many immune molecules, the seasonal rv733 and pdm09 strains had minimal effect upon assayed molecules, and the avian strains induced significant downregulation, primarily in antimicrobial response, cardiovascular and post-translational modification systems. influenza a virus (iav) is a member of the family orthomyxoviridae. iav has been responsible for numerous yearly epidemics and for at least four pandemics during the past~100 years; estimates are that more than 100 million people have died from infection during this time period [1, 2] . iav is a small enveloped virus with a genome of eight segments of negative-sense single-stranded rna that encode 10-15 proteins, depending on the virus strain [3] [4] [5] . iav is serologically categorized by two surface proteins: the hemagglutinin (ha), of which there are currently 18 types (h1-h18), and neuraminidase (na), of which there currently are 11 types (n1-11) [3, 6] . virtually all h/n combinations have been identified in water fowl [7, 8] , the generally-accepted reservoir, except for the recent identification of h17n10 and h18n11 in bats [6] , but only a small number of h/n types are known to circulate or have circulated in humans; h1n1 (1918 "spanish flu" human lung a549 cells (atcc # ccl-185, manassas, va, usa) were routinely cultured in dulbecco s modified mem (dmem) supplemented with 0.2% (w/v) glucose, non-essential amino acids, sodium pyruvate, 2 mm l-glutamine, and 10% fetal bovine serum (fbs; gibco, grand island, ny, usa). madin-darby canine kidney (mdck) cells (atcc # ccl-34) were cultured similarly, but in completed dmem supplemented with 5% fbs. cells were grown as monolayers in 5% co 2 and passaged by trypsinization 2-3 times each week. , and a/anhui/1/2013 (h7n9) were amplified, concentrated by ultracentrifugation, and titered in mdck cells by a standard plaque assay or tcid 50 procedures [13, 21] . all manipulations of live h5n1 and h7n9 viruses were performed in a respiratory bsl-3 facility, using all public health agency of canada (phac) guidelines and sops. a549 cells were infected, or sham (mock)-infected with diluent, with each of the above viruses at an moi of 5 when cells were~90% confluent. mock and infected cells were harvested at 24 h post-infection (hpi). aliquots of all infections were saved for plaque titration or tcid 50 determination to confirm infection. mock and infected cells were washed, lysed in mper solubilization buffer (pierce, waltham, ma, usa) supplemented with 1 × halt protease inhibitor (pierce) and protein concentrations determined using the bicinchoninic acid method. cell lysate protein concentrations were adjusted to 200 ng/µl, and 70 µl of each was analyzed in-house on a somalogics ® -licensed somascan ® version 1.3 platform in the manitoba centre for proteomics and systems biology as described [22, 23] . briefly, somascan ® is a new proteomic tool that uses somamers ® , dna slow off-rate modified aptamers (22) . these modified nucleotides are used because they bind to specific human proteins. somamers ® bind proteins in their native state, and are measured on dna microarray chips. somamers ® measure fm-µm protein quantities. the somascan ® version 1.3 simultaneously measures 1310 distinct proteins in up to 88 samples [22] . two biologic replicates of pr8 and rv733, and three biologic replicates of mock-, pdm09-, h5n1and h7n9-infected samples were collected at 24 hpi (=14 total samples) and were analyzed at the same time in a single somascan ® 96-well plate. results are reported as relative fluorescent units (rfus) for every protein, and these rfus are directly proportional to the quantities of each target protein in the original samples, confirmed by standard curve generation for every protein-somamer pair [22] . differences between mock and influenza a virus (iav)-infected rfu values were analyzed as described below in next section. rfu values for each of the 1310 analyzed proteins in each biologic replicate were imported into excel. values were log 2 -transformed and fold-changes determined for each protein in infected samples compared to mock samples. fold-change significances were tested both by students t-test (2 tails) and by z-score analysis, as described [10, 23] . briefly, all fold-changes found not to be significant by the t-test were re-tested by the z-score. this was done by determining the number of standard deviations each protein s value was from the population mean. any protein s z-score was deemed significant if the average z-score was >1.96σ or <−1.96 σ, the z-score satisfied this criterion in at least 2 replicates and trended > 1 2 the same direction in one or fewer replicates. thus, z-scores had to be beyond the ±1.96σ limit in both of the pr8 and rv733 analyses. after compiling all proteins deemed significant, we also analyzed levels of fold-changes of the significant proteins, and as described below, applied a fold-change cut-off of 1.50-fold dysregulation (≥1.50-fold if up-regulated, or ≤0.667-fold if down-regulated) to these proteins for added stringency and to maintain workable numbers of proteins for subsequent bioinformatics and pathway analyses. a549 cell lysates of iav-and mock-infected cells were collected 24hpi from two (pr8 and rv733) or three (mock and all other iav clones) biologic replicates and analyzed with a somalogic ® version 1.3 platform. each of 1310 protein analytes was examined and relative quantities determined. values for each of the infected samples were normalized to the mock samples from the same replicates, and these comparative values were then analyzed. more than 500 proteins were significantly dysregulated by infection (p-value < 0.05 and/or z-score > 1.96σ or <−1.96σ), and far more proteins were dysregulated by both of the h5n1 and h7n9 avian strains than by any of the h1n1 strains ( table 1) . most of the dysregulated proteins were affected <25%; thus, we considered more stringent parameters and chose fold-change cut-offs of 1.5-fold (=down-regulated to 0.667 of mock) along with significance. thus, proteins that were dysregulated >1.5-fold, but not considered significant by either p-value or z-score, because of a substantial variability in replicate values, were excluded from viruses 2019, 11, 1028 4 of 16 the subsequent analysis. using these parameters, we identified 76 proteins that were significantly dysregulated by infection with any of the tested viruses (tables 1 and 2; figure 1a ). five or fewer proteins were significantly dysregulated by rv733 and the pdm09 strains, 15 were dysregulated by pr8, with all but one being significantly upregulated, and 38 and 47 were dysregulated by h5n1 and h7n9, respectively, with the vast majority of these proteins downregulated. several immune-regulating proteins, including isg15, oas1 and stat1 were upregulated by pr8 infection (table 2) , consistent with our previous ms-based proteomic analyses [10] . thrombopoietin and neural cell adhesion molecule l1 were the only proteins upregulated by rv733 and pdm09, respectively. few proteins were upregulated by the avian iav strains in the panel of 1310 somamers. c-c motif chemokine 5 (ccl5) and il-8 (cxcl8) were both upregulated by both h5n1 and h7n9 and a few other proteins were upregulated by pr8 and h5n1 (table 2) . pairwise comparisons revealed little correlation between the identities of proteins dysregulated by most virus strains, but an r 2 value of 0.77 was determined when h5n1-dysregulated proteins were compared to h7n9-dysregulated proteins ( figure 1b ,c). many common proteins were similarly statistically downregulated by both h5n1 and h7n9 and some of these, including fibronectin and laminin, had been identified previously as downregulated by itraq-based quantitative ms [13] . several immune-regulating proteins, including isg15, oas1 and stat1 were upregulated by pr8 infection (table 2) , consistent with our previous ms-based proteomic analyses [10] . thrombopoietin and neural cell adhesion molecule l1 were the only proteins upregulated by rv733 and pdm09, respectively. few proteins were upregulated by the avian iav strains in the panel of 1310 somamers. c-c motif chemokine 5 (ccl5) and il-8 (cxcl8) were both upregulated by both h5n1 and h7n9 and a few other proteins were upregulated by pr8 and h5n1 (table 2) . pairwise comparisons revealed little correlation between the identities of proteins dysregulated by most virus strains, but an r 2 value of 0.77 was determined when h5n1-dysregulated proteins were compared to h7n9-dysregulated proteins ( figure 1b,c) . many common proteins were similarly statistically downregulated by both h5n1 and h7n9 and some of these, including fibronectin and laminin, had been identified previously as downregulated by itraq-based quantitative ms [13] . expression values for all 1310 proteins in each infection were uploaded into the ingenuity pathway analysis (ipa) tool. for this we expanded consideration of dysregulated proteins to those significantly viruses 2019, 11, 1028 7 of 16 dysregulated >1.33-fold to increase the number of analyzed molecules to nearly 100. as reflected by the numbers of dysregulated proteins induced by each virus (tables 1 and 2; figure 1a ), pr8-infected a549 cells demonstrated a moderately global positive z-score, rv733 and pdm09-infected cells showed no significant positive or negative z-score, and both h5n1-and h7n9-infected cells showed overall negative z-scores ( figure 2) . these data imply that overall, the seasonal rv733 strain and the pdm09 strain have relatively mild effect, as measured by this small set of somamers, the pr8 strain has an overall activation effect, primarily of immune-modulated and cellular movement molecules, and the h5n1 and h7n9 strains have dramatic inhibitory effects upon multiple cellular processes. ipa network analyses also revealed differences in how each virus affected common cellular networks. the four highest scoring networks, based on numbers of focus molecules, were the cellular movement, hematological system development and function, immune cell trafficking, the antimicrobial response, cell death and survival, inflammatory response, the cardiovascular system development and function, embryonic development and organismal development and the post-translational modification, protein degradation and protein synthesis networks. the cellular movement, hematological system development and function, and the immune cell trafficking network was mildly affected by pr8. no proteins in this network were affected by rv733 or pdm09. apart from ccl5 and cxcl8, which were both upregulated by both h5n1 and h7n9, many proteins in this network were significantly downregulated by the two avian iav strains ( figure 3a ). the antimicrobial response, cell death and survival inflammatory response network was most significantly affected by pr8 infection, with numerous upregulated proteins ( figure 3b ). this'network also was mildly affected by rv733 infection, but not by pdm09 infection. three focus molecules in this network (cd207, isg15 and ifnl1) were upregulated and three (igfbp7, mica and pgd) were downregulated by h5n1 infection. cd207 was also upregulated and mica also was downregulated by h7n9 infection, and additionally, lgals3bp and rspo2 were downregulated by h7n9 infection. one or more proteins in the cardiovascular system development and function, embryonic development and organismal development network were upregulated and one or more were downregulated by every virus tested. however, many more proteins (such as fstl1, igfbp4, notch and stc) were downregulated by the h5n1 and h7n9 viruses than by any of the h1n1 viruses. furthermore, hdl was slightly upregulated in every h1n1 network, but downregulated in both the h5n1 and h7n9 networks. finally, there also were substantial differences in the post-translational modification, protein degradation and protein synthesis network. many more proteins (such as ctsa, ctsv, mfge8 and tnfrsf21) were downregulated by the h5n1 and h7n9 viruses than by any of the h1n1 viruses. furthermore, cathepsin was slightly upregulated in every h1n1 network, but downregulated in both the h5n1 and h7n9 networks. various bio-functions also were examined using the ipa ® default settings for these analyses (figure 4 ). bio-function activation is assumed for z-scores > 1.96σ, and bio-function inhibition is assumed for z-scores < −1.96σ. all of these indicated bio-functions also had significant p-values. pr8 activated many bio-functions, including cellular movement categories, inflammatory response, and angiogenesis, and inhibited few bio-functions; organ inflammation and anatomical organ inflammation. various bio-functions also were examined using the ipa ® default settings for these analyses (figure 4 ). bio-function activation is assumed for z-scores > 1.96σ, and bio-function inhibition is assumed for z-scores < −1.96σ. all of these indicated bio-functions also had significant p-values. pr8 activated many bio-functions, including cellular movement categories, inflammatory response, and angiogenesis, and inhibited few bio-functions; organ inflammation and anatomical organ inflammation. the avian iav inhibited far more bio-functions, including cell survival and viability; endocytosis and engulfment, immune response and the proliferation of numerous cell types. the avian iav strains activated few bio-functions, one of which was organismal death. we then used ipa to compare various cellular canonical signaling pathways ( figure 5) . the virus strains that induced the most profound cellular responses (pr8, h5n1 and h7n9) affected many canonical pathways. pr8 uniquely significantly activated interferon signaling, which has been reported previously [10, 11] , while h5n1 uniquely inhibited gluconeogenesis and glycolysis i, and all three strains had significant effects upon multiple pathways, including acute phase response signaling, neuroinflammation, role of pattern recognition of viruses, the complement system, and autophagy. the avian strains had the most dramatic effects upon multiple canonical pathways, including notch signaling, wnt/β-catenin signaling, epithelial adherens junction signaling and regulation of epithelial-mesenchymal transition. thus, collectively and overall, the h5n1 and h7n9 avian iav strains induced more profound inhibitory cellular responses than any of the h1n1 strains, consistent with our [13] and other′s [17] studies. the avian iav inhibited far more bio-functions, including cell survival and viability; endocytosis and engulfment, immune response and the proliferation of numerous cell types. the avian iav strains activated few bio-functions, one of which was organismal death. we then used ipa to compare various cellular canonical signaling pathways ( figure 5) . the virus strains that induced the most profound cellular responses (pr8, h5n1 and h7n9) affected many canonical pathways. pr8 uniquely significantly activated interferon signaling, which has been reported previously [10, 11] , while h5n1 uniquely inhibited gluconeogenesis and glycolysis i, and all three strains had significant effects upon multiple pathways, including acute phase response signaling, neuroinflammation, role of pattern recognition of viruses, the complement system, and autophagy. the avian strains had the most dramatic effects upon multiple canonical pathways, including notch signaling, wnt/β-catenin signaling, epithelial adherens junction signaling and regulation of epithelial-mesenchymal transition. thus, collectively and overall, the h5n1 and h7n9 avian iav strains induced more profound inhibitory cellular responses than any of the h1n1 strains, consistent with our [13] and other s [17] studies. influenza a virus (iav) remains a significant human pathogen that is constantly emerging and re-emerging. the virus' genetic make-up of eight segments of single-stranded rna allows for significant genetic plasticity. like other rna viruses that lack genetic proof reading, the highly errorprone rna-dependent rna polymerase leads to a high mutation rate (=antigenic drift), and the segmented genomes allows for segment mixing during co-infections, leading to emergence of new isolates (=antigenic shift). thus, vaccines need to be reformulated each year, and the process of attempting to anticipate future isolates often leads to vaccine mismatch [24] , which may be exacerbated by an individual′s immune history [25] . a limited repertoire of anti-viral compounds that either inhibit viral uncoating or viral release, have been approved, but viral mutation quickly arises during outbreaks [26, 27] . thus, there has been increasing interest to identify host factors that the virus may require for replication and pathogenicity to complement strategies that only target the virus. a number of studies, including genome-wide rnai screens, mrna microarray screens and yeast 2-hybid assays have identified >1500 cellular targets worthy of further analysis [28] [29] [30] [31] . numerous recent studies have used quantitative ms-based assays to probe the cellular proteome after perturbation by virus infection. these include 2-dimensional differences in gel electrophoresis (2d-dige) (ex. [32, 33] ), stable isotopic labeling by amino acids in cell culture (silac) (ex. [10, 34, 35] ), isobaric tags for relative and absolute quantitation (itraq) or tandem mass tags (tmt) (ex. [13, [36] [37] [38] ), and label-free methods [39] [40] [41] . each of these non-biased techniques provides information about thousands of proteins within complex mixtures, including cell extracts (reviewed in [42] [43] [44] ). however, each of these provide only a small sampling of the entire cellular proteome. most of these methods detect and measure the most abundant proteins within the mixture. furthermore, there is less than 100% overlap between any two sample runs, whether biologic runs or technical replicates of the same sample. alternate multiplex assays that target specific proteins have been developed and include the myriadrbm ® and luminex ® platforms. many of these are limited to fewer than 100 proteins that can be simultaneously assayed and measured, although a few, such as those offered by raybiotech ® , are reported to detect and measure several hundred influenza a virus (iav) remains a significant human pathogen that is constantly emerging and re-emerging. the virus' genetic make-up of eight segments of single-stranded rna allows for significant genetic plasticity. like other rna viruses that lack genetic proof reading, the highly error-prone rna-dependent rna polymerase leads to a high mutation rate (=antigenic drift), and the segmented genomes allows for segment mixing during co-infections, leading to emergence of new isolates (=antigenic shift). thus, vaccines need to be reformulated each year, and the process of attempting to anticipate future isolates often leads to vaccine mismatch [24] , which may be exacerbated by an individual s immune history [25] . a limited repertoire of anti-viral compounds that either inhibit viral uncoating or viral release, have been approved, but viral mutation quickly arises during outbreaks [26, 27] . thus, there has been increasing interest to identify host factors that the virus may require for replication and pathogenicity to complement strategies that only target the virus. a number of studies, including genome-wide rnai screens, mrna microarray screens and yeast 2-hybid assays have identified >1500 cellular targets worthy of further analysis [28] [29] [30] [31] . numerous recent studies have used quantitative ms-based assays to probe the cellular proteome after perturbation by virus infection. these include 2-dimensional differences in gel electrophoresis (2d-dige) (ex. [32, 33] ), stable isotopic labeling by amino acids in cell culture (silac) (ex. [10, 34, 35] ), isobaric tags for relative and absolute quantitation (itraq) or tandem mass tags (tmt) (ex. [13, [36] [37] [38] ), and label-free methods [39] [40] [41] . each of these non-biased techniques provides information about thousands of proteins within complex mixtures, including cell extracts (reviewed in [42] [43] [44] ). however, each of these provide only a small sampling of the entire cellular proteome. most of these methods detect and measure the most abundant proteins within the mixture. furthermore, there is less than 100% overlap between any two sample runs, whether biologic runs or technical replicates of the same sample. alternate multiplex assays that target specific proteins have been developed and include the myriadrbm ® and luminex ® platforms. many of these are limited to fewer than 100 proteins that can be simultaneously assayed and measured, although a few, such as those offered by raybiotech ® , are reported to detect and measure several hundred proteins. thus, at the time we initiated these studies, we selected an aptamer-based assay reported to reliably detect and measure more than 1000 proteins. these slow-off-rate modified aptamers (somamers ® ) were developed by somalogics, inc., (boulder, co, usa) [18] [19] [20] , and have been used to examine cancer biomarkers [45] , alzheimer s disease biomarkers [46] and biomarkers in iav-infected clinical nasal secretions [47] . our pilot studies with the first available version, capable of detecting 1128 proteins, reliably measured differences in influenza pr8-infected a549 cells over a time course. we subsequently used the next-generation somascan version 1.3, which measures >1300 proteins, to measure zika virus-induced proteomic alterations in vero cells [23] and in u251 astrocytoma cells [48] . application of this targeted aptamer-based approach, which was designed primarily to detect low-abundance proteins, thus provides a beneficial complementary approach to the non-biased ms-based approaches that tend to measure more abundant proteins. comparative analyses of dysregulated proteins we identified in this study to proteins identified in one of our earlier ms-based studies [10, 34, 35] showed generally good agreement. two proteins (isg15 and β-2-microglobulin) were identified as upregulated by both methods, 75 proteins were determined to not be significantly regulated by both techniques and no proteins were identified as significantly upregulated by one method, but significantly downregulated by the other. thus, this newer aptamer-based multiplexed system, designed primarily to detect and measure lower abundant proteins, provides complementary data to the more commonly-used ms-based approaches and to assay numerous proteins not normally detected by quantitative ms. the current study assayed 2-3 biologic replicates of five different iav strains, each compared to mock-infected samples (a total of 14 samples). these virus strains represent a lab-adapted h1n1 strain (pr8), a mild seasonal h1n1 strain related to the a/new caledonia/20/1999-like clinical isolate (rv733), the 2009 h1n1 pandemic strain (pdm09), and two strains that show significantly higher pathology in human patients; the h5n1 "bird flu" and the h7n9 strain. all tested virus strains affected the a549 cellular proteome, upregulating some proteins and downregulating other proteins. although none of the 1310 measurable common cellular proteins was significantly dysregulated by all five viruses, some proteins were similarly dysregulated by multiple viruses. for example, ccl5 was upregulated by pr8, h5n1 and h7n9, and pgam1 was significantly downregulated by pdm09, h5n1 and h7n9. the five viruses generated three overall patterns of dysregulation, at least as measured by the 1310 proteins that can be assayed by the somascan. pr8 caused an overall general activation, primarily of antimicrobial inflammatory and immune response, as reflected by a large upregulation of more than a dozen proteins, including isg15, stat1, oas1 and downregulation only of ppid. pr8 is a highly lab culture-adapted strain passaged multiple times in mice that is highly virulent in mice, but extremely attenuated in humans. rv733 and pdm09 had very little effect in our a549 cells, as we found in a previous quantitative ms study [13] , and are relatively low virulence, despite being well adapted to growth in humans. both the h5n1 and h7n9 viruses, which demonstrate the third pattern (significant downregulation of many more proteins than the other strains, particularly in the antimicrobial response, cardiovascular, and post-translational modification networks), and significant downregulation of many common proteins, are poorly adapted to humans because of their receptor specificity, but when successfully delivered into the lower human respiratory tract, they can be highly virulent. this more profound proteomic effect by these avian-derived viruses also was observed in quantitative transcriptomic [49] and ms studies [13] . genome-wide rnai screens and mrna microarray screens identified >1500 cellular genes and proteins influenza virus may depend upon [28] [29] [30] [31] . for example, konig and colleagues identified 295 genes that were required for influenza virus replication as defined by replicase activity [29] and karlas and colleagues identified 287 genes required for replication of two different influenza viruses, including viruses 2019, 11, 1028 13 of 16 the pdm09 strain [28] . collectively, these two rnai screens, also carried out in a549 cells, identified 552 potential genes important for influenza replication, with 30 genes found in common. the low level of overlap between these various rnai screens has been previously noted [50] . we assessed potential overlap between genes identified in the two karlas and konig a549 studies with proteins we could detect and measure in a549 cells with our somascan ® panel. only four genes/proteins (jun, kpnb1, map2k3 and mdm2) overlapped in all three datasets, and of these, only kpnb1 was significantly altered according to somascan ® ; downregulated 1.48-fold by h5n1 infection. thirty-one additional genes identified by karlas are in the somascan ® panel; of these, 14 were significantly dysregulated by at least one of our tested viruses. seven proteins were significantly dysregulated by both h5n1 and h7n9, but <1.35-fold. only one protein, b2m, was dysregulated >1.35-fold; it was upregulated 2.7-fold and only by pr8 infection. forty genes identified by konig, including the four found in all three datasets, are in the somascan ® panel; of these, 12 were significantly dysregulated by at least one of our tested viruses. fgfr4 was dysregulated, but less than 20%, by three viruses; pdm09, h5n1 and h7n9. only two of the 14 proteins were dysregulated >1.5-fold; app was downregulated 3.7-fold by h5n1 and 8-fold by h7n9, and fgfr1 was downregulated 2.1-fold by h7n9 infection. the 71 genes/proteins identified by karlas and/or konig and that were present in our somascan ® panel represent proteins involved in numerous diverse functions, including cytokines, enzymes, growth factors, transmembrane receptors and many kinases, as do many of the proteins newly identified in this somascan. thus, these different methods identify a large number of cellular genes and proteins that should be more extensively analyzed as potential targets to ameliorate influenza virus infection. some of our observed dysregulated proteins also were observed in a clinical analysis using the somascan platform. marion and colleagues found some of their most significantly differentially expressed proteins were ctsd, klk7, mfge8, mapk9 and cd27 [47] . mfge8 (lactadherin) was significantly downregulated only by h5n1 and h7n9 in our study. although ctsd (cathepsin d), klk7 (kallikrein-7), mapk9 (mitogen activated protein kinase 9) and cd27 antigen were not significantly affected by any of our tested viruses, other cathepsin isoforms, ctsv (cathepsin l2) was significantly downregulated by h7n9, and ctss was significantly upregulated by pr8, klk5 was upregulated by pdm09, but only 1.4-fold, these differences probably relate to study design; marion assessed clinical nasal swabs and we examined a549 cell extracts. the a549 cell is a transformed adenocarcinoma cell line derived from an explanted tumor from a 58-year-old caucasian male. while this study provides some important information about how multiple iav strains induce changes in the cellular proteome of these cells, it will be important to perform similar assays in more physiologically-relevant primary cells to allow the comparison and possible identification of common cellular processes that might be amenable to therapeutic intervention. in conclusion, we used a targeted aptamer-based approach to complement earlier quantitative ms approaches to compare host cell responses to viruses that have differential host pathology. we found that the culture-adapted pr8 strain had an overall activation of immune molecules, the mild seasonal and pdm09 human strains had little 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an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank neil salter for expert technical assistance and gao ang for somascan analyses. we also thank nathalie bastien and yan li for use of the phac bsl3 laboratory and for providing us the a/anhui/1/2013 h7n9 isolate. the authors declare no conflict of interest. key: cord-260452-js4nr4d8 authors: yu, junyang; wu, yuzhang; wang, jingxue title: activation and role of nacht, lrr, and pyd domains-containing protein 3 inflammasome in rna viral infection date: 2017-10-31 journal: front immunol doi: 10.3389/fimmu.2017.01420 sha: doc_id: 260452 cord_uid: js4nr4d8 nacht, lrr, and pyd domains-containing protein 3 (nlrp3) inflammasome activation and effects during ribonucleic acid (rna) viral infection are the focus of a wide range of research currently. both the pathogen-associated molecule pattern derived from virions and intracellular stress molecules involved in the process of viral infection lead to activation of the nlrp3 inflammasome, which in turn triggers inflammatory responses for antiviral defense and tissue healing. however, aberrant activation of the nlrp3 inflammasome can instead support viral pathogenesis and promote disease progression. here, we summarize and expound upon the recent literature describing the molecular mechanisms underlying the activation and effects of the nlrp3 inflammasome in rna viral infection to highlight how it provides protection against rna viral infection. the ribonucleic acid (rna) virus has single-stranded (ss)rna or double-stranded (ds)rna as its genetic material. infections with rna viruses are responsible for a variety of diseases are significant threats to public health, including influenza, hepatitis, viral encephalitis, and autoimmune deficiency syndrome, to name a few of the most prominent. host antiviral defense mechanisms are critical for clearing rna viral infections and controlling the manifested diseases. the initiation of antiviral defense in response to rna viruses involves interferon (ifn) response and inflammasome activation. the rna viral infection usually leads to activation of the nacht, lrr, and pyd domainscontaining protein 3 (nlrp3) inflammasome, which is the intracellular multiprotein complex formed upon sensing pathogen-associated molecule patterns and/or damage-associated molecule patterns (1) . the inflammasome response is critical for the innate immune response's ability to initiate innate and adaptive immunity pathways during rna viral infections (2) . the nlrp3 inflammasome has been extensively investigated. its major components include pro-caspase-1 and apoptosis-associated speck-like protein containing a card (asc), in addition to the nlrp3 molecules. upon sensing activating signals, the nlrp3 oligomerize to generate a caspase-1-activating scaffold, resulting in autocleavage, and activation of pro-caspase-1. the activated caspase-1 then shears pro-interleukin (il)-1β and pro-il-18 into il-1β and il-18, leading to production of their functional forms and release from cells to mediate protective (or sometimes detrimental) inflammatory responses. the activated caspase-1, however, can also promote caspase-1-dependent pyroptosis, a type of inflammatory programmed cell death; although, the consequence of pyroptosis induced by the nlrp3 inflammasome remains to be fully elucidated in rna viral infection (3) . table 1 . this review will focus on recent advances in our knowledge of the activation mechanisms and functions of the nlrp3 inflammasome in response to such rna viral infections, discussing the related protective, and/or detrimental mechanisms that may represent manipulable targets for prevention and/or treatment. priming mechanisms of the nlrp3 inflammasome in rna viral infections nacht, lrr, and pyd domains-containing protein 3 inflammasome activation requires two types of signals. signal 1 primes the nlrp3 inflammasome and signal 2 initiates the sequence of its assembly, activation of the pro-caspase-1, and cleavage of pro-il-1β and pro-il-18. during rna viral infections, signal 1 is usually derived from the signals evoked upon pathogen recognition by toll-like receptors (tlrs) (6) and retinoic acid-inducible gene-i (rig-i)-like receptors (41) . recent research has shown that signal 1 triggering of the nlrp3 inflammasome involves transcriptiondependent and post-translation-dependent priming. the transcription-dependent priming is executed by activation of nuclear factor (nf)-κb and ifn-regulatory factor transcription factors, and subsequent up-regulation of transcription of genes encoding the major inflammasome components through the classical tlr signaling pathway: the myd88-mediated and trif-mediated pathway. the specific role of tlr7 was shown by the reduced pro-il-1β transcription in bone marrow-derived macrophages from tlr7 −/− mice in response to 24 h of iav infection (6) . another in vivo study of the iav infection model provided evidence that commensal bacteria lipopolysaccharide can provide priming signal 1 (42) . investigations of vsv infection showed that rig-i promotes the synthesis of pro-il-1β through engagement by the viral 5′-triphosphate rna (3prna). moreover, bone marrow-derived cells lacking rig-i, as well as the downstream molecules mavs and card9, showed reduced synthesis of pro-il-1β but normal caspase-1 p10 subunits when stimulated with vsv and 3prna (41) , suggesting that the rig-i/mavs/card9 signaling pathway provides the signal 1 in vsv infection. post-translation-dependent priming occurs upon the simultaneous engagement of tlrs and nod-like receptors with their ligands, even causing rapid activation of the nlrp3 inflammasome (within 15 min) (43, 44) . viral rna (vrna) moleculeinduced nlrp3 inflammasome activation also comes about by post-translational priming, but involving the rip1/caspase 8/rip3 signaling pathway (29, 45) , which may ultimately result in the deubiquitination of nlrp3 protein by brcc3 (46, 47) . rip3-deficient mice showed reduced caspase-1 activation and il-1β production following infection with vsv, sendai virus, or iav (29) . bone marrow-derived macrophages and mice lacking rip1 both displayed substantial reduction in caspase-1 cleavage and levels of il-1β and il-18 following challenge with iav (8, 29) . in various rna viral infection studies, the rip1/ rip3-mediated signaling pathway has been demonstrated to provide the endogenous signal 2 for the nlrp3 inflammasome, being derived from reactive oxygen species (ros) production by mitochondrial fission initiated by the rip1/rip3/drp pathway (29, 45, 48) . signal 2 is responsive to a wide range of stimuli [i.e., nlrp3 agonists, such as adenosine triphosphate (atp), silica, alum, and ultraviolet radiation]. however, the spectrum of different structures represented by these varied and distinctive agonists indicates that nlrp3 does not simply bind directly to each but instead relies on a common intracellular signal transmission pathway, triggered by the agonists themselves, for activation of the nlrp3 inflammasome. to date, five major signal mechanisms have been proposed as those responsible for or contributing to the nlrp3 inflammasome activation (1, 49) . in rna viral infections, in particular, viroporins and some viral functional proteins act as the stimuli for signal 2 (as shown in table 1) . viroporins are a group of virus-encoded proteins that enhance permeability of host cell membrane (through interactions with the lipid bilayer) to promote release of viral particles from cells (50) , making them a critical component of virus replication and virion release in the host system. the viroporin effect on permeability also modifies the cell's ability to regulate ion passage, disrupting ion homeostasis (51) . several viroporins, such as the iav m2 protein (6), rsv sh protein (15) , hcv p7 protein (21), sars-cov e protein (25), emcv 2b protein (27) , and rhinovirus 2b protein (32) , have been reported to activate the nlrp3 inflammasome by disturbing intracellular ionic concentrations, particularly through potassium efflux, calcium flux, and ph alteration. in the viroporin-induced nlrp3 activation itself, the host-encoded nek7 protein may contribute to the nlrp3 inflammasome assembly and activation via formation of the nlrp3-nek7 complex (52) . among the other viral proteins involved in nlrp3 inflammasome activation are iav pb1-f2 (a small protein encoded by an alternate + 1 open reading frame in the viral pb1 gene) (7) and ev71 3d protein (an rna-dependent rna polymerase) (34) . pb1-f2 activates the nlrp3 inflammasome through mitochondrial ros production, which could be a major mechanism of pathogenic inflammation induced by highly pathogenic iav strains (53) . the zbp1 protein can interact with rip3 upon sensing the viral protein pb1, and contributes to nlrp3 inflammasome activation in iav but not in vsv infection through post-translational regulation (8) . in contrast, the ev71 3d protein can directly bind to nlrp3 and asc, and promotes nlrp3 inflammasome assembly and activation (34) . plasma vrna, another common agonist for nlrp3, may bind with nlrp3 and act to subsequently activate it. although nlrp3 contains a nucleotide-binding domain (54), rna-sensing molecules have been reported to participate in the process by which vrna activates the nlrp3 inflammasome; these include the dexd/h-box helicase (dhx) family members (14, 41, 55, 56) and the 2′,5′-oligoadenylate synthetase (oas, or 2-5a)/rnase l system (30) . however, some debate exists as to the exact role of dhx33, in particular, in vsv-induced nlrp3 inflammasome activation (29) . the dhx family members contain some domains required for atp binding, hydrolysis, and nucleic acid binding and unwinding, which usually acts as a cytosolic rna sensor and triggers type i ifn response. dhx33, ddx19a, and ddx58 have been reported to regulate nlrp3 inflammasome activation. dhx33 was shown to combine directly with vrna to bind to nlrp3 through the dead domain of dhx33 and the nacht domain of nlrp3, forming a dhx33/nlrp3/asc inflammasome complex to promote nlrp3 inflammasome activation (14) . in that same study, down-regulation of dhx33 expression led to a substantial reduction in cleavage of caspase-1 and secretion of il-18 and il-1β from the human monocyte cell line, thp-1, upon stimulation with viral dsrna purified from reovirus, and produced by rsv during the replication process (14) . dhx33 has also been demonstrated to contribute to nlrp3 inflammasome activation and il-1β secretion during vsv and iav infections (30) , the process of which was found to be dependent upon the oas/rnase l system induced by ifn. oass recognize cytosolic viral dsrna and subsequently trigger synthesis of 2-5a in the presence of three atp molecules. then, rnase l is activated by 2-5a and cleaves intracellular vrna. these rnase l-mediated rna cleavage products will directly interact with dhx33 and induce a dhx33/mavs/nlrp3 complex to trigger assembly of the nlrp3 inflammasome. another study demonstrated that ddx19a binds porcine reproductive and respiration syndrome virus (prrsv) genomic rna through its atp-binding domain and c-terminal domain and directly interacts with the nlrp3 inflammasome in highly pathogenic-prrsv-infected primary porcine alveolar macrophages, dependent on the ddx19a atp-binding domain, and figure 1 | activation and effects of nacht, lrr, and pyd domainscontaining protein 3 (nlrp3) inflammasome in response to ribonucleic acid (rna) virus infections. viroporin, viral nucleic acids, and some virus-encoded functional proteins are able to activate the nlrp3 inflammasome. viroporin-mediated ion concentration change, virus replication-produced reactive oxygen species, and even viral protein direct binding promote assembly of the nlrp3 inflammasome. the cytokines interleukin (il)-1 and il-18 contain extensive downstream effector molecules and effector cells, which ultimately orchestrate innate and adaptive immunity, leading to antiviral defense, and/or inflammatory injury during rna virus infections. the nlrp3 nacht and lrr domains. knockdown of ddx19a expression led to reductions in both il-1β secretion and virus titers in the prrsv-infected porcine alveolar macrophages (56) . thus, ddx19a appears to be required for both antiviral defense and nlrp3 inflammasome activation. rig-i (ddx58) is a key intracellular sensor for cytosol ssrna and provides the transcriptional priming signal for nlrp3 inflammasome during infections with vsv (41) and iav (55) , upon recognition of the vrna. however, in primary respiratory epithelial cells, iav infection promotes rig-i binding with asc and caspase-1, implying that rig-i contributes to inflammasome assembly (55) . during the process of rna viral infection, nlrp3 inflammasome activation contributes to either enhancing antiviral defense and tissue healing, or inflammatory pathological injury, which depends mainly on the time point and extent of nlrp3 activation. the appropriate and early phase activation of the nlrp3 inflammasome in some rna viral infection systems, such those of iav, encephalomyocarditis virus, and west nile virus (wnv) (shown in table 1 ), usually provide protection, thereby decreasing mortality and viral load, as shown in virus-infected mouse models (4, 9, 16, 57) . in rhesus monkeys, neither nlrp3 inflammasome activation nor ifn response can be established successfully within 24 h following challenge with the simian immunodeficiency virus, which may be the key reason underlying the observation of viral dissemination occurring rapidly from the inoculation site to a distal site (within the first day of infection) (58) . reciprocally, when using hiv-1-specific broadly neutralizing antibodies, pgt121 is capable of inducing innate immune responses such as nlrp3 inflammasome activation in vrna-positive tissues within 24 h; in addition, viral replication is reduced in distal tissues significantly (59) . these findings imply the early nlrp3 inflammasome activation as well as ifn response play critical roles in establishment of effective antiviral defense. similarly, the administration of mc9550 (a specific nlrp3 inhibitor) on days 1, 3, and 5 but not days 3-5 following iav challenge also resulted in accelerated weight loss and mortality in iav-infected mice (9) . at 2 weeks after challenge with iav, the mortality rates among nlrp3 −/− , caspase-1 −/− (4, 5), and il-1ri −/− (57) mice are significantly higher (by 60-80%) than for the wild-type counterparts. thus, proper nlrp3 inflammasome activation in the early phase of rna viral infection protects the infected host. in contrast, if activated at a later phase or over-activated, the nlrp3 inflammasome response to rna virus results in inflammatory injury. production of il-1β and il-18, and pyroptosis are the critical and fundamental reactions directly induced by activated nlrp3 inflammasome (figure 1) . il-1β and il-18 serve to activate myriad downstream cell responses, and orchestrate innate and adaptive immunity through myd88/irak4/traf6-mediated nf-κb signaling and the jnk/p38 mitogen-activated protein kinase pathways (60-63), which may represent key events for the nlrp3 inflammasome-dependent antiviral defense. pyroptosis is a pro-inflammatory caspase-dependent programed cell death (64). nlrp3 inflammasome-dependent pyroptosis has been described in hcv-infected hepatoma cells (65) , dengue virus (dv)-infected monocytes (66) , and hiv-infected podocytes (10) . the data to date suggest that pyroptosis in viral infection leads to host injury and pathogenesis. in mice, iav-induced nlrp3 inflammasome activation initiates protective inflammation by recruitment of monocytes and neutrophils to the lung (as noted in the bronchoalveolar lavage fluid), which occurs within 3-7 days of the iav infection, and the subsequent promotion of secretion of various cytokines, such as tumor necrosis factor (tnf)-α, ifn-α, and mip-2α (4, 5) . furthermore, the impaired il-1 response by il-1ri deficiency results in impaired production of immunoglobulin m, reduced recruitment of cd4 + t cells in the bronchoalveolar lavage fluid (57) , and impaired priming of cd8 + t cells in the lung and draining mesenteric lymph nodes through the intervention of cd11b lo cd103 + dendritic cell migration and maturation (67) . in a recent study, il-18 was confirmed to be required for ifn-γ production in cd161 + vα7.2 + mucosal-associated invariant t cells, which has been observed clinically to be increased in recovered but not succumbed patients hospitalized with avian h7n9 influenza pneumonia and which implies a protective role in influenza virus infection (68) . in wnv infected mice, the il-1β produced by cd45 + cd11b + infiltrating t cells promotes the wnv-specific cd8 + t cell entry to the central neuronal system nlrp3 inflammasome induced by rna virus frontiers in immunology | www.frontiersin.org october 2017 | volume 8 | article 1420 via regulation of cxcl12-mediated t cell adhesion, eventually controlling the viral infection (16, 17) . in bv-2 mouse microglia cells infected by japanese encephalitis virus, the nlrp3 inflammasome induces production of il-1β and il-18 rapidly (within 3 h of exposure) and of tnf-α, ccl2, and il-6 later (within 6 h after exposure) (40) ; the findings suggest that the nlrp3dependent protective inflammatory response is a very early phase innate immune response against rna viral infection. in addition to initiating protective inflammation to regulate and orchestrate innate and adaptive immunity, the nlrp3 inflammasome also promotes tissue healing and reduces respiratory epithelial necrosis, which contributes to enhancing host disease tolerance (5, 69) . thus, the nlrp3 inflammasome provides protective roles through enhancing host tolerance capacity and orchestrating innate immunity and adaptive immunity during rna viral infections. aberrant nlrp3 inflammasome activation usually results in progression of viral pathogenesis and the manifested disease in host, such as the robust nlrp3 activation induced by pb1-f2 during the iav infection (7). the major pathogenic mechanism associated with pb1-f2-induced aberrant nlrp3 inflammasome activation involves excessive neutrophil influx (7, 53) . the nlrp3 activation induced by hcv promotes chronic intrahepatic inflammation in macrophages (18) and interrupts cellular lipid metabolism (20) in hcv-infected hepatoma cells, both of which contribute to chronic liver injury and liver disease progression. in hiv-infected microglia, nlrp3 inflammasome activation is involved in the occurrence of chronic neuroinflammation (12) . in addition, nlrp3-dependent pyroptosis during rna viral infection also supports the pathogenesis of virus infection diseases. hiv-induced podocyte pyroptosis could be associated with hiv-associated nephropathy (10) . nlrp3 inflammasome-mediated pyroptosis is also observed in dv-infected monocytes (66) and in hcv-infected and bystander hepatoma cells (65) , and could be associated with viral pathogenesis ( table 1) . unlike rna viruses, the dna viruses, which have a large dna genome, can encode viral homologs of some complex intracellular inflammasome-regulating molecules to regulate inflammasome activity; an example of this is the pyrin-only proteins and bcl-2 encoded by poxvirus (70, 71) . the rna viruses, in contrast, have evolved some simple yet effective strategies to inhibit activation of the nlrp3 inflammasome and evade host immunity. specifically, the proteases 3c and 2a of ev71 are able to cleave nlrp3 at the g493-l494 or q225-g226 junction (34) . furthermore, the protease 3c of ev71 is capable of degrading gasdermin d, a protein critical to the induction of pyroptosis (72) , and host failure to inhibit ev71 replication (73) . the ns1 protein of influenza virus can bind with nlrp3 directly, thereby inhibiting assembly of the nlrp3-asc-caspase-1 complex and secretion of il-1β; this inhibition is dependent on rna and the trim-25 domain of the ns1 protein (74) . the measles virus v protein also targets nlrp3, probably serving to interfere with the nlrp3 inflammasome assembly and ultimately facilitating escape from the host immune response (75) . finally, the non-structural protein 11 encoded by prrsv also antagonizes the nlrp3 inflammasome effect in the macrophages, through decreasing expression of pro-il-1β (76). nacht, lrr, and pyd domains-containing protein 3 inflammasome activation in response to rna virus is a fundamental innate immune response as well as an ifn response, which usually contributes to antivirus defense, although aberrant and inappropriate nlrp3 inflammasome response still results in the immunopathology and exaggeration of infectious disease, especially in some persistent virus infections. the activation itself is induced by different signals and probably occurs in different phases of the viral infection, triggering inflammatory response (appropriate/protective or inappropriate/detrimental), and affecting outcome of the viral infection substantially. thus, the proper regulation of nlrp3 inflammasome activation with specific nlrp3 inhibitors may have therapeutic benefit in controlling virus-related diseases and in clearance of the infecting virus. jy collecting literatures and drafting manuscript. jw design of the work, collecting literatures, drafting, and revising manuscript. yw discussing literatures, design of the work, and revising manuscript. this work was supported by grants from the national natural science foundation of china (grant no. 31670899 and 31070796) and the natural science foundation of chongqing (grant no. cstc, 2011bb5029). the inflammasomes inflammasome control of viral infection viruses and the diversity of cell death the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the intracellular sensor nlrp3 mediates key innate and healing responses to influenza a virus via the regulation of caspase-1 frontiers in immunology | www.frontiersin.org october influenza virus activates inflammasomes via its intracellular m2 ion channel activation of the nlrp3 inflammasome by iav virulence protein pb1-f2 contributes to severe pathophysiology and disease zbp1/dai is an innate sensor of influenza virus triggering the nlrp3 inflammasome and programmed cell death pathways reassessing the role of the nlrp3 inflammasome during pathogenic influenza a virus infection via temporal inhibition hiv promotes nlrp3 inflammasome complex activation in murine hiv-associated nephropathy hiv-1 tat primes and activates microglial nlrp3 inflammasome-mediated neuroinflammation hiv-1 viral protein r activates nlrp3 inflammasome in microglia: implications for hiv-1 associated neuroinflammation interleukin 18 coexpression during respiratory syncytial virus infection results in enhanced disease mediated by natural killer cells the dhx33 rna helicase senses cytosolic rna and activates the nlrp3 inflammasome human respiratory syncytial virus viroporin sh: a viral recognition pathway used by the host to signal inflammasome activation il-1beta signaling promotes cns-intrinsic immune control of west nile virus infection il-1r1 signaling regulates cxcl12-mediated t cell localization and fate within the central nervous system during west nile virus encephalitis il-1beta production through the nlrp3 inflammasome by hepatic macrophages links hepatitis c virus infection with liver inflammation and disease hcv genomic rna activates the nlrp3 inflammasome in human myeloid cells the hepatitis c virus-induced nlrp3 inflammasome activates the sterol regulatory element-binding protein (srebp) and regulates lipid metabolism the p7 viroporin of the hepatitis c virus contributes to liver inflammation by stimulating production of interleukin-1beta platelets mediate increased endothelium permeability in dengue through nlrp3-inflammasome activation resistance of interleukin-1beta-deficient mice to fatal sindbis virus encephalitis the inflammatory cytokine, interleukin-1 beta, mediates loss of astroglial glutamate transport and drives excitotoxic motor neuron injury in the spinal cord during acute viral encephalomyelitis effect of interleukin-18 on viral myocarditis: enhancement of interferon-gamma and natural killer cell activity encephalomyocarditis virus viroporin 2b activates nlrp3 inflammasome the nlrp3 inflammasome detects encephalomyocarditis virus and vesicular stomatitis virus infection rna viruses promote activation of the nlrp3 inflammasome through a rip1-rip3-drp1 signaling pathway rnase l activates the nlrp3 inflammasome during viral infections rhinovirus infection of primary cultures of human tracheal epithelium: role of icam-1 and il-1beta rhinovirus-induced calcium flux triggers nlrp3 and nlrc5 activation in bronchial cells reciprocal regulation between enterovirus 71 and the nlrp3 inflammasome ev71 3d protein binds with nlrp3 and enhances the assembly of inflammasome complex stimulation of host-defense mechanism with synthetic adjuvants and recombinant cytokines against viral infection in mice critical role for cryopyrin/nalp3 in activation of caspase-1 in response to viral infection and double-stranded rna zika virus induces inflammasome activation in the glial cell line u87-mg involvement of nlrp3 inflammasome in cvb3-induced viral myocarditis japanese encephalitis virus differentially modulates the induction of multiple pro-inflammatory mediators in human astrocytoma and astroglioma cell-lines nlrp3 inflammasome: key mediator of neuroinflammation in murine japanese encephalitis recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production microbiota regulates immune defense against respiratory tract influenza a virus infection cutting edge: tlr signaling licenses irak1 for rapid activation of the nlrp3 inflammasome irak-1 bypasses priming and directly links tlrs to rapid nlrp3 inflammasome activation caspase-8 scaffolding function and mlkl regulate nlrp3 inflammasome activation downstream of tlr3 non-transcriptional priming and deubiquitination regulate nlrp3 inflammasome activation deubiquitination of nlrp3 by brcc3 critically regulates inflammasome activity the rip1-rip3 complex initiates mitochondrial fission to fuel nlrp3 regulation of inflammasome activation viroporins: structure and biological functions nek7 is an essential mediator of nlrp3 activation downstream of potassium efflux pb1-f2 derived from avian influenza a virus h7n9 induces inflammation via activation of the nlrp3 inflammasome cryopyrin/nalp3 binds atp/datp, is an atpase, and requires atp binding to mediate inflammatory signaling type i ifn triggers rig-i/tlr3/nlrp3-dependent inflammasome activation in influenza a virus infected cells ddx19a senses viral rna and mediates nlrp3-dependent inflammasome activation interleukin-1 is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection rapid inflammasome activation following mucosal siv infection of rhesus monkeys antibodymediated protection against shiv challenge includes systemic clearance of distal virus the role of il-18 in innate immunity the il-1 family: regulators of immunity interleukin-1 (il-1) pathway molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases hepatitis c virus infection of cultured human hepatoma cells causes apoptosis and pyroptosis in both infected and bystander cells dengue virus-infected human monocytes trigger late activation of caspase-1, which mediates pro-inflammatory il-1beta secretion and pyroptosis il-1r signaling in dendritic cells replaces pattern-recognition receptors in promoting cd8(+) t cell responses to influenza a virus human mucosal-associated invariant t cells contribute to antiviral influenza immunity via il-18-dependent activation mx1 reveals innate pathways to antiviral resistance and lethal influenza disease pyrin-and card-only proteins as regulators of nlr functions inflammasomes and its importance in viral infections cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death enterovirus 71 inhibits pyroptosis through cleavage of gasdermin d the rna-and trim25-binding domains of influenza virus ns1 protein are essential for suppression of nlrp3 inflammasome-mediated interleukin-1beta secretion measles virus v protein inhibits nlrp3 inflammasome-mediated interleukin-1beta secretion the endoribonuclease activity essential for the nonstructural protein 11 of porcine reproductive and respiratory syndrome virus to inhibit nlrp3 inflammasome-mediated il-1beta induction key: cord-007853-5xnft6pd authors: lai, yanni; yan, yiwen; liao, shanghui; li, yun; ye, yi; liu, ni; zhao, fang; xu, peiping title: 3d-quantitative structure–activity relationship and antiviral effects of curcumin derivatives as potent inhibitors of influenza h1n1 neuraminidase date: 2020-04-04 journal: arch pharm res doi: 10.1007/s12272-020-01230-5 sha: doc_id: 7853 cord_uid: 5xnft6pd curcumin derivatives have been shown to inhibit replication of human influenza a viruses (iavs). however, it is not clear whether curcumin and its derivatives can inhibit neuraminidase (na) of influenza virus. in this study, a meaningful 3d quantitative structure–activity relationship model (comparative molecular field analysis r(2) = 0.997, q(2) = 0.527, s = 0.064, f = 282.663) was built to understand the chemical–biological interactions between their activities and neuraminidase. molecular docking was used to predict binding models between curcumin derivatives and neuraminidase. real-time polymerase chain reactions showed that the five active curcumin derivatives might have direct effects on viral particle infectivity in h1n1-infected lung epithelial (mdck) cells. neuraminidase activation assay showed that five active curcumin derivatives decreased h1n1-induced neuraminidase activation in mdck cells. indirect immunofluorescence assay indicated that two active curcumin derivatives (tetramethylcurcumin and curcumin) down-regulated the nucleoprotein expression. curcumin inhibited iav in vivo. the therapeutic mechanism of curcumin in the treatment of influenza viral pneumonia is related to improving the immune function of infected mice and regulating secretion of tumor necrosis-α, interleukin-6, and interferon-γ. these results indicate that curcumin derivatives inhibit iav by blocking neuraminidase in the cellular model and curcumin also has anti-iav activity in the animal model. influenza a virus (iav), a type of orthomyxoviridae virus responsible for frequent seasonal epidemics, is closely related to human health (taubenberger and morens 2008; scalera and mossad 2009) . it causes acute respiratory disease by infection of the upper respiratory tract mucosa (taubenberger and morens 2008) . the glycoproteins hemagglutinin (ha) and neuraminidase (na) are embedded in the envelope of the influenza virus, and play an important role in the invasion of host cells and viral transmission (smith et al. 2009 ). in the procession of viral shedding, neuraminidase cuts the sialic acid linkage formed between the sialic acid receptor on the surface of the host cell and the hemagglutinin to promote viral replication (nayak and jabbar 1989; ohuchi et al. 2006) . hence, neuraminidase is considered to be an important target for treatment of influenza (ohuchi et al. 2006) . currently, amantadine, oseltamivir, and zanamivir are used for treatment of influenza a (pica and palese 2013) , including inhibition of the matrix protein m2 (amantadine) and na (oseltamivir and zanamivir). m2 ion channel blockers only inhibit iav and have potential neurotoxicity (zhao et al. 2012) . neuraminidase inhibitors (nais) prevent infected cells from releasing and transmitting progeny virions during the replication cycle by binding to the active site of viral neuraminidase (belser et al. 2015) . however, in recent years, because of the widespread use of these drugs, resistant strains of iav have emerged (hurt et al. 2009; moscona 2009; cho et al. 2013) . therefore, it is urgent to abstract curcumin derivatives have been shown to inhibit replication of human influenza a viruses (iavs). however, it is not clear whether curcumin and its derivatives can inhibit neuraminidase (na) of influenza virus. in this study, a meaningful 3d quantitative structure-activity relationship model (comparative molecular field analysis r 2 = 0.997, q 2 = 0.527, s = 0.064, f = 282.663) was built to understand the chemical-biological interactions between their activities and neuraminidase. molecular docking was used to predict binding models between curcumin derivatives and neuraminidase. real-time polymerase chain reactions showed that the five active curcumin derivatives might have direct effects on viral particle infectivity in h1n1-infected lung epithelial (mdck) cells. neuraminidase activation assay showed that five active curcumin derivatives decreased h1n1-induced neuraminidase activation in mdck cells. indirect immunofluorescence assay indicated that two active curcumin derivatives (tetramethylcurcumin and curcumin) down-regulated the nucleoprotein expression. curcumin inhibited iav in vivo. the therapeutic mechanism of curcumin in the treatment of influenza viral pneumonia is related to improving the immune function of infected mice and regulating secretion of tumor necrosis-α, interleukin-6, and interferon-γ. these results indicate that curcumin derivatives inhibit iav by blocking neuraminidase in the cellular model and curcumin also has anti-iav activity in the animal model. identify new antiviral drugs that are effective against influenza viruses. due to its versatile pharmacological properties, including anti-inflammatory, anti-tumor, and anti-oxidant activities (maheshwari et al. 2006; anand et al. 2007; goel et al. 2007 ), curcumin has attracted considerable interest. in particular, curcumin is reported to have anti-influenza virus activity by interfering with the cellular signaling pathways (such as toll-like receptor2/4, p38/c-jun n-terminal kinase, mitogen-activated protein kinase, and nuclear factor-κb pathways) or with proteins required for influenza virus replication (dai et al. 2018; richart et al. 2018) . ou et al. (2013) reported that the presence of the double bonds in the central seven-carbon chain enhances the curcumin-dependent anti-iav activity and curcumin might interfere with iav entry by its interaction with the receptor binding region of hemagglutinin. chen et al. (2013) also reported that curcumin can inhibit influenza virus by disrupting integrity of the viral envelope and liposomal membranes. however, it is not clear whether curcumin and its derivatives can inhibit neuraminidase of influenza virus. in the present study, we evaluated the antiviral effects of curcumin derivatives as potent inhibitors of influenza h1n1 neuraminidase based on 3d quantitative structure-activity relationship (qsar) (duarte et al. 2019) , molecular docking (nilakantan et al. 1993 ) and biological activity evaluation both in vitro and in vivo. influenza virus strain, a/font monmouth/47(h1n1, fm1), adapted to mice, was originally obtained from chinese center for disease control and prevention. the virus was adapted to replication in the lungs of balb/c mice by eight sequential passages through mouse lungs. the inoculated virus was plaque-purified in mdck cells, then replicated in 9-day-old chicken embryos and stored at− 80 °c. the virus titer was determined as 50% tissue culture infectious dose (tcid 50 /ml) in confluent cells of 96-well microtiter plates (greiner bio-one, frickenhausen, germany). madin-darby canine kidney (mdck) cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), 100 iu/ml penicillin and 100 mg/ml streptomycin in a 5% co 2 incubator. specific-pathogen-free female nih mice weighing 13-15 g (medical experiment center of guangdong province, guangzhou, china) were used after 24 h quarantine, and were fed standard mouse chow and tap water ad libitum for the duration of the studies. the animals were housed in the guangdong association for the accreditation of laboratory animal care-accredited guangzhou university of chinese medicine (gzucm) laboratory animal research center. all experimental protocols were approved by the gzucm animal care and welfare committee. dihydrocurcumin, tetramethylcurcumin, bisdemethoxycurcumin, curcumin-d6, demethoxycurcumin, demethylcurcumin, dimethylcurcumin, curcumin, tetrahydrocurcumin, octahydrocurcumin, hexahydrocurcumin and oseltamivir carboxylate were acquired from shanghai yuanye bio-technology co. ltd. (shanghai, china) . purity was ≥ 99% by high-performance liquid chromatography (hplc). confluent mdck cells were treated with a series of graded concentrations of curcumin derivatives for 24 h. confluent mdck cells were treated with curcumin derivatives for 24 h. cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (mtt) method. the median cytotoxic concentration (cc 50 ) was calculated as the concentration of the compound that reduced the number of viable cells to 50% of the viability of the untreated controls. the maximal non-cytotoxic concentration was defined as the maximal concentration of the sample that did not exert a cytotoxic effect and resulted in > 90% viable cells. influenza virus na activity was measured by neuraminidase inhibitors screening kit. neuraminidase inhibitors screen kit (no. p0309) was purchased from beyotime institute of biotechnology co. ltd. (shanghai, china), which contained 10 ml buffer, 1 ml na, 1 ml fluorescent substrate and 1.2 ml milli-q water. we selected compounds with quantitative ic 50 values as data set, and divided the 11 compounds randomly into a training set (nine compounds) and a testing set (three compounds). we converted the ic 50 values to pic 50 values with the formula pic 50 = δlog ic 50 . we used the pic 50 values to develop the 3d-qsar model by combining the activity data with the generated hypothesis. the chemical structures and biological activities of the 11 compounds are listed in table 1 . we used to derive the comparative molecular field analysis (comfa) descriptor fields, we generated a 3d cubic lattice from the space covering the nine aligned compounds. the standard tripos force fields were used to calculate the space and electrostatic potential fields of the comfa. to establish quantitative relationships between biological activities and molecular descriptors in comfa, we used partial least squares regression analysis (golbraikh and tropsha 2002) . the leave one method was used for cross-validation analysis (golubović et al. 2018) . then, non-cross-validation was performed to obtain a conventional correlation coefficient r 2 and estimated standard errors. to enhance the predictive ability, the region focusing method was used in the comfa model (xu et al. 2016) . to predict the power of the 3d-qsar model, we verified by the test set compounds (compounds marked * in table 1 ). the qsar model was validated by the leave-n-out techniques, which helped to assess the stability of the model to changes in the training set. we performed an external validation using a testing set whose structures and activities were not considered in the model building (patel et al. 2008) . gao et al. (2011) suggested that the 3d-qsar model performed well with r test 2 > 0.6 or q test 2 > 0.5, where r test 2 is the normalized correlation coefficient of the testing set, and q test 2 is the cross-validation correlation coefficient of the testing set. to validate the predictive power of the 3d-qsar model, the activity of a three-compound testing set was predicted ( table 1 ). the comfa model was used to predict the pic 50 for each aligned conformation of the compound. to investigate further the binding patterns and the sar between curcumin derivatives and neuraminidase, we performed molecular docking. the docking procedure was performed using the surflex-dock module of sybyl-x2.1.1, and the receptor-ligand interactions were carried out by the discovery studio 2016 software. the curcumin derivatives were processed by adding polar hydrogen atoms, giving the gasteiger-huckel charge, and using maxim's genetic algorithm to perform energy minimization in the tripos force field. the x-ray structure of influenza virus neuraminidase (code: 4hzz) was taken from the rscb protein data bank (fang et al. 2011 ). the ligand-binding site of neuraminidase was defined by the reference ligand 39g. the semi-flexible method was used for docking, and the crystal structure of the original ligand as a reference for docking. the parameters in the docking process were set as follows: expansion coefficient 1, threshold parameter 0.5, and other parameters were system default values. the total score function was used to score the situation of molecular docking, which is an empirical scoring function derived from the binding energies of protein-ligand complexes and their x-ray structures. this function takes into account factors such as polarity, oseltamivir carboxylate was chosen as a positive control in the na activity inhibition assay, the ic 50 of oseltamivir carboxylate was 225.42 μm a molecules in the testing set 3d-quantitative structure-activity relationship and antiviral effects of curcumin derivatives… hydrophobicity, enthalpy, and solvation. the larger the value, the more stable the docking compound, which means better matching and binding of small molecules and large proteins (li et al 2013) . it is generally accepted that total score is > 6 for better activity, and ≥ 9 for very good activity. rt-pcr was used to assess the ability of curcumin derivatives and oseltamivir carboxylate to inhibit h1n1 virus. confluent mdck cell layers infected with 100 μl 100 tcid 50 iav (h1n1) were treated with curcumin derivatives or oseltamivir carboxylate (20 mm, non-toxic concentration, data not shown) for 24 h. then, cells were collected to detect the mrna expression level of h1n1 m gene by using iav nucleic acid test kit (rt-pcr). the primer sequences used for the detection of viral rna were 5′-ttc taa ccg agg tcg aaa cg-3′ and 5′-aca aag cgt cta cgc tgc ag-3′. cell culture supernatants were collected and treated for neuraminidase activity [expressed as relative luminescence units (mu/ml)] using the neuraminidase activity fluorometric assay kit (k732-100, biovision, milpitas, ca, usa). fluorometry was measured using polarstar optima multifunction fluorescence microplate reader (bmg, germany). confluent mdck cell layers were infected with 100 μl 100 tcid 50 iav (h1n1) for 2 h at 37 °c. after removing the virus-containing medium, the cells were treated with curcumin derivatives at maximum non-toxic doses (mntds) in dmem. cells were fixed with ice-cold acetone/methanol (40: nih mice were randomly divided into six groups, 12 in each group: normal control group (nc); placebo control group (iav-c + sterile saline, suspended in 0.5% tween 80); curcumin groups (h1n1 virus + curcumin 25, 50 and 100 mg/kg); and positive control ribavirin (70 mg/kg). the challenge inoculation of ~ 10 5 50% cell culture infectious doses (ccid 50 )/mouse was equivalent to five 50% mouse lethal challenge doses (mld 50 ). under mild anesthesia, mice were infected with 50 μl 15 mld 50 iav (sterile in pbs, ph 7.2) intranasally (without the nc group). then, mice were treated with intraperitoneal injection of curcumin (25, 50 and 100 mg/kg) or ribavirin (70 mg/kg) for 1 day prior to viral challenge and received concomitant treatment for 3 days after infection. on day 4 after infection, the mice were sacrificed to collect relevant samples, and the body weight, lung wet weight, and the degree of lung pathological changes were measured. nih mice were anesthetized in a chamber containing absorbent cotton saturated with 30 ml diethyl ether for about 30 s and infected intranasally with 2 mld 50 of iav in sterile pbs (pre-cooled). then, mice were treated with oral gavage of curcumin (25, 50 and 100 mg/kg) or ribavirin (70 mg/kg) for 1 day before infection in the protective administration group, and mice in the therapeutic administration groups were treated 6 h after infection. mice received these treatments for 12 days and were monitored daily for 15 days after infection. for the iav-c group, the mice were given saline (0.5% tween 80) only at the same intervals. fifteen days after infection, the mice were continuously observed daily for weight loss, survival, and clinical symptoms of illness (e.g., wrinkled fur, inactivity, loss of appetite, hunchback posture, and rapid shallow breathing) to assess the survival rate and mean days to death. the lung index was calculated as follows: lung index = a/b × 100, where a is the lung weight, and b is the body weight . after the mice were killed by exsanguination, the lungs were removed, homogenized, and centrifuged at 4000×g for 15 min at 4 °c. before the analysis of cytokine levels, the supernatant of the lung homogenate was stored at − 80 °c. anti-mouse enzyme-linked immunosorbent assay (elisa) kits (ebioscience, san diego, ca, usa) were used to measure the concentrations of interleukin (il)-6, tumor necrosis factor (tnf)-α and interferon (ifn)-γ in the bronchoalveolar lavage fluid. we selected 11 curcumin derivatives ( the 11 curcumin derivatives selected in the neuraminidase inhibition screening assay were used to perform the 3d qsar studies (comfa), and the comfa statistical coefficients are shown in table 2 . when the cross-validation correlation coefficient q 2 is > 0.5, the prediction model is reliable (chen et al. 2011 ). the larger q 2 is, the stronger the prediction ability is. in the comfa model, the crossvalidation coefficient q 2 was 0.527 and the best composition score was 4. partial least squares regression analysis yielded a model correlation coefficient r 2 of 0.997 and an f statistic of 282.663. the cross-validation coefficient was used to evaluate the predictive ability of the fitting equation. the higher r 2 value is, the higher the ratio of the two variables, and the better the fitting degree of the model and data; the f statistics are greater than the critical value k, that is, the effect of the regression analysis is significant. compared with the actual activities, the predicted activities of the comfa model were in general agreement with the original data ( fig. 1) , indicating that the predictive ability of the model was credible. the results of the actual and predicted pic 50 values for the training set and testing set are shown in fig. 1 . it can be seen from the figure that the actual value was close to the predicted value, and the prediction of the pic 50 value by the 3d-qsar model was reasonably accurate. the actual and predicted values were close, indicating that the model established in this study had statistical significance. the calculated and experimental values of the external verification test set were also similar (red triangle in fig. 1) , indicating that the model had a strong predictive ability and was successfully constructed. five active compounds with better inhibition of na activity in vitro were selected for further study. different colors in the comfa model represent regions of reduced or increased activity due to spatial variations of different molecules. the comfa steric field is represented as a contour map in fig. 2 . in the stereo contour map, the yellow and green regions represent areas where small and large volume groups enhance activity, respectively. we selected the most potent inhibitor, demethylcurcumin (pic 50 = 4.20), as a reference for assisted visualization. there were four green areas and five yellow areas around the composite zones. the green outline around the meta-hydroxyl group of the phenyl ring indicated where it favored the space volume, such as a meta-methoxy group of the other benzene ring. large volume groups at these positions may facilitate interactions between the ligand and its receptor, which accounted for why demethoxycurcumin activity (pic 50 = 3.70) was lower than demethylcurcumin activity (pic 50 ) = 4.20. the difference in activity between bisdemethoxycurcumin (pic 50 = 4.15) and demethylcurcumin (pic 50 = 4.20) was also reasonably explained. in addition, the green contour around the central seven carbon chain showed that the double bonds in the central seven-carbon chain may be beneficial for the interaction between the ligand and its . 1 fitness graphs between observed activity and predicted activity for the training set and the testing set compounds 3d-quantitative structure-activity relationship and antiviral effects of curcumin derivatives… receptor. for example, the activity of dihydrocurcumin (pic 50 = 3.66) and tetrahydrocurcumin (pic 50 = 3.69) was higher than that of hexahydrocurcumin (pic 50 = 1.47) and octahydrocurcumin (pic 50 = 2.81). in order to explore the binding patterns between curcumin derivatives and neuraminidase, molecular docking was performed to help understand the sars between molecules and proteins. sybyl-x2.1.1 was applied to carry out the docking studies. oseltamivir carboxylate was used as a positive control to assess the ability of other molecules to bind to na. the total score function was used to comprehensively score the situation of molecular docking, which is an empirical scoring function derived from the binding energies of protein-ligand complexes and their x-ray structures. it is generally accepted that total score is > 6 for better activity, and ≥ 9 for very good activity (golbraikh and tropsha 2002) . as shown in fig. 3 , the total scores of 11 curcumin derivatives and oseltamivir carboxylate were all > 6, indicating that these compounds were likely to have inhibited na. thus, according to na inhibitory activity assays, the binding mode between demethylcurcumin (ic 50 = 62.77 μm) and oseltamivir carboxylate (ic 50 = 225.42 μm) was further investigated in the docking study, and the top five compounds with higher ic 50 were selected for in vitro activity verification. the 3d and 2d binding models between neuraminidase and demethylcurcumin (a and b), and neuraminidase and oseltamivir carboxylate (c and d) were analyzed (fig. 4) . key residues, h bonds, van der waals forces, π-alkyl and alkyl groups were analytically labeled and visualized to understand further the details of neuraminidase binding to molecules. the chemical bonds corresponding to the residues are listed in table 3 . there were four conventional h bonds between the oseltamivir carboxylate and five residues of neuraminidase, which indicated that the binding region of this portion of the h-bond was important for the stable binding between oseltamivir carboxylate and na (fig. 4d) . the combination of demethylcurcumin with na produces five conventional h bonds (fig. 4b) , indicating that demethylcurcumin can be considered as a potential na inhibitor. in addition, there were two π-donor h bonds and one π-alkyl bond between demethylcurcumin and na, but only one alkyl bond between oseltamivir carboxylate. 3d-quantitative structure-activity relationship and antiviral effects of curcumin derivatives… to validate the neuraminidase inhibition ability for the top five compounds that showed higher ic 50 in the neuraminidase inhibition screening experiments, experiments including viral replication inhibition assay and neuraminidase inhibition assay in vitro were carried out. first, mtt cell proliferation assay was performed to measure the mntd of the compounds. the mntds for tetramethylcurcumin, demethylcurcumin, bisdemethoxycurcumin, dihydrocurcumin and curcumin were 15, 20, 20, 20 and 25 μm, respectively (fig. 5a) . the mntds of the compounds were used in the viral replication inhibition assay and neuraminidase inhibition assay. the five curcumin derivatives showed different degrees of inhibition of h1n1 m gene mrna expression (fig. 5b ). demethylcurcumin and tetramethylcurcumin significantly decreased the mrna expression levels of m gene about 4.17-fold (0.24 ± 0.07 μm) and about 3.85-fold (0.26 ± 0.07 μm) compared to mock-treated virus controls. treatment with curcumin reduced expression about 5.26fold (0.19 ± 0.03 μm). bisdemethoxycurcumin and dihydrocurcumin decreased the m gene mrna expression about 2.13-fold (0.47 ± 0.11 μm) and 1.20-fold (0.83 ± 0.09 μm), respectively. in addition, the positive drug group (oseltamivir carboxylate) almost inhibited the mrna expression of m gene, which was about 11.11-fold compared to mock-treated virus controls. furthermore, the influence of curcumin derivatives on h1n1-induced neuraminidase activation in mdck cells was analyzed by the supernatant. h1n1 infection decreased the neuraminidase activity relative to that in the mockinfected cells (fig. 5c ). compared to virus controls, tetramethylcurcumin and bisdemethoxycurcumin decreased na activity by 2.65-fold (0.37 ± 0.06 mu/ml) and 2.45-fold (0.40 ± 0.01 mu/ml), respectively, whose inhibitory effects were all better than that of the oseltamivir carboxylate group (0.44 ± 0.04 mu/ml). the other three derivatives also for 2 h at 37 °c. after removing the virus-containing medium, the cells were treated with curcumin derivatives at mnlds in dmem respectively. cells were harvested 24 h after infection and mrna expression levels of m genes were detected by real-time rt-pcr. data represent the mean ± sd of 3 separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 relative to virus group. # p < 0.05, ## p < 0.01, ### p < 0.001 relative to mock control group. (c) influence of curcumin derivatives treatment on neuraminidase activation in h1n1-infected mdck cells. mdck cells were infected with 100 μl of 100tcid 50 influenza virus a/font monmouth/47(h1n1, fm1) for 2 h at 37 °c. after removing the viruscontaining medium, the cells were treated with curcumin derivatives at mnlds in dmem respectively. twenty-four hours post-infection the cell supernatant was analyzed for neuraminidase activity (expressed as mu/ml units) using neuraminidase activity assay kit. data represent the mean ± sd of 3 separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 relative to virus group. # p < 0.05, ## p < 0.01, ### p < 0.001 relative to mock control group showed an inhibitory effect, in which curcumin, demethylcurcumin and dihydrocurcumin respectively decreased na activity by about 2.04-fold (0.48 ± 0.03 mu/ml), 1.85-fold (0.53 ± 0.06 mu/ml) and 1.81-fold (0.54 ± 0.02 mu/ml). to investigate the influence of curcumin derivatives on the reduction of the viral np, mdck cells were infected with h1n1 (100 tcid 50 ) for 2 h at 37 °c and then were treated with curcumin derivatives at mntds. np localization was detected 8 h after infection, at which time viral replication and transcription were ongoing, and newly formed viral particles began to be released and spread from infected cells. no immunofluorescence of viral np was observed in the control group, while green fluorescent signal was observed in cells infected with virus (fig. 6 ). viral np was mainly localized in the cytoplasm, with lesser amounts in the nucleus. compared to the virus group, less np was observed in the cytoplasm in cells treated with tetramethylcurcumin and curcumin. in contrast, in cells treated with demethylcurcumin, bisdemethoxycurcumin, and dihydrocurcumin, a strong green fluorescent signal was observed in the cytoplasm. these results suggested that tetramethylcurcumin and curcumin inhibited the nucleation of viral np, leading to nuclear retention of np, preventing viral assembly. mice treated with curcumin at 100 mg/kg/day remained stable in weight and showed no significant clinical symptoms, fig. 6 influence of curcumin derivatives treatment on nuclear export of viral np in h1n1infected mdck cells. mdck cells were infected with a/font monmouth/47(h1n1, fm1) at 100tcid 50 . 5 curcumin derivatives treatments were performed continuously starting after infection. eight hours postinfection np localisation was visualised using specific antibodies by immunfluorescence. np staining is shown in green. nuclei are stained by dapi (shown in blue). photographs are taken from one representative experiment. in total, three independent experiments were performed with similar results 3d-quantitative structure-activity relationship and antiviral effects of curcumin derivatives… indicating dose safety. to evaluate the therapeutic efficacy of curcumin against influenza a/font monmouth/47(h1n1, fm1) virus, a lethal murine infection model was used and all the mice were killed a few days after infection. the degree of lung pathological change of the placebo group was more serious than that of the normal control group, and the difference in lung index was significant (p < 0.01) ( table 4) . compared with the placebo control, the lung index of the ribavirin group was significantly improved, and the lung index inhibition rate was 43% (p < 0.01). however, the administration of curcumin at 100, 50, and 25 mg/kg/day improved the lung index inhibition rate at 22.42% (p < 0.05), 35.75% (p < 0.1), and 23.6% (p < 0.05), respectively. to investigate further the protective and therapeutic efficacy of curcumin against h1n1, mice were treated with curcumin at 100, 50, and 25 mg/kg/day before and after inoculation with iav. treatment with curcumin pre-infection and post-infection at all doses prolonged the average survival time and promote the death protection rate significantly, compared with placebo group (fig. 7) . it was observed that the death protection rate with pre-infection administration was dose dependent, while the death protection rate with post-infection administration at 100 mg/kg/day was the most obvious. these results showed that curcumin had a good death protection effect against iav-induced pneumonia in mice. iav infection causes an increase in pro-inflammatory cytokines, so we examined the effect of curcumin on the release of virus-induced proinflammatory cytokines. lung tissues were harvested from iav-infected mice on day 5 after infection, and several cytokines were analyzed by elisa. elevation of proinflammatory cytokines is a typical response to iav infection, so we established whether the mice were killed at 4 dpi. the lungs were removed and rinsed with sterile pbs. the lung index of mice and lung index inhibition were detected (n = 12). # p < 0.05, ## p < 0.01, ### p < 0.001, the values were compared with normal controls. * p < 0.05, ** p < 0.01, *** p < 0.001, the values were compared with placebo controls . data represent the mean ± sd of 3 separate experiments. # p < 0.05, ## p < 0.01, ### p < 0.001 relative to normal controls. * p < 0.05, ** p < 0.01, *** p < 0.001 relative to placebo controls curcumin affected the virus-induced release of pro-inflammatory cytokines. lung tissues were harvested from iavinfected mice on day 5 after infection, and several cytokines (tnf-α, il-6, and ifn-γ) were analyzed by elisa. compared to the control group, the levels of tnf-α, il-6, and ifn-γ in the lung homogenate were significantly increased (fig. 8 ). however, curcumin at different doses significantly attenuated the increases in tnf-α, il-6 and ifn-γ levels in similar lung homogenates, except for tnf-α level in mice treated with 25 mg/kg/day curcumin. influenza is a common respiratory infectious disease with high morbidity and mortality during epidemics. the emergence of adverse effects and resistance to antiviral drugs limits their clinical application (fiers et al. 2004; hurt et al. 2009; pica and palese 2013) . therefore, exploring new antiinfluenza drugs is urgently needed (suzuki et al. 2003) . curcumin, a coloring agent and spice commonly used in food (goel et al. 2007) , inhibits the replication of iav in vitro and in vivo (kannan and kolandaivel 2017; dai et al. 2018) . in this study, we used a 3d-qsar model and docking model to investigate the inhibitory effects of curcumin derivatives against neuraminidase and influenza a/font monmouth/47(h1n1, fm1) in mdck cells in vitro. then, we detected the anti-influenza virus effects of curcumin in mice and revealed that it could significantly inhibit iav and decreased tnf-α, il-6 and ifn-γ pro-inflammatory cytokines in vivo. in the present study, most of the curcumin derivatives exhibited significant inhibition of h1n1 neuraminidase. we established a meaningful 3d-qsar model (comfa) to study the sar between curcumin derivatives and neuraminidase, and predicted the activity of the ligand in the test set. in addition, establishment of the comfa model showed that the hydroxyl group at the meta-position of the benzene ring and the double bonds in the central seven-carbon chain in the curcumin derivatives may be essential for neuraminidase inhibitory activity. the docking model was also consistent with the predicted activity of qsar. curcumin inhibits iav infection (mazur et al. 2007; kannan and kolandaivel 2017) by interfering with hemagglutinin activity, while tetrahydrocurcumin is less effective in inhibiting iav infection . further sar analysis showed that the two-tone functional group as a receptor for michael addition conjugate contributes to the differential inhibition of curcuminoid. simulated docking of curcumin with the hemagglutinin structure indicates that curcumin binds to the region that constitutes the sialic acid anchoring residue, supporting the results obtained by inhibition of hemagglutinin activity. these studies indicate that the presence of the double bond in the central seven-carbon chain enhances curcumin-dependent anti-iav activity and interferes with iav entry through interaction of curcumin with the receptor-binding region of the viral hemagglutinin protein. in this study, we reported the inhibitory effects of the curcumin derivatives on iav neuraminidase. the addition of curcumin derivative after virus adsorption showed antiinfluenza activity in mdck cells. therefore, we hypothesized that the curcumin derivative inhibits the activity of na, thereby preventing the release of virions from infected cells. indeed, the inhibitory effect of curcumin derivatives on iav replication was not the same as the inhibitory effect of na expression. it can be seen that curcumin showed the best inhibitory effect on viral replication, followed by demethylcurcumin, tetramethylcurcumin, and bisdemethoxycurcumin, and dihydrocurcumin showed a weak inhibitory effect, which is consistent with the results of ou et al. (2013) . however, tetramethylcurcumin and bisdemethoxycurcumin showed a similar effect on inhibition of na, followed by curcumin, dihydrocurcumin and demethylcurcumin. these # p < 0.05, ## p < 0.01, ### p < 0.001 relative to normal controls. * p < 0.05, ** p < 0.01, *** p < 0.001 relative to placebo controls 3d-quantitative structure-activity relationship and antiviral effects of curcumin derivatives… results indicated that curcumin derivatives may inhibit iav through inhibiting many other proteins or signaling pathways, and not only neuraminidase protein. in addition, curcumin derivatives had different inhibitory effects on iav neuraminidase protein, which was relative to their structures and binding models. finally, we investigated the influence of curcumin derivatives on the reduction of viral np. two curcumin derivatives (tetramethylcurcumin and curcumin) were shown to decrease expression of np induced by iav infection. bioavailability of curcumin: problems and promises oseltamivir inhibits influenza virus replication and transmission following ocular-only aerosol inoculation of ferrets synergistic activity of baicalein with ribavirin against influenza a (h1n1) virus infections in cell culture and in mice inhibition of enveloped viruses infectivity by curcumin oseltamivir-resistant influenza viruses isolated in south korea from inhibition of curcumin on influenza a virus infection and influenzal pneumonia via oxidative stress, tlr2/4, p38/jnk mapk and nf-κb pathways integration of target discovery, drug discovery and drug delivery: a review on computational strategies a "universal" human influenza a vaccine a new protocol for predicting novel gsk-3beta atp competitive inhibitors beware of q2! curcumin as "curecumin": from kitchen to clinic 3d qsar and docking study of flavone derivatives as potent inhibitors of influenza h1n1 virus neuraminidase the anesthetic action of some polyhalogenated ethers-monte carlo method based qsar study zanamivirresistant influenza viruses with a novel neuraminidase mutation antiviral potential of natural compounds against influenza virus hemagglutinin oral administration of patchouli alcohol isolated from pogostemonis herba augments protection against influenza viral infection in mice functional and structural analysis of influenza virus neuraminidase n3 offers further insight into the mechanisms of oseltamivir resistance multiple biological activities of curcumin: a short review acetylsalicylic acid (asa) blocks influenza virus propagation via its nf-kappabinhibiting activity global transmission of oseltamivir-resistant influenza structural domains and organizational conformation involved in the sorting and transport of influenza virus transmembrane proteins new method for rapid characterization of molecular shapes: applications in drug design roles of neuraminidase in the initial stage of influenza virus infection structure-activity relationship analysis of curcumin analogues on anti-influenza virus activity 3d qsar and molecular docking studies of benzimidazole derivatives as hepatitis c virus ns5b polymerase inhibitors toward a universal influenza virus vaccine: prospects and challenges synergic effect of curcumin and its structural analogue (monoacetylcurcumin) on anti-influenza virus infection emergence of amantadine-resistant influenza a viruses: epidemiological study the first pandemic of the 21st century: a review of the 2009 pandemic variant influenza a (h1n1) virus origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic the pathology of influenza virus infections 3d-qsar analysis of a series of s-dabo derivatives as anti-hiv agents by comfa and com-sia design and synthesis of pinanamine derivatives as anti-influenza a m2 ion channel inhibitors acknowledgements this study was supported by the national natural science foundation of china (no 81470186). we thank international science editing (https ://www.inter natio nalsc ience editi ng.com) for editing this manuscript. conflict of interest the authors declare no conflict of interest. key: cord-006362-7d5wzb7p authors: van riel, debby; mittrücker, hans-willi; engels, geraldine; klingel, karin; markert, udo r.; gabriel, gülsah title: influenza pathogenicity during pregnancy in women and animal models date: 2016-07-07 journal: semin immunopathol doi: 10.1007/s00281-016-0580-2 sha: doc_id: 6362 cord_uid: 7d5wzb7p pregnant women are at the highest risk to develop severe and even fatal influenza. the high vulnerability of women against influenza a virus infections during pregnancy was repeatedly highlighted during influenza pandemics including the pandemic of this century. in 2009, mortality rates were particularly high among otherwise healthy pregnant women. however, our current understanding of the molecular mechanisms involved in severe disease development during pregnancy is still very limited. in this review, we summarize the knowledge on the clinical observations in influenza a virus-infected pregnant women. in addition, knowledge obtained from few existing experimental infections in pregnant animal models is discussed. since clinical data do not provide in-depth information on the pathogenesis of severe influenza during pregnancy, adequate animal models are urgently required that mimic clinical findings. studies in pregnant animal models will allow the dissection of involved molecular disease pathways that are key to improve patient management and care. influenza a viruses (iav) belong to the major causative agents of respiratory infections posing a considerable burden for human health. the clinical course of iav infections in healthy individuals may vary from mild self-limiting disease to severe disease, which requires hospitalization with occasional lethal outcome. the annual influenza epidemic which peaks during winter in temperate climates is estimated to cause >600 million cases globally, with 3 to 5 million cases of severe illness and up to 500,000 deaths per year worldwide (www.who.int). since iav have a zoonotic origin with an unlimited reservoir in aquatic birds, they may cross species barriers and transmit to humans leading to novel virus variants with an unpredictable outcome. recently emerged avian iav belonging to the h5n1 and h7n9 subtypes are responsible for high case fatality rates in humans and pose a future pandemic threat (www.who.int). the influenza pandemic of this century was caused by an h1n1 iav in 2009-a(h1n1 )pdm09-possessing genomes of avian, swine, and human origin [1] . it is estimated that it has caused~200,000 respiratory and an additional~80,000 cardiovascular deaths predominantly in people younger than 65 years of age [2] . mortality rates upon iav infections are highest among patients with underlying medical conditions, such as those with chronic pulmonary, cardiovascular, renal, hepatic, neuromuscular, hematologic, and metabolic disorders or in patients being obese, pregnant, or having immunodeficiencies [3] [4] [5] [6] [7] . data from previous pandemics such as the 1918 h1n1 and the 1957 h2n2 pandemic as well as from seasonal influenza epidemics revealed that pregnant women have an increased risk in developing complications upon iav infection [8] [9] [10] [11] [12] [13] . the pandemic caused by a(h1n1)pdm09 highlighted again that especially pregnant women are at the highest risk to develop severe or even fatal influenza. data from the usa show that 5 % of the a(h1n1)pdm09-related deaths were among pregnant women, although they make up only 1 % of the total population [9] . this unprecedented very severe disease outcome during pregnancy led to the revision of vaccination recommendations by the world health organization (who), setting pregnant women now as the highest priority to be vaccinated again influenza regardless of gestational age. during pregnancy, women undergo various physiological changes which might contribute to disease severity upon iav infection. these physiological changes include many metabolic, hemodynamic, and immunologic changes to allow growth and development of the fetus. metabolic changes occur, e.g., in the glucose and lipid metabolism, and hemodynamic changes include an increase of plasma volume by 1000-1600 ml and alterations in the systemic coagulation system [14] . during pregnancy, there are also specific changes in both the upper and lower respiratory tract. in the upper respiratory tract, the mucosa of the nasopharynx and oropharynx changes, including hyperemia, edema, leakage of plasma into the stroma, glandular hypersecretion, and increased mucopolysaccharide content. all these physiological changes can result in nasal congestion [15] . the lower respiratory tract is altered due to the elevation of the diaphragm by up to 4 cm and a decrease in functional residual capacity. functionally, there are changes in lung function, ventilation, and gas exchange, which lead to an increase in oxygen tension required for trans-placental oxygen transfer. furthermore, changes in the cardiovascular system result in a decrease of pulmonary vascular resistance [15, 16] . all these physiological alterations might be further challenged by respiratory viral infections, such as iav and, thus, contribute to disease severity during pregnancy. immunological changes comprise the altered differentiation status of immune cells in the uterus and the formation of a tolerogenic environment at the maternal/fetal interface that prevent rejection of the semi-allogeneic fetal tissues and thereby permit pregnancy. there are also systemic changes of the immune system, mainly caused by pregnancy hormones. these changes are reflected in increased maternal disease outcome not only upon iav infections but also in patients with hiv, malaria, and toxoplasmosis [5] [6] [7] 17] . understanding the molecular basis that mediates increased mortality upon iav infection during pregnancy is key to improve patient management and care. therefore, adequate animal models are required that reflect clinical findings and allow the identification of causative disease pathways. this knowledge will allow early diagnosis and the implementation of rapid intervention strategies to improve disease outcome during pregnancy. it is estimated that vaccination compliance among pregnant women is still below the who recommendations although it has been shown that vaccination during pregnancy protects mother and child [18, 19] . however, a thorough worldwide documentation of vaccination rates is still missing [20] . thus, there is an urgent need to increase disease awareness among this most vulnerable group to improve patient management by early diagnosis as well as by increasing the currently poor vaccination compliance among pregnant women. here, we review current knowledge on the pathogenesis of iav-associated complications during pregnancy in humans and in established animal models as a basis for mechanistic studies. pathology in iav-infected pregnant women pregnant women have an increased risk of developing iavassociated complications due to infection with seasonal, pandemic, or zoonotic influenza viruses. several studies have shown that co-morbidities were present in~30 % of the cases, of which a history of asthma was seen most frequently. other co-morbidities included diabetes, cardiac diseases, hematological disorders, and low weight. since two thirds of the complications in pregnant women could not be explained by other underlying medical conditions, pregnancy itself increased the risk of developing iav-associated complications [21, 22] . interestingly, the majority of pregnant women that develop severe influenza virus-associated disease were in their second or third trimester [21, 22] . frequent symptoms in hospitalized pregnant women include fever, cough, respiratory distress, malaise, joint pain, headache, rhinorrhea, gastrointestinal symptoms, and the development of pneumonia. the latter can lead to the development of acute respiratory distress syndrome (ards) [12, 21, [23] [24] [25] [26] . ards is a rapid onset of hypoxemic respiratory failure associated with bilateral radiographic opacities without congestive heart failure. there are many causes for the development of ards during pregnancy. besides the iavmediated pneumonia, other causes include amniotic fluid embolism, pre-eclampsia, sepsis, and aspiration pneumonia [27] . treatment of a(h1n1)pdm2009-associated ards included extracorporeal life support, also known as extracorporeal membrane oxygenation (ecmo), which resulted in a survival rate of~70 % [27] . extra-respiratory tract complications are occasionally described in pregnant women. these include the development of myocarditis after infection with a(h1n1)pdm09 [28, 29] , and the development of an encephalopathy associated with a seasonal h3n2 iav infection from which viral rnawas detected in cerebrospinal fluids [30] . as in other risk groups, the most common complication of iav in pregnant women is the development of pneumonia and ards. several studies describe the histopathology in pregnant and non-pregnant fatal cases during the a(h1n1)pdm09 pandemic. these studies did not reveal differences between the two groups, although this has not been extensively studied. in pregnant women, histopathological lesions were predominantly found in the respiratory tract, characterized by multifocal desquamation of the epithelial lining of the trachea and bronchus and inflammation of the submucosal glands. within the lungs, there was evidence of diffuse alveolar damage, characterized by extensive fibrosis, hyaline membrane formation, thickening of the alveolar septa, type ii pneumocyte hyperplasia, interstitial and alveolar edema, and an influx of many inflammatory cells [31] [32] [33] [34] . virus antigen was detected in ciliated epithelial cells as well as the submucosal glands of the bronchi and bronchioles. in the alveoli, virus antigen could be detected within the alveolar epithelial cells and alveolar macrophages [31, 32, 34] . in none of these cases, there was evidence of extra-respiratory tract replication. post-mortem histopathological examination of a 24-yearold otherwise healthy pregnant women who succumbed to a(h1n1)pdm2009 after developing ards [26] revealed severe lung damage with desquamated alveolar epithelial cells, destruction of the alveolar septae and interstitial inflammatory cells as described in other cases. additional immunohistochemical staining identified inflammatory cells which mainly consisted of cd68+ macrophages and cd3+ t lymphocytes. since the patient died 28 days after hospitalization and had received antiviral therapy, viral rna could not be detected by in situ hybridization (fig. 1) . post-mortem analysis of an h5n1 highly pathogenic avian virus (hpaiv) infected 4-month pregnant women showed similar diffuse alveolar damage characterized by focal desquamation of epithelial cells in the alveoli without any evidence of type ii pneumocyte hyperplasia, influx of macrophages, neutrophils, and few lymphocytes. no lesions were described in any other parts of the respiratory tract. virus antigen was detected in tracheal epithelial cells and alveolar epithelial cells. in contrast to reports on a(h1n1)pdm2009-infected pregnant women, upon h5n1 hpaiv infection, extrarespiratory virus spread to the placenta could be detected with virus-positive cytotrophoblastic and hofbauer cells. there, lesions included scattered foci of syncytiotrophoblast necrosis, occasionally associated with dystrophic calcification. moreover, virus was detected in the fetus with virus antigen-positive kupffer cells of the liver and pneumocytes in the lungs. virus in the fetal liver was not associated with histological lesions, but the fetal lung showed edema and a few scattered interstitial neutrophils [36] . however, the pathogenesis of iav-associated complications in pregnant women is studied to a very limited extent. a b c fig. 1 post-mortem histopathological analysis of lung biopsy material obtained from a fatal pregnant case. lung biopsy material was obtained from a 24-year-old pregnant woman without any known underlying diseases who succumbed to the 2009 h1n1 influenza virus infection after spontaneous expulsion of the fetus [26] . a severe alveolitis with numerous immunohistochemically (red) stained cd68+ macrophages (a) and cd3+ t lymphocytes (b) is observed. at this late stage of infection, influenza virus rna was not detectable anymore by radioactive in situ hybridization (he staining) (c). the histochemical stainings were performed according to protocols described before [35] important host factors for the pathogenesis of influenza in mammals including humans are the distribution of specific influenza virus receptors and nuclear transport proteins, the importin-α isoforms [37, 38] which largely determine the cell tropism of influenza viruses within the respiratory tract. unfortunately, the impact of these cellular factors has not been studied during pregnancy yet. it is therefore currently unknown whether potential alterations in the distribution of receptors or importin-α isoforms might contribute to the increased risk of developing complications during pregnancy. pregnancy causes the formation of an immune-tolerant environment in the uterus that prevents rejection of fetal tissue. moreover, pregnancy has also profound effects on the peripheral immune system outside of the uterus that might interfere with the response against iav infections [5, 6] . endocrine adaptation to pregnancy, in particular the elevated levels of estradiol and progesterone, affect cytokine levels as well as their composition and function of peripheral leukocyte populations (reviewed in [6] ). numbers of peripheral neutrophils, monocytes, as well as myeloid and plasmacytoid dendritic cells (dc) are reduced [39] . upon in vitro stimulation, both nk cells and cd4 t cells produce reduced amounts of inflammatory cytokines and chemokines. interestingly, reduced cytokine production by cd4 t cells includes both t h1 (ifn-γ, tnf-α) and t h2 cytokines (il-6, il-13) [39] arguing against a shift in the t h1 -t h2 balance during pregnancy as has been postulated from mouse studies [40] . dcs from pregnant women also display higher expression of costimulatory surface molecules, such as cd40, cd80, and cd86, than those of non-pregnant women suggesting a more mature status of dcs [41] [42] [43] . it should be mentioned that results on the phenotype and function of immune cells during pregnancy differ considerably between individual studies. our understanding of immunity during pregnancy would clearly benefit from more systematic and comprehensive approaches using advanced methodology to better define and characterize immune cell subsets. despite these changes in the peripheral immune system, vaccination studies indicate that there is no general deficit in the adaptive immune response of pregnant women to influenza virus. pregnant women still generate protective antibodies against iav, which are further transferred to the fetus [44] [45] [46] [47] . moreover, pregnant women show diminished polyclonal t cell responses but enhanced virus-specific t cell responses following influenza vaccination [48] . thus, despite significant changes in their peripheral immune system, pregnant women are still able to effectively mount an adaptive immune response against iav. currently, there is only very limited information on the immune response against iav in pregnant woman with severe iav-associated complications. as stated before, complications in pregnant women are similar to those observed in other risk groups, arguing for potentially common mechanisms that mediate disease severity. there is evidence that immunopathology strongly contributes to severe courses of iav infections [49] . high virus titers in the lung, which might represent a consequence of reduced production of type i and type iii interferons in pregnant women [50] , could cause excessive production of inflammatory cytokines and chemokines and massive infiltration of granulocytes and macrophages which subsequently cause severe tissue damage. altered production of inflammatory cytokines as well as differences in the composition and response of peripheral immune cells in pregnant women could contribute to such an exacerbated response against iav [6] . however, so far, there is no direct evidence supporting such a scenario. patients with severe iav infections show lower serum levels of igg2. healthy pregnant women also have low levels of serum igg2, and levels are further reduced in pregnant women with severe iav infections [51] [52] [53] . yet again, the contribution of low serum igg2 levels to severe courses of infection in pregnant women is not clear. overall, it is currently not known why pregnant woman are more prone to develop severe courses of disease upon iav infections. iav infections can also have an effect on the pregnancy outcome or the development of the fetus. iav infections have been associated with an increase in perinatal mortality, preterm birth, cesarean section, and a low apgar score 5 min after birth [9, [54] [55] [56] . in addition, infection with iav within the first trimester of the pregnancy increases the risk by twofold on non-chromosomal congenital anomalies, such as neural tube defects, hydrocephaly, congenital heart defects, cleft lip, digestive system defects, and limb reduction defects [57] . viral antigen has been detected in the amniotic fluid (h3n2 virus; [58] ) and in fetal tissue (h5n1 virus; [36] ). however, in the majority of cases, iav could not be isolated from the placenta or fetus [31, 59, 60] . this suggests that the effect on pregnancy outcome and fetus might represent an indirect result of iav infection of the mother, such as fever and cytokine and immune response [61] . overall, there is still a lack of detailed knowledge regarding the impact of maternal iav infection on pre-or even postnatal development in the offspring. to gain more insight into the pathogenesis of iav-associated complications during pregnancy, studies have been performed in small animal models, using mice, ferrets, and pigs. these experimental settings provide insights into events early after infection and mechanisms of diseases, which are difficult to study with materials from infected patients. experimental infections in pregnant balb/c mice at a gestational age of 12-14 days have been performed with seasonal h1n1 and a(h1n1)pdm09 iav as well as with h5n1 hpaiv. intranasal infection with 10 4 or 10 5 egg infectious dose or intraperitoneal infection with 2 × 10 6 plaque forming units of pregnant mice with a(h1n1)pdm09 iav resulted in a higher mortality compared to non-pregnant mice [62] [63] [64] . virus titers in the lungs were higher and histological lesions were more severe in pregnant mice compared to non-pregnant mice at 5 days p.i. [63] . however, in another study, virus titers in bronchoalveolar lavage (bal) fluids were comparable between pregnant and non-pregnant mice at 3 days p.i. [64] . intranasal infection of pregnant mice with a seasonal h1n1 iav did not result in increased mortality, although virus titers in the lung were higher and histological lesions were more severe compared to non-pregnant mice [63] . virus transmission to extra-respiratory tract tissues, including the placenta and fetus, was not observed in any of these studies [62] [63] [64] . the importance of viral factors in the pathogenicity of iavassociated disease during pregnancy was shown in a study which compared a wild type a(h1n1)pdm09 iav to a mouse-adapted a(h1n1)pdm09 iav that contained the d222g mutation in the hemagglutinin. infection with the mouse-adapted virus resulted in increased mortality, higher virus titers in the lung, more influenza virus-infected cells in the bronchioles and alveoli when compared to pregnant mice infected with the wild type virus [62] . a study with h5n1 hpaiv showed that in pregnant mice, virus titers in the lungs were lower compared to virus titers in non-pregnant mice at 5 days p.i. however, in pregnant mice, h5n1 hpaiv spread to extra-respiratory organs and the virus could be detected in the placenta and fetus of intranasally infected mice [65] . histological lesions in the respiratory tract of a(h1n1)pdm09-inoculated pregnant mice were observed in the bronchioles and alveoli. the bronchiolar epithelium showed accumulation of mucous and detached cilia. in the alveoli, lesions were compatible with interstitial pneumonia associated with infiltration of lymphocytes and neutrophils [63] . although histological lesions were not described in detail, the location of the lesions is consistent with those observed in humans. taken together, although the number of studies is very limited, they show that virus replication, histological lesions, and mortality are increased in pregnant mice compared to non-pregnant mice. future studies should reveal more insight into virus replication and associated lesions at different time points after infection to understand the dynamics within the mammalian host in time and the duration of infection and virus shedding. infection of mice has also been used to determine the consequences of pregnancy for the immune response against infection with pandemic a(h1n1)pdm09 [62] [63] [64] . compared to infection of mice with seasonal h1n1 strains, infection with a(h1n1)pdm09 resulted in more severe interstitial pneumonia with massive cellular infiltrations, and lung pathology was further aggravated when pregnant mice were infected [63] . compared to non-pregnant mice, pregnant mice infected with a(h1n1)pdm09 iav demonstrated enhanced accumulation of neutrophils and macrophages in lung tissue and bal fluid, and increased local no production [64] . as expected, infection with a(h1n1)pdm09 was associated with increased levels of inflammatory cytokines in the lung when compared to infection with seasonal h1n1 iav [63] . in comparison to non-pregnant mice, pregnant animals had enhanced levels of inflammatory cytokines (il-1β, il-6, tnf-α) as well as neutrophil-and monocyte-recruiting chemokines in bal fluid and lung tissue, which was consistent with the fulminant accumulation of these cells in the lung [62, 64] . in contrast to the profound changes in the innate response to iav infections, the adaptive response appeared to be less affected by pregnancy. pregnant and non-pregnant mice generated similar frequencies of influenza virus-specific cd8 + t cells and these cells showed comparable activation, as indicated by cd69 expression, and accumulation in the infected lung [64] . pregnant mice also mounted strong antibody responses against a(h1n1)pdm09, although in one study, titers of antibodies were reduced compared to non-pregnant mice [62, 64] . overall, these studies indicate that disease severity in pregnant syngenically mated mice upon a(h1n1)pdm09 iav infection is mainly due to enhanced cytokine production in the lung and massive recruitment of innate immune cells which results in severe tissue damage. intranasal inoculation of a seasonal h3n2 iav in ferrets at early, mid, and late gestation resulted in virus replication within the respiratory tract comparable to that described before for non-pregnant ferrets. virus was not transmitted to the placenta, umbilical cord, amnion, and chorion of the fetus [66, 67] . of note, intracardial inoculation of h3n2 virus did result in virus transmission to the placenta and fetus, indicating that a high virus load viremia might be crucial for virus transmission to the fetus. of note, in general, seasonal influenza viruses do not cause viremia in non-pregnant women [68] . to our knowledge, there are no studies performed in pregnant ferrets with recent seasonal iav, pandemic a(h1n1)pdm09 iav, or h5n1 hpaiv. since the pathogenesis of iav is extensively studied in this mammalian species, such studies would provide useful information on the pathogenesis of iav infections during pregnancy. in addition, the pregnant ferret model could be used to study the efficacy of vaccines and antiviral therapies. intranasal or intratracheal inoculation of young pregnant gilts, 85-90 days post insemination, with swine h1n2, swine h3n2, or a(h1n1)pdm09 iav did not result in any clinical signs or gross pathological lesions 7 days post infection. in none of the inoculated animals (n = 5 per group), there was evidence for viremia or trans-placental virus transmission [69, 70] . however, another study did reveal trans-placental transmission of a swine h1n1 virus in one out of ten pigs [71] . infection with swine h1n2, swine h3n2, and a(h1n1)pdm09 induced similar titers of influenza-specific antibodies and similar proliferative response of peripheral blood cells to stimulation with influenza virus. however, serum levels of il-6, il-10, and tnf-α were enhanced and sustained for several days in pregnant pigs infected with pandemic h1n1 suggesting that infection with this virus caused a stronger innate immune response [69, 70] . there is strong evidence that immune adaptation during pregnancy contributes to influenza disease severity. it was hypothesized that there is a contradictory demand for the maternal immune system to adapt to pregnancy in order to maintain the allogenic fetus and to simultaneously mount an immune response to clear viral infection [6] . however, available animal models reveal that the severity of the infection during pregnancy also depends on viral factors since viral pathogenicity is enhanced upon pandemic rather than seasonal iav infection. the severe and even fatal disease outcome observed in pregnant women is mostly studied and reflected in mouse models so far, although these studies are restricted to one mouse strain and infection at a gestational age of 12-14 days. the pregnant ferret and pig models may also provide complementary information on viral pathogenicity observed in humans although these models have only been used in single studies so far. in general, ferrets and to a lesser extent pigs, mimic iav-mediated respiratory pathogenesis similar to that seen in humans. therefore, pathogenesis studies in pregnant pigs and ferrets-which are both outbread-should be included in future studies. in summary, future efforts in developing animal models that compare the pathogenesis of different iav strains and associated pathogenesis, including virus replication, histological lesions, and immune responses in infected dams and their impact on the offspring are urgently required. these studies should reveal iav replication dynamics and associated immune responses. in addition, these models could be used to study the pathogenesis during pregnancy of other emerging influenza viruses and evaluate vaccine or antiviral efficacy during pregnancy. thus, these studies will allow an evidence-based risk assessment to increase disease awareness and to improve patient management and care. antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza 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during abortion or preterm delivery association of foetal wastage with influenza infection during ferret pregnancy the pregnant guinea-pig as a model for studying influenza virus infection in utero: infection of foetal tissues in organ culture and in vivo pathology of human influenza revisited the influence of experimental infection of gilts with swine h1n2 influenza a virus during the second month of gestation on the course of pregnancy, reproduction parameters and clinical status pregnancy outcome and clinical status of gilts following experimental infection by h1n2, h3n2 and h1n1pdm09 influenza a viruses during the last month of gestation transplacental transmission and neonatal infection with swine influenza virus (hsw1n1) in swine this article is a contribution to the special issue on fetomaternal cross talk and its effect on pregnancy maintenance, maternal and offspring health-guest editor: petra arck acknowledgments this work was supported by research grants from the german center for infection research (dzif), the fonds nationale de la recherche luxembourg (fnr; afr 6042498), boehringer ingelheim fonds, and the german research foundation (dfg; ga1575/3; mi476/ 5). debby van riel is supported by a fellowship from the netherlands organisation for scientific research (nwo; contract number 91614115) and the erasmus mc foundation. key: cord-278465-tjjkz16y authors: wille, michelle; lindqvist, kristine; muradrasoli, shaman; olsen, björn; järhult, josef d. title: urbanization and the dynamics of rna viruses in mallards (anas platyrhynchos) date: 2017-03-18 journal: infect genet evol doi: 10.1016/j.meegid.2017.03.019 sha: doc_id: 278465 cord_uid: tjjkz16y urbanization is intensifying worldwide, and affects the epidemiology of infectious diseases. however, the effect of urbanization on natural host-pathogen systems remains poorly understood. urban ducks occupy an interesting niche in that they directly interact with both humans and wild migratory birds, and either directly or indirectly with food production birds. here we have collected samples from mallards (anas platyrhynchos) residing in a pond in central uppsala, sweden, from january 2013 to january 2014. this artificial pond is kept ice-free during the winter months, and is a popular location where the ducks are fed, resulting in a resident population of ducks year-round. nine hundred and seventy seven (977) fecal samples were screened for rna viruses including: influenza a virus (iav), avian paramyxovirus 1, avian coronavirus (cov), and avian astrovirus (astrov). this intra-annual dataset illustrates that these rna viruses exhibit similar annual patterns to iav, suggesting similar ecological factors are at play. furthermore, in comparison to wild ducks, autumnal prevalence of iav and cov are lower in this urban population. we also demonstrate that astrov might be a larger burden to urban ducks than iav, and should be better assessed to demonstrate the degree to which wild birds contribute to the epidemiology of these viruses. the presence of economically relevant viruses in urban mallards highlights the importance of elucidating the ecology of wildlife pathogens in urban environments, which will become increasingly important for managing disease risks to wildlife, food production animals, and humans. urbanization is intensifying worldwide; most humans live in urbanized areas, and the urban human population is expected to continue to grow (united nations population fund, 2007) . within the global growth of cities, urbanization increasingly shapes the emergence and trajectory of infectious disease, both human disease and disease and parasitism in wild animals (alirol et al., 2011; neiderud, 2015) . in association with urbanization, factors affecting pathogen (and parasite) transmission in wild animals include an increase in aggregation and resource availability resulting in increased contact rates, decrease in biodiversity, modulation in host immunity and stress levels (becker and hall, 2014; becker et al., 2015; bradley and altizer, 2006; delgado and french, 2012; patz et al., 2004; penczykowski et al., 2014) . furthermore, in cities, increased contact among humans, domestic animals and wild animals may facilitate cross species spillover of (vertebrate) pathogens, with consequences for wildlife conservation, agriculture, and human health (becker et al., 2015; bradley and altizer, 2006; delgado and french, 2012; patz et al., 2004) . influenza a virus (iav) is a multi-host virus, wherein spillover between birds, humans and agricultural animals does occur, and dabbling ducks, such as those found in city parks, constitute the main reservoir host for these viruses (olsen et al., 2006; webster et al., 1992) . indeed, highly urbanized areas may contain canals and large city parks with ponds housing a wide variety of wild and semi-domestic birds. rna viruses such as iav have a low pathogenicity phenotype in their natural hosts (olsen et al., 2006) , but have large negative socioeconomic consequences when they spillover into food production animals and humans (alexander and brown, 2009; fao, 2005 fao, , 2012 . for example, the most recent remerging highly pathogenic iav h5n8, which was transported globally by waterfowl, resulted in the culling of hundreds of thousands of chickens and turkeys, and is a risk to human health given the reassortment potential (european food safety authority, 2014; lee et al., 2014; pasick et al., 2015; verhagen et al., 2015; wu et al., 2014) . dabbling ducks are a host for a number of rna viruses, including avian coronavirus [cov] , avian paramyxovirus type 1 [apmv-1], and emerging evidence suggests they may also be hosts for an array of avian astroviruses [astrov] (e.g. chu et al., 2012; ramey et al., 2013; tolf et al., 2013b; wille et al., 2015; wille et al., 2016b) . these viruses do not cause signs of disease in their wildlife hosts, but have closely related forms causing morbidity and mortality in poultry, such as infectious bronchitis (cov) (e.g. domanska-blicharz et al., 2014; jackwood et al., 2012; zhuang et al., 2015) , newcastle disease (apmv-1) (e.g. alexander, 2011; jindal et al., 2009; ramey et al., 2013; snoeck et al., 2013; tolf et al., 2013b) , duck hepatitis (astrov) or avian nephritis (astrov) (e.g. chu et al., 2012; pantin-jackwood et al., 2011) . these viruses have been assessed, to various degrees, in wild migrating waterfowl. in sweden, and globally, the ecology of iav is well described in wild waterfowl, where up to 30% of mallards (anas platyrhynchos) are infected during the autumn migration (latorre-margalef et al., 2014; olsen et al., 2006) . recent studies have been instrumental in starting to describe dynamics and ecology of ampv-1 and cov in wild birds; 9-12% of migrating mallards have cov infections, compared to a lower prevalence (2%) of ampv-1 towards the end of the migratory season in sweden (tolf et al., 2013b; wille et al., 2015) . most apmv-1 is detected during iav studies where agglutinating agents are detected after culture that are not iav (e.g. jindal et al., 2009; ramey et al., 2013) , so few true prevalence estimates exist. beyond these viruses, we have a limited understanding of the virodiversity in waterfowl; astroviruses for example have only recently been assessed in wild birds, and the results of a single study suggest that waterfowl may be important in the epidemiology of these viruses (chu et al., 2012) . given that waterfowl are hosts for both multi-host viruses and viruses that cause morbidity and mortality in food production birds, combined with the increased contact between waterfowl and humans in urban areas, dynamics of these viruses in urban bird populations should be explored. in this study, we followed the dynamics of rna viruses at a pond utilized year round by mallards, located in the centre of sweden's fourth largest city. this pond is on the same migratory route as wild mallards assessed for these viruses in southern sweden, allowing a comparison between urban and wild ducks on a limited spatial scale (latorre-margalef et al., 2014; tolf et al., 2013a; tolf et al., 2013b; wille et al., 2015; wille et al., 2016b) . thus, this intra-annual dataset allows us to add to the natural history of iav, cov, ampv-1, and the rarely assessed astrov. furthermore, we aim to elucidate if less frequently studied rna viruses follow intra-annual cycles similar to that of the intensively studied iav. in context of iav, and to a lesser degree cov and apmv-1, an assessment of virus prevalence and diversity in an urban population will further allow us to assess if dynamics in wild birds are reflected in an urban setting. an urban population of mallards residing in the artificial pond "svandammen" in the centre of the city of uppsala, sweden (59°51′ 16″n, 17°38′25″e) were sampled. the pond is kept ice-free during the winter months, and is a popular location where the ducks are fed, resulting in a resident population of ducks year-round. this pond has a largely constant population size between 300 and 600 individuals through the autumn and winter, with fewer birds occupying the pond during breeding in the summer months ( fig. a.1) . the low population count in may is likely the result of unfavorable conditions on the day of the count and sampling. slightly higher population counts in the winter, when most of the city ponds are frozen, likely represent the congregation of birds from ponds across uppsala to utilize this ice-free habitat ( fig. a.1) . two sampling strategies were employed: following capture, freshly deposited feces were collected from a single-use cardboard box, or, due to difficulties in capturing birds, freshly deposited feces were collected from the ground around the perimeter of the pond. samples were collected with a sterile tipped applicator, and were placed in virus transport media (vtm) and stored at − 70°c within 2-6 h of collection. ethical approval for trapping and sampling was obtained from the uppsala animal ethical committee (reference number c228/12), a permit was obtained from the city of uppsala to capture, and a permit from swedish museum of natural history to ring birds. viral rna was extracted from pooled vtm samples, containing 4 samples per pool, with the magnatrix 8000 extraction robot (magnetic biosolutions, sweden) and vet viral na kit (nordiag asa, oslo, norway). the rna extraction was performed by the molecular diagnostics department at the swedish national veterinary institute. positive pools were re-extractioned individually using the maxwell 16 instrument and viral total nucleic acid purification kit (promega, madison, usa). following extraction, samples were assayed by real time reverse transcriptase pcr (rrt-pcr) for iav, cov, and ampv-1 using previously published methods. briefly, iav was screened using a rrt-pcr assay targeting a short region of the matrix gene (spackman et al., 2002) and a pan-coronavirus rrt-pcr assay targeting the rna-dependant rna polymerase (rdrp) gene (muradrasoli et al., 2009 ) using the iscript one step rt-pcr kit (biorad, hercules, usa). a rrt-pcr targeting the matrix (m) gene (tolf et al., 2013b; wise et al., 2004) with the one step rt-pcr kit (qiagen, hilden, germany) was employed to screen for apmv-1. a cycle threshold (ct) cutoff of 40 was used for all screens. to screen for astrov, cdna was synthesized using superscript iii (invitrogen) and random hexamers (invitrogen) followed by a nested pcr targeting the rdrp (chu et al., 2012; chu et al., 2008) using taq polymerase (qiagen). samples positive for iav were propagated in 10-11 day old embryonated chicken eggs. eggs were inoculated via the allantoic route, and allantoic fluid was harvested two days following inoculation. the fluid was assayed for the presence of iav using a haemagglutination assay. rna was extracted from positive samples as previously described. egg isolation and extractions from allantoic fluid were performed by the molecular diagnostics department at the swedish national veterinary institute. full length ha, na, and m sequences were generated as described in wille et al. (2013) , and two samples were additionally deep sequenced in-house at the swedish national veterinary institute (virus 540/h10n3 and 816/h1n1). a fragment of the cov rdrp was sequenced as described in wille et al. (2016b) . the rdrp fragment generated during screening of astrov was used and subsequently cloned with pgem-t easy vector system (promega). all pcr products were purified by the wizard clean-up system (promega) and all sequencing was completed at macrogen (the netherlands). in the case of astroviruses, 3-5 clones of each sample were sequenced. resulting sequences were aligned using the mafft algorithm (katoh et al., 2009 ) within geneious 7 (biomatters, new zealand). phylogenetic models were determined in mega 6 (tamura et al., 2011) , and maximum likelihood trees were built using phyml (guindon and gascuel, 2003) implemented in seaview (gouy et al., 2010) and bootstrapped 10,000 times. reference sequences for phylogenetic analysis comprised of the top blast hits for each sequence generated in this study, as well as similar sequences from sweden. outgroup sequences were added to root all trees. all sequences generated in this study have been deposited in genbank under the accession numbers ky320400-39. seasonal prevalence for each virus was estimated using generalized additive models (gams) with binomial errors including a spline function of month using the mgcv package in r (r development core team, 2008). the best order polynomial was evaluated through akaike information criterion (aic) and given similar aic values the least complex model was selected (table a.1) . prevalence estimates of iav, cov and apmv-1 from this study were compared to those from wille et al. (2015) , wherein prevalence for these viruses was estimated in wild migratory mallards across the autumn season. we compared data from sept-dec, which represents large sample sizes in both studies. prevalence data were compared with fisher exact tests for the four rna viruses for each month. pvalues of b0.05 were taken to indicate a significant difference in the compared proportions. over the course of 13 months, 977 samples were collected from mallards. most of the samples collected were freshly deposited feces from the ground (n = 912), though 65 samples were fecal samples collected from captured birds. during the autumn months 100 samples were collected each month, with smaller sample sizes in the spring and winter (fig. a.1 ). prevalence of iav, apmv-1 and cov were low, with an overall prevalence of 1%, 1%, and 0.3% respectively, across the intra-annual sampling regime. as expected, prevalence for iav peaked during the autumn months, with only a single positive fecal sampled collected from the ground outside this period, in april. seasonal prevalence of other rna viruses mirrored patterns of iav, with a prevalence peak in the autumn through to the early winter, as well as a detection in april; cov in particular had a very similar prevalence curve to iav in both the temporal trend and amplitude. prevalence of apmv-1 was low, even in the autumn, with a single detection in august, november and december. interestingly, both iav and cov were detected in april, and this did not represent a co-infected sample (fig. 1) . in comparing prevalence [sept-dec] between our urban dataset and a wild bird dataset from southern sweden using the same qpcr methods (wille et al., 2015) , autumnal prevalence for iav (p b 0.0010) and cov (p b 0.0010) is significantly different, where prevalence for both these viruses is lower in urban mallards (fig. 2) . a more detailed comparison suggests that this effect is strongest in oct/nov (p b 0.0010) for iav and sept/oct (p = 0.0036, p = 0.0049) for cov. total autumnal prevalence, and monthly comparisons are not significantly different between these datasets for apmv-1, but this is driven by sample size constraints (fig. 2) . timing of prevalence peaks do vary across years, that is the prevalence peak may occur in a different month across years. however, the difference in prevalence between urban and wild ducks does not appear to be driven by a temporal mismatch in prevalence peaks. rather, the overall amplitude of the prevalence curves across the entire autumn for wild ducks and urban ducks are different, where the urban ducks consistently had lower prevalence for iav and cov (fig. 2) . overall for both iav and cov there was detected diversity, irrespective of lower prevalence compared to the wild migratory bird system. despite few iav detections, five subtype combinations were detected: h1n1, h3n8, h6n4, h10n3 and h10n4. furthermore h3 and h10, representing group 2 ha, were detected earlier in the season (september), followed by h6 and h1, group 1 ha viruses, in october and december, respectively (table a. 2). genetically the ha segment of h6 and h10 were similar to viruses previously detected in europe, including sweden (fig. a.4-a.5) . specifically the ha segment of h6 falls into a mixed clade containing viruses from europe, asia and north america (fig. a.4) . the ha of h10 is similar to sequences from sweden, the netherlands, as well as egypt, and the republic of georgia (fig. a.5) . the ha segment of both h3 and h1 fall into clades dominated by asian viruses (fig. 3, fig. a.2-a.3) . the h1n1 virus is further interesting as the na segment and the m segment are also most similar to asian viruses (fig. 3) ; as compared to the n3, n4 and n8 sequences and the m segment of the other viruses (table a .3, fig. a.6 ). all n4 sequences were identical, despite being detected in two different months and with different ha types (h10n4 in september and h6n4 in october) ( table a. 3). finally, the m segment of all viruses except h1n1 were highly similar (fig. a.6) . similarly, a diversity of cov rdrp fragments was present in this population (fig. 4) . all viruses were identified as gammacoronaviruses, and fell into the clade dominated by wild bird viruses and those recovered from domestic ducks. some sequences generated here were very similar to those from mallards migrating through southern sweden in 2011 (virus 485, 937). but, virus 271 and 665 were most similar to sequences from waterfowl coronaviruses isolated in hong kong. most interestingly, virus 271 and 665 were identical, despite being isolated 6 months apart (14 april and 13 october 2014, respectively), suggesting rdrp sequences falling into this clade were present in sweden despite not being previously detected (fig. 4) . we were unable to sequence apmv-1 given we were unable to culture these viruses and the original material had high ct values. against expectation, the highest rna virus prevalence in this study was that of astrov (fig. 1d) . furthermore, we detected astrov in 9/13 months, making it more pervasive than iav in this population. however, prevalence did follow the general seasonal trend where most detections occurred in the autumn migration period (fig. 1d) . additionally, 5/6 co-infected samples in the entire dataset were co-infected with astrov (astrov:flu = 3, astrov:cov = 2, cov:apmv-1 = 1). as with the other viruses, there was a diversity of astrov in the population (fig. 5) . indeed, we identified viruses in all three branches of the avian astrov tree. the virus detected in group 2that is viruses similar to avian nephritis virus -(virus 313) was the outgroup to this clade, suggesting undiscovered diversity, potentially in the wild bird reservoir. we similarly found an outgroup virus to group 3 viruses (virus 509), which are wild bird astroviruses detected thus far only in waterfowl. two other viruses also fell into group 3. most of the viruses sequenced were group 1 viruses, both group 1.2 and 1.3, and most viruses were similar to duck hepatitis viruses (eu669004 duck hepatitis virus 3 and eu669003 duck hepatitis virus 2). viruses were also similar to turkey astrovirus 2 (virus 137; e.g. dq066581) and chicken astrovirus (virus 553; e.g. eu668998). our preliminary findings suggest that group 1.2 was more common early in the year (february, april, august) and group 1.3 was more common towards the end of the study (september, november, december, january) (fig. 5b) , however a larger dataset is needed to confirm this putative trend. urbanization is intensifying world-wide, directly affecting interactions between humans and wild animals, in particular wild animals utilizing urban environments. in this study we aimed to characterize the dynamics of four avian rna viruses in wild birds utilizing an urban environment. these viruses, while causing no apparent clinical signs in wild waterfowl, are closely related to or have pathogenic variants, which may cause significant morbidity and mortality in poultry. in wild birds, especially waterfowl, iav has been intensively assessed, and we found that in an urban environment, annual dynamics of iav was similar to the global consensus. that is, very low prevalence in the spring and summer, with a higher prevalence in autumn and early winter when birds are migrating (olsen et al., 2006) . in this study prevalence is lower in urban ducks than wild migrating ducks, here significantly lower than wild ducks utilizing a stop-over site in southern sweden (latorre-margalef et al., 2014; wille et al., 2015) . there is some evidence that prevalence of iav might be lower in urban and sentinel populations, but this still needs more expansive assessment. verhagen et al. (2012) demonstrated that iav prevalence is inversely correlated with urbanization, and urban mallard prevalence is only 1.9%, which corroborates our findings. however, the bird species composition and temporal sampling between urban and rural areas were mismatched (verhagen et al., 2012) . in another study, in eastern canada, prevalence of largely non-migratory urban ducks in the city of st. john's, newfoundland, was 7.2%, with higher prevalence only reported when samples sizes were small (huang et al., 2013a) . one hypothesis for low viral prevalence in urban areas is host population structure and migratory propensity. concentrated resources presented in urban environments influence host migration and among/between species contact rates (altizer et al., 2011; bradley and altizer, 2006) . specifically, a larger proportion of urban ducks are non-migratory, although local movements do occur, particularly during breeding. due to a more sedentary lifestyle, following the initial input of susceptible ducklings after breeding, there is limited immigration, representing input of susceptible individuals across the autumn. in contrast, at a migratory stopover location such as ottenby, there is continual input of new individuals across the season representing both susceptible and infected birds (latorre-margalef et al., 2014) . the continual immigration creates a constant pool of susceptible birds and input of diverse ha subtypes. emigration allows for the removal of recovered birds from the system, allowing for higher viral prevalence across the autumn migration (altizer et al., 2011; avril et al., 2016) . lack of migration is also a feature of sentinel ducks, and prevalence and viral diversity was low in sentinel ducks being assessed on lake constance (globig et al., 2009; globig et al., 2012) and adult sentinel ducks in sweden (tolf et al., 2013a ). an interesting parallel is iav dynamics in africa where iav seasonality in fig. 2 . comparisons of autumnal prevalence for (a) iav, (b) cov and (c) apmv-1 between urban ducks (this study) and wild migratory ducks (wille et al., 2015) . mean and 95% confidence intervals shown for prevalence, and asterisks indicate a significant difference. "total" here is the combined number of viruses detected/total samples screened for the months of september, october, november and december. muted, and prevalence is very low. the putative driver is different life history strategies of waterfowl, wherein the classical patterns of waterfowl aggregation and migration in temperate regions are less pronounced, with only the subset of palearctic breeding waterfowl exhibiting long distance migration; afro-tropical waterfowl are resident or partial migrants likely due to more abundant resources. furthermore, an increase in iav prevalence was correlated to the influx of palearctic migrants (gaidet et al., 2012; gaidet et al., 2007) . in terms of population structure, urban ponds in uppsala are utilized by a single dabbling duck species -mallards; other dabbling duck and waterfowl species are absent limiting the breadth and size of the host reservoir. finally, urban mallards have access to more resources than their wild conspecifics due to supplemental feeding, which in turn may allow these individuals to mount a more efficient antiviral response (chandra, 1999; hall et al., 2009) , at both the innate (antiviral genes more highly unregulated) (barber et al., 2008; vanderven et al., 2012) and acquired level (length of antibody life) (magor, 2011) . unfortunately, there are few studies assessing the antiviral response to iav, and those that do exist are largely focussed on the response to highly pathogenic iav (barber et al., 2008; huang et al., 2013b; vanderven et al., 2012) . interestingly, despite the potential for an improved immune response, some studies suggest that an increase of resource may increase transmission potential and pathogen transmission (becker and hall, 2014; penczykowski et al., 2014) . despite empirical studies suggesting lower prevalence of iav in urban systems, however limited, theoretical studies imply that pathogen prevalence should be higher in these conditions (e.g. hall et al., 2014) . these theoretical studies have been verified by empirical work. for example, in monarch butterflies (danaus plexippus) that have lost migratory behaviour there is an increase in infection risk of a protozoan parasite (satterfield et al., 2015) . the reason for this conflict is unknown, however, one hypothesis is that these studies utilize a chronic disease model, whereas influenza is an acute infectious disease, and dynamics are driven largely by the herd immunity of the population (latorre-margalef et al., 2014; van dijk et al., 2014; wille et al., 2016a) . there are a number of factors which may be important drivers in dynamics of diseases in urban environments, including the relationship between provisioning, stress, pollution and immune response which affect susceptibility and ability to fight infection, however these are challenging to disentangle (becker et al., 2015; bradley and altizer, 2006; delgado and french, 2012; patz et al., 2004) , and these factors in relation to rna virus dynamics need to be assessed. while iav has been intensively assessed, we are only starting to explore dynamics of other avian rna viruses. indeed, this is the first intraannual dataset exploring dynamics of cov, apmv-1 and astrov and, furthermore, the first comparison between wild and urban settings for cov and apmv-1. it is also the second study assessing astrov in wild birds (chu et al., 2012) . given the economic implications of these viruses, our limited understanding of the dynamics and ecology of these viruses in wild birds is disquieting. perhaps unsurprising, overall annual trends in prevalence were similar for all viruses, and shared ecological drivers, such as those identified for iav (van dijk et al., 2014) , are the most parsimonious explanation for the shared patterns in long term dynamics of these viruses. that is, increased prevalence due to input of immunologically naïve birds into the system after breeding and aggregating of birds for autumn migration, and decreased prevalence in the winter following an increase in herd immunity (latorre-margalef et al., 2014; olsen et al., 2006; van dijk et al., 2014) . this dataset provides further evidence of the importance of waterfowl, urban or wild, in the epidemiology of cov and apmv-1. it is only within the last 10 years that cov have been assessed in wild birds and these viruses have largely been assessed using single time point studies, across a range of species, and using an array of different screening methods (e.g. chu et al., 2011; muradrasoli et al., 2010; wille et al., 2016b) . given the long history of apmv-1 research (alexander et al., 2012) , and "accidental" isolation of this virus in iav studies (e.g. jindal et al., 2009; ramey et al., 2013) , it is known that these viruses are present in waterfowl, however accurate prevalence estimates are still rare (tolf et al., 2013b; wille et al., 2015) . not all economically relevant avian rna viruses have been assessed in wild birds, and astroviruses are such an example. most strikingly, in this population, astrov might be more pervasive than iav, which has long been thought to be one of the most important rna viruses in wild waterfowl. not only was prevalence of astrov higher than iav, viruses were detected over a longer temporal interval. these viruses are particularly interesting due to the importance in poultry including chickens, turkeys and ducks, but the overall lack of assessment in wild birds leads to limited understanding of the epidemiology and ecology beyond food production birds. furthermore, this study corroborates chu et al. (2012) in that wild birds appear to contribute to the epidemiology of chicken or turkey "adapted" astrovirus strains. not only was the overall prevalence trend conserved across all viruses, the prevalence difference between wild and urban birds was also conserved for cov. this relationship between cov and iav, illustrated here by similar trends in urbanization could be due to a mutualistic relationship, that is prevalence of cov in waterfowl has been shown to be higher given infection with iav in wild migrating mallards (wille et al., 2015) . prevalence for apmv-1 was not significantly different between this urban population and a wild migratory population, however this could be driven by sample size constraintsthat is for a disease with a prevalence of b2% a much larger sample size is required to adequately assess prevalence with confidence (hoye et al., 2010) . furthermore, given the scarcity of prevalence studies in wild birds and diversity of methods used, it is not certain if this trend in apmv-1 is due to methodological constraints or whether there are different drivers of apmv-1 ecology. indeed, there might be an inverse relationship between apmv-1 and iav prevalence, where apmv-1 prevalence increases when iav prevalence decreases in wild mallards (tolf et al., 2013b; wille et al., 2015) . despite the factors associated with urbanization, this overall seasonal trend for these viruses, likely driven by shared ecological factors, remains clear. this study highlights our limited understanding of rna virus dynamics in birds in general, and more specifically, viruses in mallards. mallards are one of the most common avian species on the planet, which is owed to the fact that they are able to adapt to environments disturbed by human activities, and are a common sight in many cities (cramp and simmons, 1977; drilling et al., 2002) . mallards and other dabbling ducks are the natural reservoir for iav, are known to harbour high prevalence of iav in the wild, and may be implicated in the spread of highly pathogenic iav (latorre-margalef et al., 2014; van dijk et al., 2015; verhagen et al., 2015) . indeed, the h1n1 iav isolated in this study was more similar to viruses isolated in asia than europe suggesting long distance dispersal prior to circulation in this urban duck pond. this study was undertaken in 2014, prior to the influx of highly pathogenic h5n8 which were carried by apparently healthy birds (verhagen et al., 2015) . given these urban mallards harbour "asian" iav there is certainly concern for zoonotic spillover. furthermore, it is not a stretch to imagine that mallards may also be reservoirs and important in the spread and dynamics of other economically relevant rna viruses such as cov, apmv-1, and astrov. of all pathogens, rna viruses are the most likely to be zoonotic (woolhouse and gowtage-sequeria, 2005) , and it is in environments where humans are in close proximity to a high density of birds that zoonotic spillover is most likely to occur. for example, live-bird markets are central in the transmission of avian influenza viruses from birds to humans (wan et al., 2011) . the role of urban ducks, given low virus prevalence, is uncertain, however, to better understand the zoonotic risk a better understanding of the rna virus diversity and wildlife-pathogen dynamics in urban landscapes is crucial. newcastle disease in the european union the long view: a selective review of 40 years of newcastle disease research history of highly pathogenic avian influenza urbanisation and infectious diseases in a globalised world animal migration and infectious disease risk capturing individual-level parameters of influenza a virus dynamics in wild ducks using multistate models association of rig-i with innate immunity of ducks to influenza too much of a good thing: resource provisioning alters infectious disease dynamics in wildlife linking anthropogenic resources to wildlife-pathogen dynamics: a review and meta-analysis urbanization and the ecology of wildlife diseases nutrition and immunology: from the clinic to cellular biology and back again avian coronavirus in wild aquatic birds a novel group of avian astroviruses in wild aquatic birds novel astroviruses in insectivorous bats mallard (anas platyrhynchos) parasite-bird interactions in urban areas: current evidence and emerging questions detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations birds of north america online highly pathogenic avian influenza a subtype h5n8 economic and social impacts of avian influenza, fao emergency centre for transboundary animal diseases operations h5n1 highly pathogenic avian influenza global review. 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rna-dependent rna polymerase gene analysis of worldwide newcastle disease virus isolates representing different virulence types and their phylogenetic relationship with other members of the paramyxoviridae host range and emerging and reemerging pathogens novel reassortant influenza a(h5n8) viruses in domestic ducks, eastern china genomic analysis and surveillance of the coronavirus dominant in ducks in china we acknowledge the contribution of jon hessman and carolina stigwall to sample collection. positive controls were kindly provided by siamak zohari (sva) and camille lebarbenchon (inserm, reunion).ethical approval for trapping and sampling was obtained from the uppsala animal ethical committee (reference number c228/12), and a permit was obtained from the city of uppsala to capture, and swedish museum of natural history to ring birds. this work was supported by the swedish research council (grant number 2016-02606) vr and the swedish research council formas (grant number 211-2013-1320). supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2017.03.019. key: cord-102586-mx534xbx authors: javaid, w.; ehni, j.; gonzalez-reiche, a. s.; carreno, j. m.; hirsch, e.; tan, j.; khan, z.; kriti, d.; ly, t.; kranitzky, b.; barnett, b.; cera, f.; prespa, l.; moss, m.; albrecht, r. a.; mustafa, a.; herbison, i.; hernandez, m. m.; pak, t.; alshammary, h.; sebra, r.; smith, m.; krammer, f.; gitman, m.; sordillo, e. m.; simon, v.; van bakel, h. title: real time investigation of a large nosocomial influenza a outbreak informed by genomic epidemiology date: 2020-05-15 journal: nan doi: 10.1101/2020.05.10.20096693 sha: doc_id: 102586 cord_uid: mx534xbx background: nosocomial respiratory virus outbreaks represent serious public health challenges. rapid and precise identification of cases and tracing of transmission chains is critical to end outbreaks and to inform prevention measures. methods: we combined conventional surveillance with influenza a virus (iav) genome sequencing to identify and contain a large iav outbreak in a metropolitan healthcare system. a total of 381 individuals, including 91 inpatients and 290 health care workers (hcws), were included in the investigation. results: during a 12-day period in early 2019, infection preventionists identified 89 hcws and 18 inpatients as cases of influenza-like illness (ili), using an amended definition, without the requirement for fever. sequencing of iav genomes from available nasopharyngeal (np) specimens identified 66 individuals infected with a nearly identical strain of influenza a h1n1 (43 hcws, 17 inpatients, and 6 with unspecified affiliation). all hcws infected with the outbreak strain had received the seasonal influenza virus vaccination. characterization of five representative outbreak viral isolates did not show antigenic drift. in conjunction with iav genome sequencing, mining of electronic records pinpointed the origin of the outbreak as a single patient and a few interactions in the emergency department that occurred one day prior to the index ili cluster. conclusions: we used precision surveillance to identify and control a large nosocomial iav outbreak, mapping the source of the outbreak to a single patient rather than hcws as initially assumed based on conventional epidemiology. these findings have important ramifications for more effective prevention strategies to curb nosocomial respiratory virus outbreaks. nosocomial outbreaks of pathogens represent major challenges for health care providers and 63 institutions. it is critical for hospitals and health systems to not only quickly identify infected cases but 64 also determine the source of the outbreak in order to mitigate the threat to patients and health care 65 workers (hcws). nosocomial influenza virus outbreaks have been described worldwide (1-3); 66 children, the elderly, institutionalized and immuno-compromised patients are particularly vulnerable. in 67 some instances, nosocomial outbreaks have been caused by hcws who work while ill (4). influenza virus is a single-stranded, negative sense, segmented rna virus that causes an acute here we report the integration of our conventional infection prevention measures with precision 82 surveillance, including sequencing the genome of iav from clinical biospecimens and data mining of 83 electronic medical records (emrs), to successfully identify and control a large nosocomial iav 84 outbreak affecting both inpatients and hcws. epidemiology of the nosocomial influenza outbreak. in early 2019, symptoms suggestive of 88 influenza like illness (ili) were first observed in several hcws as well as in inpatients receiving critical 89 care at hospital a in nyc. at the direction of the infection prevention department, the hospital's 90 incident command system was activated. an extensive outbreak investigation was started, which 91 included mandatory staff symptom checks and testing of all inpatients with any respiratory symptoms, 92 regardless of fever status. enhanced cleaning of patient care areas and clinical staff workspaces was over the course of the hospital-wide outbreak investigation, a total of 381 individuals (91 inpatients 95 and 290 hcws) were screened by regular body temperature checks, symptom surveys and/or 96 molecular diagnostic testing for iav, ibv and respiratory syncytial virus (rsv). a total of 18 inpatients 97 (19.8%) and 89 hcws (29.7%) included in the epidemiological investigation tested positive for iav 98 ( figure 1a ). subtyping of iav from the nasopharyngeal (np) samples collected during the epidemiological 100 investigation (n=104), the routine influenza surveillance at hospital a (n=150) and hospital b 101 (n=231), revealed a stark increase of iav/h1 at day 4, 5 and 6 of the investigation ( figure 1b) . of 102 note, all the samples from inpatients and hcws included in the investigation that we successfully 103 subtyped harbored iav/h1n1, suggesting a single transmission chain. the 89 positive hcws were distributed across 29 different work assignment categories (figure 1c ), 105 predominantly front line care providers, including 24 resident physicians (residents, fellows, or interns), 106 16 registered nurses, 8 patient care assistants, and 6 attending physicians. eighty-seven of these 89 107 hcws (>90%) had been vaccinated with the quatrivalent seasonal influenza virus vaccine two to five 108 months (average: 108 days) prior to being tested positive for iav ( figure 1d) . importantly, these 109 infected hcws presented various, and mostly minor, clinical symptoms, and most individuals would 110 not have been classified as having "influenza-like illness" given the lack of fever ( figure 1e ). because 111 of this altered influenza disease manifestation in vaccinated hcws, the case definition was amended 112 early in the context of our investigation. genomics of the nosocomial influenza virus outbreak. in order to determine whether there was 115 transmission of a single iav strain or there were several independent introductions into the hospital 116 system, we performed next generation sequencing (ngs) of iav from the np specimens that were 117 banked following the initial diagnostic testing. as part of our institution's pathogen surveillance program, we routinely sequence influenza virus from a subset of the patients seeking care at our 119 hospitals (termed "surveillance"). thus, in addition to cases identified in the outbreak investigation, we 120 included surveillance samples obtained from the general patient population seeking care at our 121 hospitals as part of our institution's pathogen surveillance program in order to determine potential 122 community circulating strains. complete genomic sequences were obtained from 214 iav isolates (figure 2a) , including 126 from 124 hospital a (investigation and surveillance) and 88 from hospital b (surveillance only). pairwise 125 comparison of these viral genomic sequences showed a large cluster of 66 viral isolates that differed 126 by no more than 3 single-nucleotide variants (snvs), indicating that a single virus clone was 127 responsible for a large portion of the nosocomial outbreak ( figure 2b) . additionally, our analyses 128 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 15, 2020 . . https://doi.org/10.1101 /2020 indicated that other independent introductions of iav h1n1 strains, with limited forward transmissions, 129 had caused smaller clusters of ili at both hospital a and hospital b. we also noted two small 130 independent clusters due to transmission of iav h3n2 viruses. correlating virus genomic sequences with the timing and the source of these isolates showed that all 132 of the virus isolates obtained on day 0 and most of virus isolates on day 1 of the infection prevention 133 investigation were distinct from the viral isolate that caused the large outbreak. although two hcws 134 were tested positive for iav on day 0, their viruses were different from the one that caused the 135 outbreak, and not associated with any nosocomial transmission. all hcws infected with the outbreak 136 virus had received the seasonal influenza virus vaccine. the first isolate that clustered with the 137 outbreak virus strain was obtained from a patient identified on day 1 of the investigation ( figure 2c ). altogether, the genomic analyses of available clinical influenza isolates showed that cases identified 139 by the conventional epidemiological investigation encompassed patients and hcws who together cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020 . . https://doi.org/10.1101 /2020 sample matched the outbreak strain, and we used the pathospot framework (https://pathospot.org) 162 to query various electronic hospital records in order to create a timeline ( figure 4a ). the data showed that 4 of the 9 initial nosocomial iav cases were seen (patients p1, p2, and p3) or 164 worked (hcw1) in the emergency department (ed) on the same day (day -1), during overlapping 165 time periods. the 3 patients were admitted from the ed to different wards and had no other shared 166 interactions with hcws, indicating that the common exposure most likely occurred in the ed. similarly, because one of the patients who acquired nosocomial iav did not have direct contact with 168 hcw1 and had already been transferred out prior to the time hcw1 was present in the ed, the 169 evidence indicates hcw1 was exposed during that work shift rather than being the primary case. in contrast, patient p3, the putative primary case for this outbreak, was brought to the ed in the 171 morning of day -1, several hours before p1 and p2, and interacted with hcw1. p3 was admitted to a 172 medical unit the same day ( figure 4a , grey) with fever, shaking chills, dyspnea, and abdominal pain, but developed systemic inflammatory response syndrome and was transferred to the icu where the 174 patient was intubated, a procedure that can generate significant aerosols (7) because blood cultures 175 of p3 grew gram-negative bacteria, the diagnosis of iav was delayed. however, the patient remained 176 febrile despite antibiotics prompting diagnostic testing for influenza virus on day 3. the next three early cases (hcw2, p4 and p5) most likely acquired infection in the icu from p3, 178 although p4 and p5 had further overlapping stays following transfer to the same medical/surgical 179 inpatient unit from the icu. our data suggests that cases p6 and p7, who were admitted through the 180 ed several days after the start of the outbreak, acquired iav infection from ed hcws who had been 181 exposed to p3 and became iav-test positive in the days thereafter. the interaction network based on available contact records ( figure cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. since we routinely sequence influenza virus isolates from patients receiving care at our health system 219 as part of our pathogen surveillance program, we could compare the strains from the outbreak 220 investigation conducted at hospital a to the strains found in the surveillance of hospital a as well as 221 hospital b (figure 3) . these additional data allowed us to not only identify previously unrecognized 222 smaller transmission events (four inpatients and two hcws at hospital a and six patients at hospital 223 b) but also ascertain that there was a large (in number) but limited (in time) outbreak of h1n1 in our 224 health system (figures 2 and 3) . importantly, this specific h1n1 virus outbreak strain did not spread 225 further in the community. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. . https://doi.org/10. 1101 /2020 a limitation of our study is that we did not have access to biospecimens from 22 hcws whose tests 227 were performed at laboratories outside our health system. additionally, only partial viral genomes 228 could be retrieved from two of the available biospecimens linked to the epidemiological outbreak 229 investigation. however, we were able to obtain viral genomes for all the patients identified during the 230 first three days of the outbreak, providing a solid foundation for the reconstruction of the transmission 231 chain. our data suggest that the outbreak began in the ed most likely through introduction of the virus by a 233 single patient, who had received aggressive resuscitative care and was subsequently transferred to 234 the intensive care unit of hospital a (figure 4) . a possible solution to mitigate such risks to hcws 235 and patients in the future is to enhance screening and isolation of patients coming into the ed with 236 any respiratory symptoms, even when an alternative diagnosis seems to be the predominant . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. among these 381 individuals, a total of 89 hcws and 18 patients were found to be infected with iav. a high incidence of cases was found among hcws, including interns, residents, fellows, and rotators, 267 as well as nursing and associated fields. clusters were found in several areas inside the hospital 268 including intensive care units and several medical/surgical floors and an inpatient psychiatric unit. prophylaxis with oseltamivir was offered to exposed hcws and inpatients. treatment with oseltamivir 270 was provided to infected inpatients and hcws, in addition to extended sick leave for hcws who 271 remained afebrile but symptomatic. there was no mortality reported in association with the outbreak. one patient required readmission secondary to ili. we educated staff throughout the hospital 273 including the trainees on symptoms associate with influenza and encouraged all to report to their 274 supervisors if they had any symptoms. mandatory symptom checks at beginning of each shift were 275 also implemented. anyone symptomatic was encouraged to get tested. we were able to contain this 276 outbreak from detection to eradication within 10 days, with incredible collaboration between many the time frame from which surveillance and investigation samples were included covered a total of 27 284 days, starting from six days before the investigation to seven days after the 12 days long outbreak 285 investigation. viral rna was extracted from 280µl of np-utm using the qiaamp viral rna minikit 286 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. were then used to construct a multi-genome alignment of isolates in each cluster using parsnp. (9)pairwise distances between genomes were then calculated as the number of single nucleotide 319 variants (snvs) between genome alignments in each cluster for further analysis. to identify 320 transmission events we used the pathospot heatmap view (fig. 2) . we set a threshold of ≤3 snvs 321 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020 . . https://doi.org/10.1101 /2020 to identify potential transmissions. next, the dendro-timeline view was used to perform phylogenetic 322 analysis of outbreak isolates, and to reconstruct the early outbreak timeline based on the full 323 admission/transfer/discharge (adt) history for each patient obtained from the electronic medical 324 record. finally, the adt history was combined with patient-hcw interaction data to reconstruct a 325 network of all known contacts in cytoscape.(10). in order to confirm phylogenetic grouping, we inferred a second ml analysis at the whole genome 340 level for all strains that belonged to the highest supported clade that contained the outbreak isolates. hemagglutination inhibition (hi) assay. these assays were performed as described before (12, 13). briefly, serum samples from eight healthy study participants receiving the seasonal influenza virus 355 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. palese for guidance and many thought provoking discussions. we are indebted to dr. c. cordoncardo for continued support of the pathogen surveillance program. we would like to thank the staff, the nursing leadership, including christine mahoney and maria we gratefully acknowledge the authors, originating and submitting laboratories of sequences from 379 gisaid's epiflu (www.gisaid.org) that were used as background for our phylogenetic inferences. the 380 list of authors and submitting laboratories is shown in table s1 . (s10od018522 and s10od026880) and institutional seed funds. this manuscript was edited by life science editors. 386 387 388 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020 . . https://doi.org/10.1101 /2020 authors contributions: cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020 . . https://doi.org/10.1101 /2020 cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 15, 2020. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 15, 2020 . . https://doi.org/10.1101 /2020 nosocomial transmission of influenza: a retrospective cross-467 sectional study using next generation sequencing at a hospital in england hospital-acquired influenza infections detected by a surveillance system over six 472 seasons characterisation of 474 nosocomial and community-acquired influenza in a large university hospital during two 475 consecutive influenza seasons nosocomial outbreak of 478 influenza a h3n2 in an inpatient oncology unit related to health care workers presenting 479 to work while ill donnell 482 mr. 2019. precision surveillance for viral respiratory pathogens: virome capture 483 sequencing for the detection and genomic characterization of severe acute respiratory 484 infection in uganda use of whole sequencing in the investigation of a nosocomial influenza virus outbreak aerosol generating 490 procedures and risk of transmission of acute respiratory infections to healthcare workers: 491 a systematic review origins of the 2009 h1n1 influenza pandemic in swine in 495 mexico. elife 5 the harvest suite for rapid core-497 genome alignment and visualization of thousands of intraspecific microbial genomes cytoscape: a software environment for integrated models of 501 biomolecular interaction networks raxml version 8: a tool for phylogenetic analysis and post-analysis 503 of large phylogenies antigenic sites in influenza 506 h1 hemagglutinin display species-specific immunodominance key: cord-003772-1345qct4 authors: kummer, susann; avinoam, ori; kräusslich, hans-georg title: ifitm3 clusters on virus containing endosomes and lysosomes early in the influenza a infection of human airway epithelial cells date: 2019-06-12 journal: viruses doi: 10.3390/v11060548 sha: doc_id: 3772 cord_uid: 1345qct4 interferon-induced transmembrane proteins (ifitms) have been shown to strongly affect influenza a virus (iav) infectivity in tissue culture. moreover, polymorphisms in ifitm3 have been associated with the severity of the disease in humans. ifitm3 appears to act early in the infection, but its mechanism of action and potential interactions with incoming iav structures are not yet defined. here, we visualized endogenous ifitm3 interactions with iav in the human lung epithelial cell line a549 and in primary human airway epithelial cells employing stimulated emission depletion super-resolution microscopy. by applying an iterative approach for the cluster definition and computational cluster analysis, we found that ifitm3 reorganizes into clusters as iav infection progresses. ifitm3 cluster formation started at 2-3 h post infection and increased over time to finally coat iav-containing endosomal vesicles. this iav-induced phenotype was due to the endosomal recruitment of ifitm3 rather than to an overall increase in the ifitm3 abundance. while the iav-induced ifitm3 clustering and localization to endosomal vesicles was comparable in primary human airway epithelial cells and the human lung epithelial cell line a549, the endogenous ifitm3 signal was higher in primary cells. moreover, we observed ifitm3 signals adjacent to iav-containing recycling endosomes. influenza a virus (iav) is the major cause for a contagious illness of the upper and lower respiratory tract during the seasonal influenza epidemics with peaks in fall and winter for each hemisphere [1] [2] [3] . besides the acute risk through circulating human specific strains, the zoonotic reservoir (e.g., birds, swine) poses a constant threat of new influenza pandemics [4] [5] [6] [7] . iav hijacks cellular import mechanisms to enter the host cell, thereby making use of multiple entry routes. the binding of iav by the sialylated receptor [8, 9] is followed by clathrin-mediated endocytosis [10, 11] or micropinocytosis [12] . it is reported that iav entry is cell-type dependent [13] and that subtypes with a filamentous particle shape preferentially enter host cells via macropinocytosis [14] . the trimeric surface glycoprotein hemagglutinin (ha) is a key factor for several steps during viral entry via endocytosis [15, 16] . irrespective of the entry mechanism, iav exploits the non-linear endosomal pathway with its multitude of branches and needs to pass different stages of the endocytic machinery, which is assembled and constantly renewed around the internalized virus particles [17] [18] [19] . endosomal trafficking to the perinuclear region is crucial for ph-dependent membrane fusion [20] [21] [22] , followed by the release of the viral genome into the cytosol and nuclear import [23] . the iav genome is composed of eight single rna strands in a negative sense orientation [24] , and each segment is complexed with the nucleoprotein np [15, 24] , forming ribonucleoparticles (rnps; [25, 26] ). viral genome replication, splicing and transcription then occur in the nucleus. iav replication is strongly inhibited by type 1 interferons already at the early stage, with mx1 being identified as a crucial interferon-induced host protein blocking iav replication [27] . genome-wide sirna screens have identified many host factors modulating influenza virus infection [28] [29] [30] [31] [32] . brass et al., 2009 [32] and shapira et al., 2009 [33] reported the strong inhibition of iav infection by interferon-induced transmembrane proteins (ifitms). the ifitm variants 1-3 can be induced by interferon i and ii. their localization is cell type-or tissue-dependent and changes with the expression level with a preference for cytoplasmic vesicles in most monolayer cell lines [34] [35] [36] [37] [38] . besides iav, ifitm proteins have also been reported to confer a basal resistance to members of the flaviviridae (dengue and west nile virus) [39] , bunyaviridae (rift valley fever virus) [40] , filoviridae (ebola virus) [41] , coronaviridae (sars) and retroviridae (hiv) [32] , showing an unusually broad activity against a wide variety of enveloped and some non-enveloped viruses [32] . however, murine leukemia virus (mlv) is not restricted by ifitm3 despite being an enveloped virus [32] . the relevance of the ifitm3-mediated inhibition of iav infection received strong support when it was shown that ifitm3 polymorphisms correlated with the severity of iav disease in human infection [42] . the relevance of the ifitm3 rs12252-c polymorphism for severe iav infection appears to be population-dependent [43] [44] [45] [46] [47] [48] , and a second ifitm3 single nucleotide polymorphism, rs34481144-a, was also reported to influence the severity of iav infection in humans [49] . however, the mechanism of the ifitm-mediated inhibition of iav infection remains under discussion. ifitms have been suggested to disrupt viral membrane fusion [50, 51] by altering cellular membrane properties such as fluidity and curvature [38, 52, 53] . it has also been discussed that ifitms alter the lipid/protein composition of acidic intracellular compartments such as endosomes and lysosome [37, 54] . the lipid composition-based models of the ifitm3 function cannot easily explain the lack of antiviral effects on viruses like amphotropic mlv or arenaviruses [55, 56] , which also enter by endocytosis and endosomal fusion. in the case of iav, it was recently reported that ifitm3 elevates the level of cholesterol on late endosomes and lysosomes, thereby restricting early iav infection [57] . other hypotheses suggest that ifitm3 directly interferes with the iav fusion pore formation or redirects iav containing endosomes to a non-productive pathway [41] . it was further demonstrated that the inhibition of the hemagglutinin-mediated membrane fusion required the amphipathic helix of ifitm3 [58] . most findings about the antiviral action and localization of ifitm3 are based on studies using expression plasmids creating an over-expression and/or focusing at the late stages of infection (>12 h post-infection) [37, 38, 51] . due to the enormous protein load in the overexpression situation, it is far more difficult to determine the subtle differences in the localization of individual molecules, and thus it is possible that these changes may be overlooked. in addition, proteins whose expression is enhanced artificially are more prone to be degraded via the lysosomal pathway. in this regard, it is difficult to judge whether this is a natural re-localization as part of the antiviral defense or just a degradation via lysosomes, particularly in the late stages of infection. we therefore investigated the role of endogenous ifitm3 in the viral uptake at an early stage. we analyzed the localization of endogenous ifitm3 through the course of iav infection using confocal and super-resolution (sted) microscopy. we observed a strong clustering of ifitm3 early after iav infection, which was distinct from the interferon alpha induced ifitm3 up-regulation. moreover, a two-color sted nanoscopy revealed a close proximity of ifitm3 with the iav np protein in rab11 positive compartments within a time range of 1-6 h after the iav addition. a549 and mdck cells were maintained in dulbecco's modified eagle medium (dmem) (invitrogen, karlsruhe, germany), supplemented with 10% foetal calf serum (fcs) (invitrogen) and penicillin (200 u/ml)/streptomycin sulphate (100 µg/ml) (capricorn scientific gmbh, ebsdorfergrund, germany). human small airway epithelial cells (hsaepcs) were obtained from promocell (c-12642) and grown in a primary cell basal medium (promocell) without antibiotics. four days after thawing, the hsaepcs were seeded for immunofluorescence. the ifitm3 knock-down in a549 cells was induced by crispr-cas9 gene editing using a single guide rna targeting the polyadenylation side of ifitm [59, 60] . a549 ifitm3 knock-down cells were selected by puromycin resistance (10 µg/ml). the ifitm3 expression was checked by real-time quantitative pcr, as described by zhao et al., 2013 [61] using the 7500 fast real time pcr system (applied biosystems, lifetechnologies, sigma-aldrich, steinheim, germany) and by immunofluorescence, as noted below ( figure s5 ). in general, the cells subjected to immunofluorescence were seeded in 24 well plates on 10 mm glass cover slips. for immunoblotting, cells were seeded in 6 well plates. human interferon alpha (hifnα) (sigma-aldrich, steinheim, germany) was used at 1000 u/ml for 24 h prior to infection. amantadine (sigma-aldrich) and bafilomycin a1 (merck, darmstadt, germany) were applied to the cells at 5 µm and 10 nm concentrations 30 min prior to infection. the cells were treated with mouse anti-hifnα (catalogue no. ab11408, abcam, berlin, germany). influenza a virus a/hong kong/1/1968 (hk/1/68), a/puerto rico/8/1934 (pr/8/34) and a/regensburg/ d6/2009 (r/d6/09) were recovered in a hek 293t-mdck-coculture using the eight plasmid reverse genetics systems, as previously described [62] . in live-cell experiments, cell mask™ orange plasma membrane stain (catalogue no. c10045, life technologies, sigma-aldrich, steinheim, germany) was directly added to the cell supernatant prior to the virus purification, following the manufacturer's guidelines. the virus stocks were titrated, making use of a standard plaque assay with minor changes, as previously described [63] . briefly, serial 10-fold dilutions of each virus stock were prepared in dmem supplemented with 0.3% bovine serum albumin (bsa, sigma-aldrich, steinheim, germany), 20 mm hepes (sigma-aldrich, steinheim, germany), penicillin (200 u/ml)/streptomycin sulphate (100 µg/ml) (capricorn scientific gmbh, ebsdorfergrund, germany) and tpck-trypsin (2 µg/ml) (sigma-aldrich, steinheim, germany). mdck cell monolayers were incubated with 200 µl of each dilution for 1 h at 37 • c in a 6 well plate format. then, the cells were overlaid with 2 ml of 2.4% avicel (fmc) in aqua dest. and 2 × dmem (merck, darmstadt, germany) in a 1:1 ratio. after 5 days, the overlay was removed, the cells were fixed with 1 ml/well of 70% ethanol for 30 min at room temperature and stained with 1 ml/well of 0.3% crystal violet (sigma-aldrich, steinheim, germany) in 20% methanol for 10 min at room temperature. the cells were fixed using 4% paraformaldehyde (pfa) in 1 × phosphate buffered saline (pbs) for 10 min at 37 • c and subsequently treated with 0.05% triton-x100 in pbs for 5 min. blocking was performed using 5% bsa in pbs for 10 min. the following primary antibodies were used for immunofluorescence: mouse anti-np (catalogue no. mab8257, merck, darmstadt, germany), rabbit anti-ifitm3 (catalogue no. 11714-1-ap, proteintech, manchester, united kingdom), mouse anti-eea1 (catalogue no. 610457, bd transduction laboratoriestm, san jose, ca, usa), mouse anti-rab7 (catalogue no. ab50533, abcam, berlin, germany), mouse anti-rab11 (catalogue no. sc-6565, santa cruz, dallas, texas, usa), and mouse anti-lamp1 (catalogue no. ab25630, abcam, berlin, germany). the cells were incubated with the indicated primary antibodies in a blocking solution for 1 h at room temperature, and washed three times in pbs. the fixation, cellular membrane permeabilisation, and blocking were repeated between the first and secondary antibody incubation, as described above. for sted microscopy, goat anti-mouse star red (catalogue no. 2-0002-011-2, abberior, göttingen, germany) and goat anti-rabbit atto594 (catalogue no. abin964988, antibodies-online gmbh) were used as the secondary antibodies. for the triple staining, including the non-diffracted 488 nm channel, donkey anti-goat alexa 488 (catalogue no. ab150129, abcam, berlin, germany) was used. the secondary antibody staining was performed for 1 h at room temperature under exclusion of light. after repeated washing in pbs, cover slips were mounted on the objective slides using mowiol. whole-cell lysates were prepared using a 6 × lysis buffer (10% sucrose, 0.1% bromophenol blue, 5 mm edta ph 8.0, 200 mm tris ph 8.8, 10% sds, and 2% beta-mercaptoethanol). protein samples were resolved by 12.5% sds-page at 160 volt for 1.5 h. then, the proteins were transferred to immobilon-fl membranes (merck, darmstadt, germany). the membranes were blocked in li-cor blocking buffer for 30 min at room temperature and probed with first antibodies (rabbit anti-ifitm3, catalogue no. 11714-1-ap, proteintech, manchester, united kingdom, and rabbit anti-actin, catalogue no. a2066, sigma-aldrich, steinheim, germany, mouse anti-tubulin no. t5168, sigma-aldrich, steinheim, germany, or mouse anti-gapdh, catalogue no. sc365062, santa cruz, dallas, texas, usa) in a blocking solution diluted 1:5000 at 8 • c overnight. after three repeated washings in 1 × tbst for 15 min, the membranes were incubated with a secondary donkey anti-mouse 800 antibody (catalogue no. 926-32212, li-cor) or secondary donkey anti-mouse 700 antibody (catalogue no. 925-68072, li-cor, lincoln, nebraska usa), for 1 h at room temperature in the dark. the protein bands were visualized using the li-cor odyssey scanner (li-cor, lincoln, nebraska usa). fluorescence imaging was performed using a two-colour-sted microscopy instrumentation (abberior instruments gmbh, göttingen, germany). for confocal and sted microscopy, a 100× olympus uplansapo (na 1.4) oil immersion objective was used. for excitation (λ= 594 nm and λ = 640 nm), a nominal laser power of 20% was applied, and for sted (λ = 775 nm, max. power = 1.2 w), a nominal laser power of 70 % was applied. the pixel size was set to 60 nm (confocal) and 15 nm (non-diffracted), respectively. minor adjustments of contrast and brightness of the acquired images as well as the richardson-lucy deconvolution with a regularisation parameter of 0.001 (stopped after 30 iterations) were carried out with the imspector software (16.1.7098-win64-aifpgav3, abberior instruments gmbh, göttingen, germany). the 2d object counter and coloc2 plugin in fiji software (https://fiji.sc/, 64-bit version)were used for the cluster and mander's coefficient correlation (mcc) analyses, respectively. mcc was performed using the costes regression threshold. the defined objects were assigned as a cluster being larger than 10 5 nm 2 based on sted images with approximately 50 nm resolution. all graphs show the mean values of three independent experiments plotted with the standard error calculated as a two tailed unpaired t-test. a statistical analysis was performed using graphpad prism software (version 5). to visualize the subcellular localization of endogenous ifitm3, we performed immunofluorescence microscopy of the uninfected and iav infected a549 cells. the basal ifitm3 levels of the uninfected cells showed weak cytosolic signals upon immunofluorescence staining. following infection with iav a/hong kong/1/1968 (hk/1/68) for 24 h, most cells exhibited strong and clustered ifitm3 signals in the extranuclear space ( figure 1a ). the ifitm3 signal increase and clustering correlated with the overall dim np signals in the extranuclear space and no nuclear np signals in the respective cells. in contrast, the productively iav infected a549 cells (exhibiting a strong nuclear np signal) in the same field of view did not exhibit an increased ifitm3 signal intensity or clustering ( figure 1a , right panels). given that we used a relatively high moi of one and that dim np signals were generally detected in these cells, these observations strongly argue that iav infection was abortive in the cells exhibiting an early accumulation of ifitm3. ifitm3 is induced by type 1 interferon [34] . therefore, we analyzed its distribution upon the treatment of a549 cells with human interferon alpha (hifnα) for 24 h and compared this to a549 cells which had been pretreated with hifnα and subsequently infected with iav ( figure 1b ). the cells treated for 24 h with hifnα exhibited an increased ifitm3 signal intensity in the extranuclear ifitm3 is induced by type 1 interferon [34] . therefore, we analyzed its distribution upon the treatment of a549 cells with human interferon alpha (hifnα) for 24 h and compared this to a549 cells which had been pretreated with hifnα and subsequently infected with iav ( figure 1b ). the cells treated for 24 h with hifnα exhibited an increased ifitm3 signal intensity in the extranuclear space, both in the uninfected a549 cells and in the iav-infected cells with a dim or absent np signal. importantly, the productively infected cells exhibiting strong nuclear np signals again exhibited a weak ifitm3 signal, even after the pretreatment with hifnα. comparing the non-treated and hifnα-pretreated iav-infected a549 cells lacking a strong nuclear np signal, we observed an increased ifitm3 signal in both cases, partially colocalizing with vesicular structures. vesicular localization appeared more obvious in the absence of a hifnα pretreatment with a less diffuse cytosolic signal. blocking ifnα with a neutralizing antibody during the iav infection of a549 cells resulted in increased infection rates, and a concomitant minimal increase of the ifitm3 signal ( figure 1c ). to determine whether an iav-induced viral membrane fusion and genome uncoating are required for the observed ifitm3 signal increase upon iav infection, we performed experiments in the presence of bafilomycin a1, specifically inhibiting endosomal acidification, or in the presence of amantadine, specifically blocking the tetrameric m2 channel of iav, thereby preventing genome uncoating. in both cases, no increase in the ifitm3 signal intensity and no ifitm3 clustering were observed ( figure 1d ,e). these results indicate that iav-induced membrane fusion and genome uncoating are required for an ifitm3 signal increase in a549 cells. the mechanism by which ifitm3 impedes iav infection was divergent in previous studies, and inhibition was mostly described upon ifitm3 over-expression [37, 38, 51] . it was demonstrated that an ifitm3 knockdown in a549 cells increases infection rates [37] . to determine whether endogenous ifitm3 is relevant for iav infection in a549 cells under the experimental conditions of our study, we performed iav infection in a549 ifitm3 knock-down cells ( figure s1a ,b). the ifitm3 expression was reduced by a factor of 10 (on the mrna level) in these cells ( figure s1c ). the iav infection rate was significantly increased in the knock-down situation compared to the a549 wildtype cells (figure 2a ). viruses 2019, 11, x for peer review 6 of 17 space, both in the uninfected a549 cells and in the iav-infected cells with a dim or absent np signal. importantly, the productively infected cells exhibiting strong nuclear np signals again exhibited a weak ifitm3 signal, even after the pretreatment with hifnα. comparing the non-treated and hifnαpretreated iav-infected a549 cells lacking a strong nuclear np signal, we observed an increased ifitm3 signal in both cases, partially colocalizing with vesicular structures. vesicular localization appeared more obvious in the absence of a hifnα pretreatment with a less diffuse cytosolic signal. blocking ifnα with a neutralizing antibody during the iav infection of a549 cells resulted in increased infection rates, and a concomitant minimal increase of the ifitm3 signal ( figure 1c ). to determine whether an iav-induced viral membrane fusion and genome uncoating are required for the observed ifitm3 signal increase upon iav infection, we performed experiments in the presence of bafilomycin a1, specifically inhibiting endosomal acidification, or in the presence of amantadine, specifically blocking the tetrameric m2 channel of iav, thereby preventing genome uncoating. in both cases, no increase in the ifitm3 signal intensity and no ifitm3 clustering were observed ( figure 1d ,e). these results indicate that iav-induced membrane fusion and genome uncoating are required for an ifitm3 signal increase in a549 cells. the mechanism by which ifitm3 impedes iav infection was divergent in previous studies, and inhibition was mostly described upon ifitm3 over-expression [37, 38, 51] . it was demonstrated that an ifitm3 knockdown in a549 cells increases infection rates [37] . to determine whether endogenous ifitm3 is relevant for iav infection in a549 cells under the experimental conditions of our study, we performed iav infection in a549 ifitm3 knock-down cells ( figure s1a ,b). the ifitm3 expression was reduced by a factor of 10 (on the mrna level) in these cells ( figure s1c ). the iav infection rate was significantly increased in the knock-down situation compared to the a549 wildtype cells ( figure 2a ). the increase in the ifitm3 signal intensity in a549 cells upon iav infection ( figure 1a ) and interferon treatment ( figure 1b ) could be caused either by higher expression levels or by ifitm3 clustering, yielding viruses 2019, 11, 548 7 of 18 a higher density of the signal. to distinguish between these possibilities, we determined the ifitm3 abundance by an immunoblot analysis of the a549 cells at different time points after iav infection or interferon treatment, respectively. no significant increase in the ifitm3 expression was observed during the first 6 h of iav infection ( figure 2b) , indicating that the signal changes observed by fluorescence microscopy (examples are shown in figure 3 ) during this period were caused by the re-localization of constitutively expressed ifitm3 rather than by an induced ifitm3 expression. a strong increase in the ifitm3 expression levels was observed at late time points (24 h p.i.; figure s2b ), in accordance with previous studies [32] . an increased ifitm3 expression was also observed following the interferon treatment, but starting already at 6 h after the interferon addition ( figure s2 ), as previously reported [32] . accordingly, the immunofluorescence phenotype after the interferon treatment ( figure 1b) is likely to be caused by an increased expression rather than by clustering. the increase in the ifitm3 signal intensity in a549 cells upon iav infection ( figure 1a ) and interferon treatment ( figure 1b ) could be caused either by higher expression levels or by ifitm3 clustering, yielding a higher density of the signal. to distinguish between these possibilities, we determined the ifitm3 abundance by an immunoblot analysis of the a549 cells at different time points after iav infection or interferon treatment, respectively. no significant increase in the ifitm3 expression was observed during the first 6 h of iav infection ( figure 2b) , indicating that the signal changes observed by fluorescence microscopy (examples are shown in figure 3 ) during this period were caused by the re-localization of constitutively expressed ifitm3 rather than by an induced ifitm3 expression. a strong increase in the ifitm3 expression levels was observed at late time points (24 h p.i.; figure s2b ), in accordance with previous studies [32] . an increased ifitm3 expression was also observed following the interferon treatment, but starting already at 6 h after the interferon addition ( figure s2 ), as previously reported [32] . accordingly, the immunofluorescence phenotype after the interferon treatment ( figure 1b) is likely to be caused by an increased expression rather than by clustering. to unravel the distribution pattern of ifitm3 in a549 cells at early stages of infection, we made use of super-resolution microscopy. the blurring effect of diffraction limited imaging can obfuscate the potential clustering of ifitm3. stimulated emission depletion (sted) super-resolution microscopy was therefore applied to resolve ifitm3 clusters in early iav-infected a549 cells. cells infected with iav hk/1/68 were fixed at 1-6 h post infection (h p.i.), stained with anti-ifitm3 and subjected to confocal and sted microscopy (resolution < 50 nm) of the same region ( figure 3a ). an increased ifitm3 signal intensity was again observed in the early phase of the iav infection, and individual ifitm3 clusters could be resolved by sted microscopy. we computationally analysed the ifitm3 subcellular localization based on raw sted images using a 2d cluster analysis (objects counter). the clusters were defined as extranuclear ifitm3 signal accumulations with a size of > 10 5 nm 2 . the threshold was chosen by an iterative approach searching for the proportion of clusters, with the critical size being altered upon the infection progression. the analysis of uninfected and hk/1/68-infected a549 cells at different time points revealed a very low proportion of large clusters in uninfected cells or up to 2 h p.i. at 3 h p.i. and later, we observed a significant increase in cluster abundance ( figure 3b although a549 cells are an established model cell line for iav research, adaptation to cell-culture conditions may have occurred; hence, we verified our results in primary human respiratory epithelial cells. to determine whether iav infection also causes ifitm3 clustering in primary cells, we infected human small airway epithelial cells (hsaepcs) with iav pr/8/34 for different periods of time and performed an indirect immunofluorescence analysis for ifitm3 and np (from incoming iav particles) using confocal and sted microscopy ( figure 4 , uninfected example see figure s5 ). the iav infected hsaepcs revealed a co-localization of ifitm3 and iav np signals at apparently vesicular structures as early as 1 h p.i. (figure 4a , white arrowheads). at later time points (3 h p.i.), this was more obvious, with a clear ifitm3 clustering on the np-containing vesicular structures ( figure 4b ). ifitm3 often exhibited a ring-like appearance, suggesting the coating of endosomal vesicles, and this phenotype became more obvious at later time points (6 h p.i; figure 4c ). some ifitm3-positive vesicles exhibited strong np signals (e.g., figure 4b ), suggesting ifitm3-coated vesicles carrying multiple iav particles. a strong ifitm3 clustering with a ring-like appearance indicating vesicle coating was observed in both iav-infected a549 cells ( figure 5a ) and hsaepcs at 10 h p.i. (figure 5c ; white arrowheads indicating ring-like structures). in the case of the a549 cells, the ifitm3 signals in uninfected cells were weak and mostly diffuse. upon iav infection, > 90% of cells lacking a strong nuclear np signal displayed ifitm3 clustering (e.g., the right cell in figure 5a ), while this was only observed in < 10% of cells with a strong nuclear np ( figure 3b ). given the high infection rate in this cell line and the fact that cytoplasmic np was also observed in cells lacking the bright nuclear np of replicating iav ( figure 1a) , we suggest that early ifitm3 clustering at vesicular structures correlates with an abortive infection in this cell type, and that only cells that do not induce the vesicular clustering of ifitm3 become infected. in general, the ifitm3 signals were stronger in hsaepcs compared to a549 cells ( figure 5a,c) , and ifitm3 clusters apparently coating cytoplasmic vesicles were also present in productively iav-infected cells exhibiting a strong nuclear np signal in this case ( figure 5d ). no ifitm3 clustering at vesicular structures was observed in hsaepcs in the absence of iav infection, while the overall ifitm3 signal intensity was much higher in these primary cells compared to a549 cells without infection ( figure s5 ). a strong ifitm3 clustering with a ring-like appearance indicating vesicle coating was observed in both iav-infected a549 cells ( figure 5a ) and hsaepcs at 10 h p.i. (figure 5c ; white arrowheads indicating ring-like structures). in the case of the a549 cells, the ifitm3 signals in uninfected cells were weak and mostly diffuse. upon iav infection, > 90% of cells lacking a strong nuclear np signal displayed ifitm3 clustering (e.g., the right cell in figure 5a ), while this was only observed in < 10% of cells with a strong nuclear np ( figure 3b ). given the high infection rate in this cell line and the fact that cytoplasmic np was also observed in cells lacking the bright nuclear np of replicating iav (figure 1a) , we suggest that early ifitm3 clustering at vesicular structures correlates with an abortive infection in this cell type, and that only cells that do not induce the vesicular clustering of ifitm3 become infected. in general, the ifitm3 signals were stronger in hsaepcs compared to a549 cells ( figure 5a,c) , and ifitm3 clusters apparently coating cytoplasmic vesicles were also present in productively iav-infected cells exhibiting a strong nuclear np signal in this case ( figure 5d ). no ifitm3 clustering at vesicular structures was observed in hsaepcs in the absence of iav infection, while the overall ifitm3 signal intensity was much higher in these primary cells compared to a549 cells without infection ( figure s5 ). viruses 2019, 11, x for peer review 10 of 17 the ring-like appearance of ifitm3 clusters suggested the vesicular recruitment of this protein to iav-carrying endosomal structures. we therefore analysed whether ifitm3 clusters co-localize with early endosomal (eea1), late endosomal (rab7), or lysosomal (lamp1) marker proteins at early stages of iav infection in a549 cells ( figure 6 , figure s3 ). we observed typical staining for early and late endosomes and for lysosomes with a weak ifitm3 signal in uninfected cells. the ifitm3 signal intensity increased at 1 h and 6 h p.i., as seen in the previous experiments, with some apparent colocalization with the endosomal marker proteins. to determine the degree of co-localization, we calculated the mander's correlation coefficient (mcc) [64] . only ifitm3 clusters defined as objects with a size of > 10 5 nm 2 were included in this analysis. we observed a highly significant increase of ifitm3 cluster co-localization with early endosomes at 1 h p.i., which was subsequently lost in the course of infection ( figure 6a ). in contrast, a significant increase of co-localization with the late endosomal marker ( figure 6b ) and some increased co-localization with the lysosomal marker ( figure 6c ) was seen at 6 h, but not at 1 h p.i. these results suggest that ifitm3 clustering on iav carrying endosomes already occurs early after endocytosis, prior to the ph-induced fusion and release from late endosomes, and probably persists as the endosomes mature. the ring-like appearance of ifitm3 clusters suggested the vesicular recruitment of this protein to iav-carrying endosomal structures. we therefore analysed whether ifitm3 clusters co-localize with early endosomal (eea1), late endosomal (rab7), or lysosomal (lamp1) marker proteins at early stages of iav infection in a549 cells ( figure 6 , figure s3 ). we observed typical staining for early and late endosomes and for lysosomes with a weak ifitm3 signal in uninfected cells. the ifitm3 signal intensity increased at 1 h and 6 h p.i., as seen in the previous experiments, with some apparent co-localization with the endosomal marker proteins. to determine the degree of co-localization, we calculated the mander's correlation coefficient (mcc) [64] . only ifitm3 clusters defined as objects with a size of > 10 5 nm 2 were included in this analysis. we observed a highly significant increase of ifitm3 cluster co-localization with early endosomes at 1 h p.i., which was subsequently lost in the course of infection ( figure 6a ). in contrast, a significant increase of co-localization with the late endosomal marker ( figure 6b ) and some increased co-localization with the lysosomal marker ( figure 6c ) was seen at 6 h, but not at 1 h p.i. these results suggest that ifitm3 clustering on iav carrying endosomes already occurs early after endocytosis, prior to the ph-induced fusion and release from late endosomes, and probably persists as the endosomes mature. endocytosed material is generally sorted to different destinations: reusable ligands and receptors are returned to the cell surface via recycling endosomes, while the material destined for degradation traffic to lysosomes [65] . previous studies reported a substantial co-localization between virus-containing compartments and rme-1 that is thought to be associated with sorting and recycling endosomes [66] . furthermore, recent reports have suggested that rab11-associated recycling endosomes are required for vrnp trafficking, assembly, and virion budding at the cellular surface in the late phase of iav infection [67] [68] [69] [70] . to determine whether iav and ifitm3 clustering can be detected on recycling endosomes, we compared uninfected and iav-infected a549 cells at different time points. some co-localization of np with ifitm3 and rab11 signals was observed on apparently vesicular structures in the extranuclear space of iav infected a549 cells at 1-6 h p.i. (figure 7a-c) . however, the co-localization of ifitm3 and rab11 was already present in the uninfected a549 cells, with no significant change of co-localization during the course of infection, as shown by the mcc analysis ( figure s4 ). the uptake of iav particles into recycling endosomes is expected to lead to an abortive infection, as recycling endosomes do not undergo acidification during trafficking. it is likely that rare events of an enhanced endocytosed material is generally sorted to different destinations: reusable ligands and receptors are returned to the cell surface via recycling endosomes, while the material destined for degradation traffic to lysosomes [65] . previous studies reported a substantial co-localization between virus-containing compartments and rme-1 that is thought to be associated with sorting and recycling endosomes [66] . furthermore, recent reports have suggested that rab11-associated recycling endosomes are required for vrnp trafficking, assembly, and virion budding at the cellular surface in the late phase of iav infection [67] [68] [69] [70] . to determine whether iav and ifitm3 clustering can be detected on recycling endosomes, we compared uninfected and iav-infected a549 cells at different time points. some co-localization of np with ifitm3 and rab11 signals was observed on apparently vesicular structures in the extranuclear space of iav infected a549 cells at 1-6 h p.i. (figure 7a-c) . however, the co-localization of ifitm3 and rab11 was already present in the uninfected a549 cells, with no significant change of co-localization during the course of infection, as shown by the mcc analysis ( figure s4 ). the uptake of iav particles into recycling endosomes is expected to lead to an abortive infection, as recycling endosomes do not undergo acidification during trafficking. it is likely that rare events of an enhanced ifitm3 and rab11 the ifitm3-mediated inhibition of iav infection has been suggested to be caused by changes in the membrane tension resulting in a block of fusion pore formation [71] , and may be due to constitutively expressed protein or interferon induction [38, 41, 57, 72] . applying confocal and superresolution imaging of endogenous ifitm3, we show that pre-existing ifitm3 clusters at early and late endosomal structures carry iav at early time points of infection, prior to interferon induction. as shown previously [32] and in our study ( figure s2 ), interferon alpha led to increased protein levels of ifitm3 at later time points. on the other hand, no increase in the ifitm3 protein levels was observed up to 6 h p.i. in iav infected a549 cells. a semi-automated image analysis of iav a/hk/1/68 (pdmh3n2) and iav a/r/d6/2009 (pdmh1n1) infected a549 cells revealed ifitm3 clustering early after infection in both cases, with few differences in kinetics, which may reflect strain specific replication kinetics [73] . the cd225 domain of ifitm3, which is composed of the intermembrane domain 1 and the intracellular loop, contains two phenylalanine residues mediating the physical association between ifitms; these residues are strongly connected with the antiviral function [39] . combining this finding with our observation of the increasing levels of larger ifitm3 clusters, we suggest a direct relation between cluster formation and ifitm3`s antiviral activity on endosomal vesicles. the ifitm3 clustering on apparently vesicular structures started early after infection (3-5 h p.i.), with an initial co-localization of ifitm3 with an early endosomal marker and a later co-localization with a late endosomal marker, reflecting the early to late endosomal pathway of iav. it appears likely, therefore, that ifitm3 the ifitm3-mediated inhibition of iav infection has been suggested to be caused by changes in the membrane tension resulting in a block of fusion pore formation [71] , and may be due to constitutively expressed protein or interferon induction [38, 41, 57, 72] . applying confocal and super-resolution imaging of endogenous ifitm3, we show that pre-existing ifitm3 clusters at early and late endosomal structures carry iav at early time points of infection, prior to interferon induction. as shown previously [32] and in our study ( figure s2 ), interferon alpha led to increased protein levels of ifitm3 at later time points. on the other hand, no increase in the ifitm3 protein levels was observed up to 6 h p.i. in iav infected a549 cells. a semi-automated image analysis of iav a/hk/1/68 (pdmh3n2) and iav a/r/d6/2009 (pdmh1n1) infected a549 cells revealed ifitm3 clustering early after infection in both cases, with few differences in kinetics, which may reflect strain specific replication kinetics [73] . the cd225 domain of ifitm3, which is composed of the intermembrane domain 1 and the intracellular loop, contains two phenylalanine residues mediating the physical association between ifitms; these residues are strongly connected with the antiviral function [39] . combining this finding with our observation of the increasing levels of larger ifitm3 clusters, we suggest a direct relation between cluster formation and ifitm3's antiviral activity on endosomal vesicles. the ifitm3 clustering on apparently vesicular structures started early after infection (3-5 h p.i.), with an initial co-localization of ifitm3 with an early endosomal marker and a later co-localization with a late endosomal marker, reflecting the early to late endosomal pathway of iav. it appears likely, therefore, that ifitm3 clusters, initially recruited to early endosomes and then coating endosomal vesicles through their trafficking pathway, may mediate the antiviral activity of ifitm3 and block the release of vrnps. this hypothesis is further supported by the identification of a critical sorting signal that is essential for the ifitm3 localization to endosomes and its anti-viral activity [74, 75] . similar to iav, arena-(including lasv, lymphocytic choriomeningitis virus, and macv) and alphaviruses (including the chikungunya virus, sindbis virus, and venezuelan encephalitis virus) also fuse from late endosomes and lysosomes at a similar acidic ph, but are not restricted by ifitm3 [51] . it will be of interest, therefore, whether a similar ifitm3 clustering can be observed on endosomal vesicles carrying these viruses or whether they can escape from ifitm3 clustering. importantly, a similar phenotype was observed in human primary respiratory cells from healthy donor tissue (hsaepcs). the basal levels of ifitm3 were much higher in these primary cells, compared to a549 cells in the absence of iav infection. however, ifitm3 clustering was not observed in the naïve primary cell population, but rapidly increased again upon iav infection and was even stronger than in a549 cells. ifitm3 clustering on cytoplasmic vesicles was strongly induced in both a549 cells and hsaepcs exhibiting weak extranuclear signals for iav np, which most likely reflects an incoming input virus. in contrast, no such ifitm3 clusters were seen in productively infected a549 cells with a strong nuclear np signal, and we speculate that infection of this cell type mainly occurs in cells that have not managed to block iav entry by inducing ifitm3 clustering. alternatively, ifitm3 clusters may be abrogated once productive infection has occurred and may thus no longer be visible in these cells. live-cell microscopy using labelled ifitm3 will be required to distinguish between these possibilities. a different phenotype was observed in primary hsaepcs, where ifitm3 clustering on vesicular structures was also observed in productively infected cells with a strong nuclear signal. despite having a much higher basal level, ifitm3 clustering in hsaepcs does not appear, therefore, to efficiently block iav infection in these primary cells. from our findings we assume two putative and presumably additive mechanisms for ifitm3 mediated antiviral action: (i) the first contact with iav particles after an endocytic uptake in (early) endosomes (role of recycling endosomes is discussed below) triggers the continuous recruitment of pre-existing ifitm3 proteins to iav containing compartments as a fast antiviral defense in the initial phase of infection. (ii) ifitm3 protein levels are increased by interferon signaling, further propagating its antiviral effect and possibly synergizing with additional ifn-induced antiviral factors [76] . continuing studies aim to identify the modalities of the direct or indirect interaction between iav and ifitm3. we have to admit that deficient particles might have the potential to fuse with the plasma membrane but fail to replicate. an abortive and ineffective infection might trigger the ifitm3 activation and recruitment to endosomes in the same way as can be seen for an efficient iav infection. various members of the endosomal network fulfil crucial functions in the viral entry, trafficking, and genome release. rab11 is mainly assigned to recycling endosomes [67] . we made the observation that rab11-positive vesicles loaded with ifitm3 often carried iav np early during infection. previous reports using live-cell imaging suggested that ifitm3 mediates the directed re-localization of viral particles to lysosomes [77] . our results argue that the internalization of iav via recycling endosomes may trap iav in a non-productive pathway, as the membrane fusion is precluded in recycling endosomes having only a mildly acidic ph of 6.5 [78] . it is conceivable that both mechanisms exist synergistically. in general, viruses being restricted by ifitm3 fuse at a lower ph in late endosomes or lysosomes [51] . ifitm3 on recycling endosomes is thus unlikely to restrict the endosomal entry of iav (or of other viruses described to be restricted by ifitms), but may be an important defense against other pathogens entering via recycling endosomes [78] . supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/6/548/s1, figure s1 : ifitm3 knock-down validation, figure s2 : ifitm3 abundance in the absence and presence of hifnα and upon iav infection (24 h p.i.) determined by western blotting, figure s3 : ifitm3 proteins cover early, late and recycling endosomes, 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signal essential for the antiviral action of ifitm3 phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (ifitm3) dually regulates its endocytosis and ubiquitination klf5 is involved in regulation of ifitm1, 2, and 3 genes during h5n1 virus infection in a549 cells hang, h.c. ifitm3 directly engages and shuttles incoming virus particles to lysosomes emerging roles of recycling endosomes we thank kathleen börner (university hospital, heidelberg) for advice using crispr-cas9 gene editing, marino zerial (max planck institute of molecular cell biology and genetics, dresden) for the prab11_egfp plasmid and jürgen stech (friedrich-loeffler-institute, insel riems) for the recovery plasmid system of influenza a/hong kong/1/1968, a/puerto rico/8/1934 and a/regensburg/d6/2009. we are grateful to vibor laketa (idip, heidelberg), bärbel glass and vera sonntag-buck (technical assistance) and carola sparn (student assistance) for excellent assistance. the authors declare no conflict of interest. key: cord-300423-q2i328sz authors: bai, lei; zhao, yongliang; dong, jiazhen; liang, simeng; guo, ming; liu, xinjin; wang, xin; huang, zhixiang; sun, xiaoyi; zhang, zhen; dong, lianghui; liu, qianyun; zheng, yucheng; niu, danping; xiang, min; song, kun; ye, jiajie; zheng, wenchao; tang, zhidong; tang, mingliang; zhou, yu; shen, chao; dai, ming; zhou, li; chen, yu; yan, huan; lan, ke; xu, ke title: co-infection of influenza a virus enhances sars-cov-2 infectivity date: 2020-10-14 journal: biorxiv doi: 10.1101/2020.10.14.335893 sha: doc_id: 300423 cord_uid: q2i328sz the upcoming flu season in the northern hemisphere merging with the current covid-19 pandemic raises a potentially severe threat to public health. through experimental co-infection of iav with either pseudotyped or sars-cov-2 live virus, we found that iav pre-infection significantly promoted the infectivity of sars-cov-2 in a broad range of cell types. remarkably, increased sars-cov-2 viral load and more severe lung damage were observed in mice co-infected with iav in vivo. moreover, such enhancement of sars-cov-2 infectivity was not seen with several other viruses probably due to a unique iav segment as an inducer to elevate ace2 expression. this study illustrates that iav has a special nature to aggravate sars-cov-2 infection, and prevention of iav is of great significance during the covid-19 pandemic. the outbreak of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) at 31 the end of 2019 has become pandemic worldwide. up to date, there had been more than 36 32 million confirmed infected cases and 1 million deaths globally (https://covid19.who.int/). 33 the ending time and the final severity of the current covid-19 pandemic wave are still 34 uncertain. meanwhile, the upcoming seasonal influenza merging with the current pandemic 35 might bring more challenges and pose a bigger threat to public health. there are many 36 debates on whether seasonal flu would impact the severity of the covid-19 pandemic and 37 whether massive influenza vaccination is necessary for the coming winter. however, no 38 experimental evidence is available concerning iav and sars-cov-2 co-infection. 39 it is well known that disease symptoms from sars-cov-2 and iav infections are quite 40 similar, such as fever, cough, pneumonia, acute respiratory distress syndrome, etc(1, 2). 41 moreover, both sars-cov-2 and iav are airborne transmitted pathogens that infect the 42 same human tissues such as the respiratory tract, nasal, bronchial, and alveolar epithelial 43 cultures (3, 4) . besides, alveolar type ii cells (at2 pneumocytes) appeared to be 44 preferentially infected by sars-cov-2, which were also the primary site of iav 45 replication(5, 6). therefore, the overlap of the covid-19 pandemic and seasonal influenza 46 would pose a large population under the high risks of co-occurrent infection by these two 47 viruses(7). 48 unfortunately, during the last winter flu season in the southern hemisphere, there was 49 little epidemiological evidence about the interaction between covid-19 and flu, probably 50 due to a low iav infection rate resulted from social distancing (8, 9) . a case report showed 51 that three out of four sars-cov-2 and iav co-infected patients rapidly develop to 52 respiratory deterioration(10). on the contrary, other reports only observed mild symptoms 53 in limited co-infection outpatients(11). thus, the clinical co-infection outcomes are still 54 unclear when a large population will face the threats of both viruses. 55 in this study, we tested whether iav infection could affect the subsequent sars-cov-2 56 infection in both infected cells and mice. the results demonstrate that the pre-infection of 57 iav strongly enhances the infectivity of sars-cov-2 by boosting viral entry in the cells 58 and by elevating viral load plus more severe lung damage in infected mice. these data 59 suggest a clear auxo-action of iav on sars-cov-2 infection, which implies the great 60 importance of influenza virus and sars-cov-2 co-infection to public health. iav promotes sars-cov-2 virus infectivity. 63 to study the interaction between iav and sars-cov-2, a549 (a hypotriploid alveolar a549 was converted to be highly sensitive (up to 10,000-fold) against the psars-cov-2 72 virus after different doses of iav infections (from low moi of 0.01 to high moi of 1, also 73 shown by psars-cov-2 with mcherry reporter in fig. s1 ). in contrast, the pre-infection 74 of iav had no impacts on pseudotyped vsv particles bearing vsv-g protein (fig.1c ). we 75 further tested more cell lines to show that the enhancement of the psars-cov-2 infectivity 76 by iav was a general effect although the increased folds were different (lower basal level 77 of infectivity, higher enhancement fold) (fig.1d ). to validate the above results, we substituted the psars-cov-2 with the sars-cov-2 79 live (experimental scheme shown in fig.1e ). we found that the pre-infection of iav 80 strongly increased the copy numbers of the sars-cov-2 genome (e and n genes) in both 81 cell lysates and supernatants of a549 (~15 folds) (fig.1f) . notably, in calu-3 (fig.1g ) and 82 nhbe ( fig.1h ) cells that are initially susceptible to sars-cov-2, iav pre-infection could 83 further increase >5 folds of sars-cov-2 infectivity. collectively, these data suggest an auxo-action of iav on sars-cov-2 in a broad range 85 of cell types. 87 load and more severe lung damage. 88 the hace2 transgenic mice were applied to study the interaction between iav and 89 sars-cov-2 in vivo. mice were infected with 3x10 5 pfu of sars-cov-2 with or without 90 2000 pfu of iav pre-infection and were then sacrificed two days later after sars-cov-2 91 infection (the experimental scheme is shown in fig. 2a) . the viral rna genome copies 92 from lung homogenates confirmed that sars-cov-2 efficiently infected both groups 93 (more than 4x10 8 n gene copies) (fig. 2b) , while the influenza np gene was only detected 94 in iav pre-infection group (fig. 2b ). intriguingly, a significant increase in sars-cov-2 95 viral load (12.9-fold increase in e gene and 6.6-fold increase in n gene) was observed in 96 lung homogenates from co-infection mice compared to that from sars-cov-2 single-97 infected mice (fig. 2c ). the histological data in fig. 2d further illustrated that iav and 98 sars-cov-2 co-infection induced more severe lung pathologic changes with massive 99 infiltrating cells and obvious alveolar necrosis as compared to sars-cov-2 single 100 infection or mock infection. iav components specifically facilitate the entry process of sars-cov-2. 102 we further tested if several other viruses on hand had similar effects to promote sarscatl were increased around three folds (a549 in fig. 4a , calu-3 in fig. s2 ). an obvious 119 switch of intracellular ace2 expression was triggered at 12 h post-iav-infection (fig. 4c ). in the meantime, influenza np, mx1, and isg54 increased accordingly confirming a 121 successful infection of iav (fig. 4b ). the data indicated that iav permitted increased sars-cov-2 infection through the up-133 regulation of ace2 expression. enhanced sars-cov-2 infectivity is independent of ifn signaling. ace2 was reported to be an interferon-stimulated gene (isg) in human airway epithelial 136 cells (19) . iav infection will also stimulate type i ifn signaling. we, therefore, tested 137 whether the augment of ace2 expression is dependent on ifn or not. for this, cells were the a/wsn/33 virus was generated by reverse genetics as previously described (25). all the mrna levels were normalized by β-actin in the same cell. the relative number of sars-cov-2 viral genome copy number were determined using ifnα for 12 hours. cells were then infected with psars-cov-2 for another 24 hours 455 followed by measuring luciferase activity and mrna expression levels of indicated genes. the data of mrna levels were expressed as fold changes relative to non-treatment cells. figure s1 . iav facilitates the entry process of psars-cov-2 (fig.1) . figure s2 . iav infection induces elevated ace2 expression (fig.4) . figure s3 . enhanced sars-cov-2 infection is independent of ifn signaling (fig.5) . figure s4 . iav facilitates viral entry of wt or mutant sars-cov-2. mutations in the spike protein of middle east respiratory syndrome 333 structural and functional basis of sars-cov-2 entry by using human ace2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 338 receptor sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by 340 a clinically proven protease inhibitor furin, a potential therapeutic target for covid-19. iscience could an endo-lysosomal ion channel be the achilles heel of sars-cov2? 343 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected 348 in specific cell subsets across tissues human antibody responses after dengue virus infection are highly cross-reactive to zika virus key: cord-003639-bjtxf1y8 authors: vahey, michael d; fletcher, daniel a title: influenza a virus surface proteins are organized to help penetrate host mucus date: 2019-05-14 journal: nan doi: 10.7554/elife.43764 sha: doc_id: 3639 cord_uid: bjtxf1y8 influenza a virus (iav) enters cells by binding to sialic acid on the cell surface. to accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (ha) and the receptor-cleaving protein neuraminidase (na). although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. using site-specific fluorescent labeling and super-resolution microscopy, we show that ha and na are asymmetrically distributed on the surface of filamentous viruses, creating a spatial organization of binding and cleaving activities that causes viruses to step consistently away from their na-rich pole. this brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus’s conflicting needs to both penetrate mucus and stably attach to the underlying cells, potentially contributing to the prevalence of the filamentous phenotype in clinical isolates of iav. for a virus to infect a cell, it must reach receptors on the cell surface while avoiding neutralization or clearance by 'decoy' receptors in the surrounding environment. for viruses that bind to sialic acid, a receptor that is abundant both on the surface of cells and in the secreted extracellular mucosal environment, attachment and detachment is controlled by receptor-binding and receptor-destroying activities on the viral surface (rosenthal et al., 1998; zeng et al., 2008) . influenza a viruses (iavs), respiratory pathogens that contribute to seasonal flu and have pandemic potential, achieve this balance with hemagglutinin (ha)-mediated receptor binding and neuraminidase (na)-catalyzed receptor destruction (air and laver, 1989; skehel and wiley, 2000) . although the importance and genetic basis of the functional balance between ha and na in iav has been thoroughly characterized (wagner et al., 2002; xu et al., 2012; yen et al., 2011) , it is becoming clear that transmissibility of influenza viruses within and between hosts depends on factors beyond the sequence of these two genes (chou et al., 2011; herfst et al., 2012; neumann and kawaoka, 2015) . mucosal barriers present the first line of defense against iav infection. to infect the underlying epithelium, viral particles must first pass through a~1-10 mm thick layer of mucus that is being steadily transported towards the pharynx where it can be swallowed, neutralizing any virus immobilized within it (bustamante-marin and ostrowski, 2017; wanner et al., 1996; zanin et al., 2016) . viruses that bind too tightly to sialic acid will pass through the mucus barrier very slowly, and will thus be unable to reach the surface of an airway epithelial cell before mucociliary clearance. in contrast, viruses that bind only very weakly to sialic acid (or which rapidly destroy it through excessive na activity [cohen et al., 2013; yang et al., 2014] ) will quickly penetrate mucus, but may be unable to stably attach to the surface of the underlying epithelium once it is reached. adaptations that help the virus to overcome both of these conflicting challenges, such as changes in virus morphology or a particular spatial organization of envelope proteins, could be evolutionarily favored during in vivo replication. interestingly, one feature of iav that tends to diverge when clinical isolates are cultured in a laboratory environment, or when animals are infected with laboratory-grown strains, is particle morphology. while clinical isolates of iav -samples adapted to transmission in a mucosal environmentform filamentous particles with a consistent diameter but widely varying length, laboratory-adapted strains tend to produce more uniform, spherical particles (badham and rossman, 2016; chu, 1949; dadonaite et al., 2016; seladi-schulman et al., 2013) . recent evidence from the 2009 pandemic suggests that filamentous morphology, conferred by the virus's m segment, may play a role in transmission (campbell et al., 2014; lakdawala et al., 2011) . however, whether or not virus morphology contributes directly to virus transmission -and if so, how -remains unclear. similarly, although the two major envelope proteins of iav, ha and na, have been observed by electron microscopy to cluster non-uniformly on both the viral and pre-viral envelope (calder et al., 2010; harris et al., 2006; leser and lamb, 2017) , whether and how the spatial organization of ha and na affects virus transmission also remains unclear. motivated by these observations, we reasoned that virus shape, together with the packaging and organization of ha and na in the viral membrane, could influence the balance of attachment and detachment in ways that promote efficient virus penetration through mucus. to test this idea, we sought to characterize the organization of proteins in filamentous iav particles while simultaneously observing their engagement with sialic acid -a measurement that requires a non-destructive approach. to make this measurement possible, we recently developed strains of influenza a virus that are amenable to fluorescence microscopy through site-specific tags introduced into the viral genome (vahey and fletcher, 2019) . here we show that filamentous particles frequently contain asymmetric distributions of ha and na in their membranes, and that this distinctive organization biases the diffusion of these particles in a persistent direction over distances of several microns. by enhancing the effective diffusion of a viral particle without reducing the stability of its attachment to the viral receptor, this mechanism could promote virus penetration across mucosal barriers. ha and na are distributed asymmetrically on the surface of iav particles we first sought to characterize the organization and dynamics of proteins in the viral membrane. by labeling ha and na, along with the viral nucleoprotein, np, we are able to measure features of virus organization on intact, infectious particles that corroborate and extend previous observations made using electron microscopy (calder et al., 2010; chlanda et al., 2015; harris et al., 2006; leser and lamb, 2017) . for these experiments, we use a tagged variant of the strain a/wsn/1933 with m1 from a/udorn/1972, which differs from wsn m1 at six residues and confers filamentous morphology (elleman and barclay, 2004) . consistent with our prior observations, this virus produces filamentous particles that vary widely in size, from sub-diffraction limited spots to particles > 10 mm in length (vahey and fletcher, 2019) . in viruses with fluorescently-labeled ha and na, we observe a pronounced tendency for na to be enriched at one end of the virus ( figure 1a , inset). to quantify this enrichment, we measured the relative intensities of ha and na within~400 nm of each viral pole in filamentous particles > 1 mm in length. across 20344 such particles, approximately one third have na intensities at least 5-fold higher on one pole than on the other ( figure 1a, left) . similarly, viruses with labeled np (the most abundant protein in vrnp complexes and a proxy for the virus genome) reveal individual foci that localize to one end of the virus. aligning filamentous particles with polarized np foci (4805 in total) reveals a tendency for na to colocalize with np ( figure 1a , right). for the subset of these particles > 4.5 mm in length (540 in total), we created a composite image showing the average densities of viral proteins within 2 mm of each viral pole (figure 1-figure supplement 1). on average, na is enriched~2 x relative to ha at the np-containing pole in these particles, as compared to the middle region of the particle and the opposite pole ( approximately one third of filamentous particles have na intensities five-fold higher on one pole than the other, as indicated by the vertical dashed lines (distributions are symmetric in this case because poles are designated randomly). inset images show a particle figure 1 continued on next page filamentous particles do not recover fluorescence in either ha or na over tens of minutes ( figure 1b) . to investigate the finer details of protein organization and to determine if na in fluorescent viruses is clustered, as suggested by electron microscopy (calder et al., 2010; harris et al., 2006) , we use two-color stochastic optical reconstruction microscopy (storm) to reconstruct images of ha and na with resolution~10x better than the diffraction limit (~30 nm compared to~300 nm; figure 1-figure supplement 2). at this resolution, we find that the tendency of na to concentrate at one of the viral poles is pronounced even in particles smaller than 300 nm in length ( figure 1c) . we also find that the na seen at low levels along the length of filamentous viruses without super-resolution imaging is actually organized into small na clusters ( figure 1d ). additionally, ha and na distributions in these particles are modestly anti-correlated ( figure 1e ), suggesting that receptor-binding and receptor-destroying activities may be spatially segregated on some regions of the virus. collectively, these measurements present a picture of a variegated iav envelope whose spatial organization is stable and coupled to the presence and location of the viral genome, with~70% of npcontaining viruses having na biased to the proximal pole. we next investigated whether spatial organization of the iav envelope could have functional significance for virus binding and detachment. as a first test of this idea, we compared the spatial organization of viruses that were released from the cell surface with those that remained attached after challenging the virus with the neuraminidase inhibitor (nai) oseltamivir carboxylate (he et al., 1999) (materials and methods). to quantify virus spatial organization, we defined ha-na polarity as the separation between the center of masses for ha and na divided by the virus length. interestingly, viruses that escaped the cell surface under nai challenge showed higher ha-na polarity (0.155; 95% confidence interval, ci = 0.139-0.171) than viruses released in the absence of nai challenge, both before (0.123; 95% ci = 0.106-0.140) and after (0.100; 95% ci = 0.090-0.110) facilitating virus release by treating with exogenous sialidase ( figure 1f & g). in comparison, viruses treated with both nai and exogenous sialidase have polarities similar to the untreated virus (0.124; 95% ci = 0.111-0.136). these results suggest that viruses with na concentrated at one of the viral poles may be more effective at navigating environments rich in sialic acid. with na enrichment of~5.6 fold (~31 st percentile of particles). the plot to the right shows the same comparison for particles aligned to their np-foci, designated as pole 1 (distributions are determined from a total of 4805 particles with polarized np distributions). the prevalence of particles with na ratios > 1 indicates a tendency for na to be enriched at the virus's genome-containing pole. inset images show a particle with na enrichment of~3.3 fold (~48 th percentile of particles based on na polarity). (see also (e) distribution of ha-na spatial correlation coefficients from regions of 24 filamentous particles following reconstruction at~30 nm resolution. (f) to compare populations of virus that are able to detach from infected cell to those that remain bound to the cell surface with or without nais, we collect virus in two separate stages: first, we harvest virus from media at 16hpi, followed by the addition of media supplemented with an exogenous, oseltamivir-resistant bacterial neuraminidase. after one hour of treatment with exogenous na, we harvest virus that had previously remained bound to the cell surface. polarized viruses step persistently away from their na-rich pole to directly test how na polarization might affect virus motion, we characterized interactions between fluorescently-labeled viruses and sialic acid coated coverslips, where the well-defined geometry and density of sialic acid allow straightforward analysis of virus diffusion (materials and methods) (figure 2a ). because our approach to fluorescently labeling na preserves its activity (figure 2-figure supplement 1) and viruses harboring fluorophores on both ha and na preserve~85% of their infectivity (vahey and fletcher, 2019) , we could carry out functional assays with the virus and at the same time visualize ha and na distributions on the viral membrane. surprisingly, the motion we observed did not resemble randomly-oriented diffusion of the viral particle, but rather persistent, brownian ratchet-like diffusion, in which filamentous particles with polarized distributions of na exhibited directed mobility away from their na-rich pole ( figure 2b , video 1). labeling coverslips with fluorescein-labeled erythrina cristagalli lectin (ecl), which binds specifically to the terminal galactose exposed following sialic acid cleavage (iglesias et al., 1982) , revealed the history of virus trajectories and confirmed that virus motion is accompanied by receptor destruction ( figure 2b ). aligning the trajectories of mobile particles (defined as those with diffusion coefficients > 100 nm 2 /s, comprising~68% of the population; figure 2c ) to the orientation of their ha-na axis reveals that directional mobility can persist for several microns, many times the length of the particle itself ( figure 2d ). consistent with the observation that polarized distributions of ha and na serve as a determinant for persistent motion and enhanced diffusion, we find that non-mobile viruses have, on average, less polarized distributions of ha and na than mobile ones ( figure 2e ). to determine if polarized distributions of na were necessary for persistent directional mobility, we disrupted the spatial organization of viral surface proteins by removing the cytoplasmic tail of na (residues 2-6; figure figure 3a ), indicating that spatial organization of na at the poles is necessary for persistent mobility of iav. correlations in the direction of successive steps in a random walk will increase the rate at which the walker explores its environment, analogous to how more rigid polymer chains typically have larger end-to-end distances than those with equivalent contour length but lower bending rigidity. to quantitatively compare the rate at which the less persistent nadct viruses explore their environment to that of the more persistent wildtype virus, we determined mean squared displacements from observed trajectories of mobile viruses (diffusion coefficients > 100 nm 2 /s over the observation period) and simulated random walks in which the number of steps, the size of each step, and the time interval between steps all match the observed data, but the direction of each step is uncorrelated with previous steps ( figure 3b ). from this analysis, we estimate that the diffusion coefficients of mobile viruses are enhanced approximately five-fold as a result of directional correlations ( figure 3b , left). in contrast, the diffusion of mobile nadct viruses is only modestly influenced by directional correlations (~1.5 fold increase; figure 3b , right). to further quantify the difference between viruses with wildtype na and nadct, we tracked the displacement of particles between subsequent frames acquired 30 s apart and plotted the distribution of the stepping angle relative to the orientation of the virus's ha-na axis. viruses with wildtype na step in the direction of increasing ha (with the na pole at the rear) roughly twice as frequently as the opposite direction, while nadct viruses exhibit no correlation between orientation and stepping direction ( figure 3c ). additionally, despite having lower amounts of na in the viral membrane, nadct viruses left significantly larger trails of cleaved receptors than their wildtype counterparts ( results demonstrate that the spatial organization and mobility of na on the viral surface both play an important role in determining where and when it cleaves substrate. computational modeling suggests a mechanism for persistent directional mobility to investigate the mechanism of this persistent directional mobility, we modeled the diffusion of filamentous viruses with defined spatial organization of ha and na as the viruses bind to sialic acid on a two-dimensional surface (appendix, figure 3 -figure supplement 4). in this model, virus diffusion is constrained by the tens of attachments the has form with sialic acid at any instant in time (english and hammer, 2004; xu and shaw, 2016) . at the interface between ha-rich and na-rich regions of the viral membrane, like those seen in fluorescence images ( figure 1a -d), na will periodically gain access to, and hydrolyze, sialic acid as the position of the virus thermally fluctuates. the binding partners available to has located close to this interface will therefore be asymmetrically distributed, with more potential binding partners located further away from areas of higher na density. this concept is illustrated most clearly in the case where the virus is constrained to a one-dimensional path. as this case illustrates, the sialic acid density along the length of the virus is lowest in close proximity to na (figure 3-figure supplement 5a-c). when na is confined to a single pole, this results in a gradient in sialic acid density along the length of the particle. since the number of available binding partners is higher in one direction than another, newly-formed ha-sialic acid bonds will tend to be offset from previous bonds in this direction. this leads to the persistent motion we observe experimentally as well as in our simulations (video 2). consistent with this mechanism, persistent motion of the virus is lost when na is no longer localized to a single virus pole and the sialic acid distribution beneath the particle becomes symmetric (figure 3-figure supplement 5b-d). we note that virions with symmetric distributions of na also occur in the virus population and would not be expected to exhibit persistent motion (for example, those particles with na polar ratios of~1 in figure 1a ). since virus motion is dependent on the virus's ability to establish distinct regions with and without sialic acid, we expect that dynamic distributions of either na or sialic acid will alter virus mobility. consistent with our experimental observations with the nadct virus, allowing na to freely diffuse eliminates directional bias in virus motion in both experiments ( figure 3c ) and simulations ( figure 3d ). however, allowing surface-bound sialic acid to diffuse can either enhance or suppress directed motion, depending on the kinetic parameters of ha and na, the diffusion coefficient of sialic acid (d sa ), and the size of the virus (appendix). although our experimental data corresponds to the case where sialic acid is immobilized (d sa~0 ), our simulations predict that polarized iav bound to slowly-diffusing transmembrane glycoproteins or gel-forming mucins (d sa <0.01 mm 2 /s) will exhibit enhanced directed mobility, likely due to the biased spatial distributions of sialic acid binding partners that are available to has distal to the virus's na-rich pole. in contrast, increasing the rate of sialic acid diffusion further (d sa >0.01 mm 2 /s) is expected to suppress persistent motion, by flattening the sialic acid gradient that is created beneath the virus ( figure 3e, video 3) . finally, we find that increasing the length of the virus in simulations increases the number of ha-sialic acid interactions, reducing virus mobility but increasing directional persistence (figure 3-figure supplement 6). thus, the organization of proteins on the viral surface, the size of the virus, and the nature of the receptor to which the virus is bound can each influence the persistence of a virus's motion. although our results focus on a two-dimensional geometry in which a virus adheres to a flat surface decorated with sialic acid (a model for the cell surface), we reasoned that they should generalize to three-dimensional systems, such as the secreted mucus gel through which iav must penetrate to reach naïve cells to infect. to test this prediction, we cultured calu-3 cells at an air-liquid interface, resulting in a~1-10 mm thick gel of secreted mucus overlaying the apical surface of the cells ( figure 4a ). adding virus with labeled ha and na to the mucus, followed by fixation and labeling with ecl, revealed tracks similar to those we observed on two-dimensional surfaces ( figure 4b ). this suggests that the asymmetry of na and ha biases the direction of virus diffusion in three dimensions, producing trails of cleaved sialic acid that can reach several microns in length as the virus moves ( figure 4c ). this directional mobility is less prevalent in viruses with more uniformly distributed na, though they nonetheless are capable of creating swaths of ecl-staining within the mucus that lack clear directionality ( figure 4d ). similar to our observations on idealized two-dimensional surfaces ( figure 3c ), we find that viruses in mucus exhibit a slight tendency to align their ha-na axis (captured at the moment of fixation) with the displacement of the virus relative to the center of ecl labeling ( figure 4e) . these results suggest that the spatial organization of ha and na on the virus surface may also promote penetration of polarized iav particles through mucus barriers in vivo. advances in electron microscopy over the past several decades have presented an increasingly detailed picture of the morphology and organization of influenza a virus. this has revealed organizational features of filamentous iav whose origins and functional significance remain unclear. by using site-specific fluorescent labeling to measure the organization of a virus while preserving its function, we are able to corroborate key observations of envelope protein non-uniformity obtained from electron microscopy and extend them to dynamic observations of both protein motion on the surface of the virus, as well as the directionally-persistent motion of influenza a virus particles as they engage with the virus receptor, sialic acid. these observations demonstrate that the morphology of a virus, the spatial organization of proteins in its membrane, and constraints on the diffusion of these proteins collectively confer the tendency for viral particles to exhibit persistent directional mobility, enhancing the virus's rate of diffusion without diminishing its strength of adhesion. importantly, our simulations show that this feature of filamentous morphology would not be limited to the extremely large particles whose length is easily measured using diffraction limited microscopy, but rather extends to capsule-shaped particles < 200 nm in length (figure 3-figure supplement 6) , which are known to be produced by filamentous strains of iav (calder et al., 2010) . in contrast, this effect may be suppressed in spherical particles, due both to the absence of a clear axis for polarization and video 3. simulations of polarized filamentous viruses on surfaces with freely-diffusing sialic acid (green). simulations correspond to d sa = 10 à7 mm 2 /s, d sa = 10 à5 mm 2 /s, and d sa = 10 à1 mm 2 /s, as plotted in figure 3e . their ability to roll on two-dimensional surfaces. however, super-resolution live imaging will be required to confirm these predictions experimentally. although more work is needed to understand if these characteristics of iav organization enhance infectivity and are adaptive during in vivo replication, a simple model of viral transport suggests how the directional viral mobility we observe could contribute during host-to-host transmission. on first entering the respiratory tract, viruses must diffuse through the mucosal barrier to infect the underlying airway epithelial cells. competing with this process is mucocilliary clearance, which carries particles bound to mucus towards the pharynx where they are neutralized (figure 4-figure supplement 1a ). if the transport of a virus through a mucosal barrier is modeled as a first passage problem with a time limit imposed by the rate of mucociliary clearance, a five-fold increase in the diffusion coefficient (as predicted in figure 3b ) would lead to a proportional reduction in the first passage time. more dramatically, it would also increase the fraction of particles that reach the cell surface when the rate of mucociliary clearance is high and would normally prevent most particles from reaching the cell surface. our experiments on two-dimensional sialic acid-coated surfaces suggest a diffusion coefficient of~800 nm 2 /s for polarized viruses; adapting this value for a one-dimensional first passage model suggests that persistent motion increases the number of particles that reach the epithelium before being cleared by several orders of magnitude (appendix, figure 4-figure supplement 1b) . although there are multiple ways that a virus's passage through mucus can be accelerated (reducing its size, decreasing the number of has on its surface, increasing the number of nas, or making the spatial distribution of na more uniform), each of these changes would likely reduce binding stability once the virus has managed to reach the cell surface. in contrast, the polarized distributions of na on the viral surface that we observe increase virus diffusion without compromising binding stability. balancing the need to robustly attach to the cell surface with the need to escape immobilization in host mucus is a fundamental challenge for influenza and other viruses that use sialic acid to enter cells. previous work has shown that influenza particles are not immobilized during their interactions with sialic acid (guo et al., 2018; sakai et al., 2017) . additionally, work characterizing the diffusion of influenza c virus, in which receptor-binding and receptor-destroying activities are combined in a single protein (hef), has shown that the destruction of receptors on a 2d surface prevents virions from retracing their steps (sakai et al., 2018) . our results show that influenza a virus accomplishes a similar feat through a different mechanism -asymmetric distribution of receptor-binding and receptor-destroying activities on the viral surface that results in persistent motion while maintaining stable attachment. the persistent directional mobility shown here orients filamentous virus motion parallel to its major axis, where it could further enhance the anomalous diffusion coefficients observed for cylindrical nanoparticles in mucus (yu et al., 2016) . analogous to the combined importance of active motility and cell shape in mucosal bacteria (bartlett et al., 2017; sycuro et al., 2010) , the directed mobility and cylindrical shape of iav could help to explain the virus's ability to penetrate host mucus and contribute to the prevalence of filamentous morphology in clinical isolates of iav. strains of influenza a virus amenable to site specific labeling on ha, na, and np were designed and characterized as described previously (vahey and fletcher, 2019) . briefly, viruses expressing ha with five consecutive glycine residues following the signal sequence (for labeling via sortase a [theile et al., 2013] ), na with a c-terminal ybbr tag (for labeling via sfp [yin et al., 2006] ), and np with a c-terminal tetracysteine motif (for labeling via direct binding of the biarsenical dye flash [griffin et al., 1998 ]) were rescued using reverse genetics (hoffmann et al., 2000) by transfecting co-cultures of hek293t and mdck-ii cells with plasmids encoding each of the eight genomic segments under the control of bidirectional promoters. cells used in this work were obtained and authenticated by the uc berkeley cell culture facility and tested negative for mycoplasma. all viruses used in this work are derived from a/wsn/1933, with the wsn m1 gene replaced by that of a/udorn/1972, to establish the filamentous phenotype. mdck-ii cells used to propagate virus were maintained in dmem supplemented with 10% fetal bovine serum (thermo fisher, 10438026) and 1x penicillin/streptomycin (thermo fisher, 15140122) . prior to infection, confluent monolayers of cells were washed once with pbs, and serum-containing growth media was replaced with virus growth media (mem, 0.25% bsa, 1 mg/ml tpck-treated trypsin, and penicillin/streptomycin). viruses used for experiments were collected from cells following infection at moi~1 and 16 hr of growth at 37˚c in virus growth media. media containing virus was collected, centrifuged at 2000 â g for five minutes to remove cell debris, and treated with 10mu/ml soluble sialidase (from c. perfringens, sigma n2876) to ensure that viruses were well dispersed. viruses were labeled in solution for 90 min at room temperature using ntc buffer (100 nm nacl, 20 mm tris ph 7.6, 5 mm cacl 2 ) supplemented with 5 mm mgcl 2 , sortase a (180 mm enzyme, 50 mm clpetgg peptide) and sfp synthase (5 mm enzyme, 5 mm coa probe). following labeling, capto core 700 beads (ge healthcare;~1:1 resin volume to sample volume) were used to remove residual dyes and enzymes from the solution of labeled virus. for labeling np with the biarsenical dye flash, viruses were immobilized on coverslips, washed in ntc buffer, and incubated with 2 mm flash for 30 min at room temperature. to qualitatively evaluate the mobility of ha and na on the virion surface ( figure 1b, figure 3 -figure supplement 2b), unfixed, immobilized viruses were imaged using total internal reflectance (tirf) microscopy. filamentous virus of sufficient length (>5 mm) were positioned within the field of view such that when the field stop was closed, only approximately half of the virus was exposed to illumination. this half of the virus was then bleached using maximum laser power and then imaged at lower power at 30 s intervals as the field stop was opened to monitor recovery. representative results are shown in figure 1b and figure 3 -figure supplement 2b. samples for storm imaging were prepared by binding labeled virus to antibody or sialic acid (i.e. fetuin) functionalized coverslips for one hour at 4˚c, followed by the incubation of dragon greenlabeled 220nm-diameter streptavidin coated beads. viruses and beads were then fixed to the surfaces with 4% paraformaldehyde in pbs and washed 3x with buffer containing 1m tris ph 8.0, 5% glucose, and 140 mm b-mercaptoethanol. following these washes, the buffer was supplemented with glucose oxidase and catalase to final concentrations of 0.6 mg/ml and 0.035 mg/ml, respectively, and mounted on the microscope for imaging. storm data was acquired in the following sequence. first, an image of the dragon green beads (serving as fiducial marks), ha (labeled with cf568-conjugated peptide and srta), and na (labeled with af647-coa and sfp) was acquired, to enable registration. next, a sequence of storm images was acquired using a 640 nm laser at full power, with acquisition of the ha channel via a 560 nm laser at low power every 50 frames to correct for drift. after collecting 15000-35000 frames in this way, we performed storm imaging on cf568-ha using a 560 nm laser. to correct for drift, we acquire an image every 50 frames using a 405 nm laser and 575/20 nm emission filter; these settings allow us to image the dragon green beads, while simultaneously accelerating blinking of the cf568 dye. for reconstructions of ha, we acquire 25000-35000 frames. for quantification and localization of blinking events, we use the imagej plugin thunderstorm (ovesný et al., 2014) , combined with custom matlab scripts for additional drift correction and removal of fluorophores that remain in the 'on' state for more than one frame. this analysis results in a list of coordinates for each localization that we then use to reconstruct images of virus at varying resolutions. reconstructed storm images (e.g. figure 1c & d) are displayed by representing each localization as a gaussian with a standard deviation of 30 nm. challenge experiments with the neuraminidase inhibitor oseltamivir are performed as described previously (vahey and fletcher, 2019) . the analysis from figure 1e uses an image dataset from vahey and fletcher (2019) , reanalyzed to measure the spatial organization of ha and na on the surface of released virus particles. we infect a polarized monolayer of mdck cells grown on a collagen gel at moi of 1-3. after incubating cells with virus for one hour at 37˚c, we wash to remove excess virus, replacing media with virus growth media supplemented with or without a specified concentration of oseltamivir carboxylate (toronto research chemicals o700980), but without tpcktreated trypsin. at 16 hr post infection, we remove the virus containing media for labeling and imaging, and replace with media supplemented with 1 u/ml nani from c. perfringens (sigma n2876). after treating with this exogenous sialidase for one hour at 37˚c, we again collect cell culture media for virus labeling and imaging. to measure the ha-na polarization on viruses released in these experiments, we segment filamentous viruses with lengths > 1 mm and measure the intensity-weighted centroid (i.e. 'center of mass') for both the ha and na channels. the vector connecting the na centroid to the ha centroid defines the orientation of ha-na polarity. dividing the magnitude of this vector by the total particle length gives the ha-na polarity metric plotted throughout this work. coverslips presenting sialic acid for virus attachment were prepared as described previously (vahey and fletcher, 2019) . briefly, nh2-peg-oh (rapp polymere, 122000-2) supplemented with 2.5 mole-percent nh2-peg-biotin (rapp polymere, 133000-25-20) was conjugated to silanized coverslips for subsequent attachment of sialylated proteins. following pegylation, custom pdms chambers were attached to coverslips, and chambers were incubated for 10 min at room temperature with streptavidin at 50 mg/ml in 150 mm nacl, 25 mm hepes, ph 7.2, and washed 5x in the same buffer. fetuin (sigma f3004) labeled with nhs-biotin was then added at 100 nm and incubated~30 min at room temperature. coverslips were then washed 5x in ntc buffer and equilibrated to 4˚c in preparation for virus binding. for surfaces functionalized in this way (piehler et al., 2000) , we expect a peg density of~0.75 molecules/nm 2 , corresponding to roughly one peg-biotin per 7 nm x 7 nm area on the surface. if each biotin is bound by one streptavidin tetramer and subsequently one molecule of biotinylated fetuin (with~10 sialic acid residues per molecule), the surface density of sialic acid will be~0.2 sa/nm 2 . viruses with ha and na enzymatically labeled as described previously were bound to coverslips equilibrated to 4˚c for one hour on ice. immediately before imaging, excess virus was washed with pre-chilled ntc, and the sample was mounted on the microscope stage. after allowing the chamber to equilibrate to room temperature, the buffer in the chamber was exchanged to na buffer (100 mm nacl, 50 mm mes ph 6.5, 5 mm cacl 2 ), and timelapse recordings were collected using tirf microscopy at 30 s intervals. to visualize trails of cleaved sialic acid, fluorescein-labeled erythrina cristagalli lectin (ecl; vector labs fl-1141) was added to each well at a concentration of 5 mg/ml in ntc buffer with 10 mg/ml bsa and incubated for 30 min at room temperature. during this incubation period, rapid multivalent binding of ecl to cleaved sialic acid on the viral surface effectively blocks further motion of the virus. images of ecl trails were acquired using tirf microscopy without washing unbound ecl from the chamber. to analyze images of viruses, we separately segment each image according to the intensity in both the ha and na channels. merging the two sets of segmented images allows us to determine the position of each virus (i.e. the centroid of the combined ha and na masks) as well as their morphological features. to determine the ha-na polarization of each virus, we calculate the distance between the centroid of ha and na intensity for a particular virus, divided by that virus's length. the data in figure 2 and figure 3 is compiled by extracting these features for each virus in each frame of a timelapse recording. to analyze the trails of cleaved sialic acid left by mobile viruses, we segment images of samples labeled with fluorescent ecl according to the intensity of lectin staining. because the virus itself contains high densities of sialic acid on its surface, we use a bandpass threshold to specifically quantify ecl bound to processed glycans on the coverslip (which produces a signal brighter than the background, but dimmer than the virus). although viruses dissociate from some of the tracks, those that remain bound to the surface allow us to connect intensity of ecl labeling on the surface to intensities of ha and na on the virus that generated the track. this data is plotted in figure 3 -figure supplement 3, with and without normalization to na intensity. for assays using the fluorogenic neuraminidase substrate munana, 10 ml of solution containing labeled virus was diluted into 40 ml of na buffer (100 mm nacl, 50 mm mes ph 6.5, 5 mm cacl 2 ) and incubated in a test tube at 37˚c. at time points of 0, 30, 60, 120, and 180 min, aliquots were collected and na was inactivated by adding sodium carbonate to a final concentration of 100 mm, and fluorescence was measured by imaging a fixed volume of sample on a confocal microscope using excitation at 405 nm. the rate of turnover was then determined from the slope of the intensity versus time plot with the signal at 0 min subtracted from each timepoint. because strains with wildtype na and nadct produce different titers of virus that also differ in their morphology and na composition, we normalized samples to total na content ( figure 3-figure supplement 3a) , measured by immobilizing viruses on coverslips, imaging them, and integrating the total na intensity associated with the two samples. this yields an estimated 6-fold difference in total na content between virus with wildtype na and viruses with nadct. calu-3 cells grown on plastic dishes for fewer than 10 passages (dmem, 10% fbs, 1x penicillin/ streptomycin, supplemented with 1 mm sodium pyruvate (thermo fisher, 11360070) and 1x nonessential amino acids (thermo fisher, 11140076)) were split at 80% confluence and seeded onto 6 mm transwell supports at 50000 cells per insert. approximately 3 days after seeding, media from the apical compartment was removed (the 'airlift') and cells were provided with fresh media in the basal compartment every other day until being collected for experiments, 8-12 days following the airlift. cells cultured in this way differentiated into a secretory phenotype, producing a layer of mucus~1-10 mm thick over the apical surface of the monolayer ( figure 4a ). to bind virus without washing away secreted mucus, 5-10 ml of labeled virus was added to the apical side of each transwell insert (enough to just coat the surface), and immediately removed, leaving~1 ml of residual virus-containing solution that is approximately evenly distributed on the surface of the monolayer. the cells were then returned to the incubator for 3-6 hr, allowing virus to bind and diffuse, and allowing some of the residual moisture to dry before the cells are collected and fixed on ice for 20 min using 4% paraformaldehyde in pbs supplemented with 1 mm cacl 2 . following fixation, cells and mucus were labeled with muc5ac monoclonal antibody (45m1; ma5-12178 thermofisher) and erythrina cristagalli lectin labeled with fitc (5 mg/ml; vector laboratories, fl-1141). following labeling, the transwell insert was carefully excised with a razor blade and inverted onto a coverslip for imaging. to quantify alignment between the ha-na axis of a virus particle and the direction of its displacement from the associated ecl track ( figure 4e ), fluorescent images of ha, na, and ecl were segmented in both ha (to identify virus particles) and ecl (to identify regions of cleaved sialic acid) channels. for each segmented virus particle, we calculate the ha-na axis (defined as the vector displacement between the center of masses for ha and na) and the ecl displacement (defined as the vector displacement between the center of masses for ha and ecl) ( figure 4b , right). cases where multiple particles contact the same ecl track are excluded from analysis. replicates referenced throughout this paper refer to biological replicates, defined as virus collected from separate infected cell cultures, labeled, and assayed as indicated. no statistical methods were used to predetermine sample size. image data was excluded from analysis in rare cases if the sample drifted on the microscope stage, or if coverslip preparations showed non-specific virus binding. statistical tests and the number of replicates used in specific cases are described in figure captions. all statistical tests were performed in matlab r2017b using the statistics and machine learning toolbox. confidence intervals for the data in figure 1g were calculated using a critical value of the student's t distribution of 3.182 (95% confidence interval for n-1 = 3 degrees of freedom). data availability all data generated or analysed during this study are included in the manuscript and supporting files. for the typical time steps used in our simulations (~0.2 s), this amounts to instantaneous cleavage of any sa residues within reach of na. using these three probabilities, we calculate at each instant in time the number and location of each point of attachment between the virus and the surface and we remove any cleaved sialic acid residues for the remainder of the simulation. note that we do not prohibit multiple sas from binding to a single ha centroid; although this would be inaccurate at high densities of sa, under the conditions of these simulations it results in each ha centroid being bound to 0,1, or 2 sas at any particular instant in time -reasonable values for a trimeric molecule. to model diffusion of the virus, we use expressions derived for dilute suspensions of cylindrical rods (brenner, 1974) : axisymmetric, rotations about j do not appreciably change the number or location of ha and na molecules that are proximal to the surface. as a result, we do not expect that omitting this rotational degree of freedom from our simulations would qualitatively affect the results for cylindrical, axisymmetric particles. however, these simplifying approximations could not be extended to a spherical particle, which could rotate more freely on the surface, altering the number and location of na molecules capable of cleaving sialic acid. in this model, rectification of virus motion comes from asymmetries in the location of ha relative to the sa that are available for binding. at the boundary between regions where na activity has cleaved sa and regions where it has not, ha molecules will have more potential binding partners in the direction of the uncleaved region. the constraints introduced by binding to these sas will leave the virus with greater likelihood to step in the direction of the uncleaved region. although this asymmetrically distributed freedom of motion will only apply to a subset of all has, over the course of~10 6 simulation steps, it is sufficient to bias the direction of virus diffusion. this establishes a form of brownian ratchet, where the input energy could derive from the hydrolysis of sialic acid by na. any change in na organization that gets rid of this asymmetry in the orientation of the ha-na interface (e.g. placing na at both poles, in a single band away from the poles, spatially separated from ha, or in a uniform distribution over the surface of the virus) abolishes the rectified motion ( figure 3 -figure supplement 5). so far, these simulations have used static distributions of sa. allowing sa to diffuse on the surface will act to dissipate steep gradients in receptor density created by the activity of na on the virus, resulting in shallower gradients that extend beyond the localized distributions of na on the viral surface. the extent of this concentration gradient in available receptors will depend on the balance between the catalytic rate of na, the rate of diffusion of sa, and the size and rate of motion of the virus. these relationships can be parameterized using a nondimensional dahmkohler number: where k cat is the catalytic rate of na as before (~100 s à1 ), d sa is the diffusion coefficient of sialic acid on the surface, and l is a characteristic length for the reaction-diffusion system. taking l~100 nm, k cat~1 00 s à1 , and d sa~0 .01-0.1 mm 2 /s (reasonable values for large glycoproteins [freeman et al., 2018; jiang et al., 2015] ) gives da~10-100. the larger this value is, the better able a virus would be to maintain gradients in sialic acid density to support directional mobility. to test this prediction, we performed simulations where the diffusion coefficient of sa is varied while k cat remains constant and new sa residues are generated at the simulation boundary every time one is cleaved (to prevent complete removal of sa when its diffusion coefficient is high). consistent with the prediction, simulations of virus motion at da~100 or greater show preferential diffusion along the axis of the virus, while simulations at da < 100 do not ( figure 3e ). interestingly, for iav this transition corresponds to a sialic acid diffusion coefficient of~0.01 mm 2 /s, characteristic of some membrane-anchored glycoproteins (jacobson et al., 1984) , but considerably slower than measured values for gangliosides (komura et al., 2016) . although sialic acid diffusion can attenuate persistent virus motion if it is too rapid, it can also increase the rate of motion relative to immobile sialic acid by allowing has distal to the ha-na interface to also sample from asymmetric distributions of receptors ( figure 3e) . thus, the nature of the sialic acid receptors to which a virus is bound will likely influence how the virus moves -with greater persistent directional mobility on mucin gels of anchored glycoproteins, and with less persistence once it reaches glycolipids closer to the cell surface. the neuraminidase of influenza virus filamentous influenza viruses a periplasmic polymer curves vibrio cholerae and promotes pathogenesis biophysical measurement of the balance of influenza a hemagglutinin and neuraminidase activities rheology of a dilute suspension of axisymmetric brownian particles cilia and mucociliary clearance structural organization of a filamentous influenza a virus the m segment of the 2009 pandemic influenza virus confers increased neuraminidase activity, filamentous morphology, and efficient contact transmissibility to a/puerto rico/8/1934-based reassortant viruses structural analysis of the roles of influenza a virus membrane-associated proteins in assembly and morphology the m segment of the 2009 new pandemic h1n1 influenza virus is critical for its high transmission efficiency in the guinea pig model filamentous forms 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influenza a virus promotes escape from host cells functional balance between haemagglutinin and neuraminidase in influenza virus infections mucociliary clearance in the airways functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 h1n1 influenza pandemic a simple model of multivalent adhesion and its application to influenza infection a beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic h1n1 influenza virus in ferrets site-specific protein labeling by sfp phosphopantetheinyl transferase rotation-facilitated rapid transport of nanorods in mucosal tissues the interaction between respiratory pathogens and mucus structure of coronavirus hemagglutininesterase offers insight into corona and influenza virus evolution the authors would like to acknowledge members of the fletcher lab for feedback and technical consultation. this work was supported by the nih r01 gm114671 (daf), the immunotherapeutics and vaccine research initiative at uc berkeley (daf), and the chan zuckerberg biohub (daf). mdv was funded by a burroughs wellcome fund casi fellowship. daf is a chan zuckerberg biohub investigator. we wanted to develop simulations that would provide insight into the mechanisms of the directional mobility of influenza a virus. because individual viruses are highly variable (differing in shape, composition, and organization) and the dynamics of a cylindrical particle close to a surface are complex, we did not seek to develop a model that quantitatively predicts the rates of virus migration that we observe experimentally. instead, we focused on predicting the general phenomenon of organization-dependent directional mobility, and how this mobility changes when characteristics of the virus or its receptors (i.e. sialic acid, sa) are altered.modeling ha-sa binding and na-sa cleavage we model sialic acid coated surfaces as a uniform random distribution of sa at a density of 0.02 /nm 2 . although this value is lower than the estimated density in our experiments as well as on the surface of a cell (~0.1-1 sa/nm 2 ) (rosenberg and einstein, 1972) , it serves as a reasonable approximation given that has do not bind all sa glycoforms with similar affinity, and also that not all sas on the surface of fetuin may be accessible for binding after the protein is immobilized on the surface. for ha and na distributions on the surface of the virus, we use a triangular lattice with a spacing of 15 nm between neighboring spikes, with 84 has and 16 nas proximal to the surface for a filamentous virus 300 nm long. although we do not explicitly account for the trimeric/tetrameric nature of ha/na, this should have relatively little effect on our results since each ha can bind more than one sa and each na can cleave multiple sas. except in simulations where na is allowed to diffuse on the viral surface (modeling the behavior of nadct that we observe experimentally, figure 3 -figure supplement 2b), the locations of ha and na on simulated viruses are static, translating and rotating with the virus as a rigid object. although this is consistent with our measurements of protein mobility on the viral surface ( figure 1b) , it does not account for rotation of the virus around its minor axis, which could occur in our experiments.at each time step in a simulation, we identify ha-sa and na-sa pairs that are within a radius b of each other; we define these sa as being available for either binding (if close to ha) or cleavage (if close to na). the radius b, which we take to be 7.5 nm, models the flexibility of the carbohydrate and protein backbone to which sa is attached, along with the potential flexibility of ha and na on the surface of the virus. for ha binding kinetics, we used k on = 400-1000 m à1 s à1 and k off = 1 s à1 , based on previous measurements (sieben et al., 2012; takemoto et al., 1996) . the probabilities of a particular ha-sa pair within a radius b of each other forming (p on ) or losing (p off ) a bond in a time interval dt are therefore approximated by:the factor of 1000 in the denominator of the expression for p on converts the volume accessible to an ha molecule to units of liters. since the concentration of sialic acid within a binding radius is high relative to the k m for na (>10 mm versus 100 mm-1mm [benton et al., 2015] ), the probability of an na-sa pair resulting in cleavage is determined from the catalytic rate constant (k cat~1 00 s à1 ) as: once the virus has formed attachments to sa on the surface, it's translational and rotational motion becomes constrained. to account for this constraint, after calculating the translational and rotational increments during a given time interval, we only accept the resulting values if they do not result in extension of any ha-sa bonds beyond their allowable radius, b; otherwise, the simulation time does not advance, and we resample the langevin parameters {xx, xy, xq} to take another trial step. as the number of bonds increases, the possible steps that satisfy the constraints of all bonds becomes smaller, resulting in immobilization of the virus. in comparison, in the time it would take a single ha-sa pair to dissociate (1/k off~1 s), the typical displacement of an unconstrained virus would be~1 mm, several orders of magnitude larger than would be tolerated by the constraints of the bond. to reduce the number of times we must resample at each time step, we reduce the translational and rotational diffusion coefficients 100-fold to dampen the virus's motion.this formulation makes several simplifying approximations, motivated by the cylindrical geometry of viral particles. in particular, we only account for rotations about the virus' major axis, q, and not around the angles j or y (figure 3-figure supplement 4a ). for a cylindrical particle interacting with a flat surface, rotations about the angle y would reduce the contact area between the particle and the surface, and would not be favored. this is consistent with our experiments, in which we do not observe filamentous particles pivoting on the surface around the virus's poles. in contrast, rotations around the angle j would be permissible, although we are unable to detect them experimentally. because the particles we model are increasing the length of the virus increases the number of potential ha-sa interactions, which we would expect to reduce virus mobility if the kinetics of ha-sa binding are held constant. this trend is supported by simulations; varying particle length (185 nm, 285 nm, or 385 nm) while keeping ha-sa binding kinetics constant dramatically reduces particle mobility (figure 3-figure supplement 6, top row) , whereas holding the average number of ha-sa bonds constant results in slightly higher persistent directional mobility for larger particles (figure 3-figure supplement 6, bottom row) . in both cases, simulations show a greater tendency for longer viruses to maintain constant orientation. thus, although particle size and ha-sa binding kinetics both alter virus motion quantitatively, the qualitative features of directional mobility described throughout this work are preserved in particles approaching the lower end of the length distributions observed for filamentous iav. the diffusion of viral particles through mucus can be modeled as a one-dimensional problem, where particles initially binds to the distal part of a mucus gel with thickness d and diffuse until reaching the surface of the underlying epithelial cells. solving the diffusion equation with reflecting boundary conditions at x = d (modeling the tendency of viruses to stay bound to the mucus layer) and absorbing boundary conditions at x = 0 (modeling transfer of the viral particle from the mucus gel to the cell surface) gives the probability density of particles within the mucus gel as a function of time:integrating this expression over x gives the probability that a virus will remain in the mucus gel at time t, and subtracting this quantity from one gives the probability, p(t), that a virus will have reached the cell surface by time t:this expression allows us to estimate the survival probability of a virus in the presence of mucociliary clearance by evaluating p(t = l/u), where l (~10 cm) is the distance a bound particle must be carried to be neutralized and u (~100 mm/s) is the velocity of mucociliary transport. this will depend on the virus diffusion coefficient, d virus , which we estimate from the data in figure 3 and the relationship d virus = msd/4 t. this gives a value of~800 nm 2 /s in two dimensions or~400 nm 2 /s in one dimension, which we can substitute into the expressions above. the results of these calculations are plotted in figure 4 -figure supplement 1, where we compare the first passage probability of viruses with five-fold differences in their diffusion coefficients in a mucus layer that is 2 mm thick. for the parameters listed here, the timescale for mucociliary clearance (~20 min) is substantially shorter than the timescale for virus diffusion (~2 hr), and the percentage of successful particles (defined for simplicity as those that reach the epithelial surface) is extremely sensitive to their diffusion coefficient: the five-fold decrease in d virus that we estimate would result from the loss of persistent motion would lead to > 1000 fold decrease in the number of successful particles (figure 4-figure supplement 1b) . this calculation assumes that binding of viruses to the cell surface is immediate and irreversibleassumptions that are reasonable if the cell surface is densely decorated with sialic acid and if viruses that come in contact with it are rapidly endocytosed. however, regardless of how interactions between viruses and the underlying epithelium are modeled, the rate at which viruses encounter the epithelium -our primary focus, and a prerequisite for productive infection -will always be inversely proportional to the virus's diffusion coefficient. although this simple model provides only a rough estimate, this analysis, which is based on reasonable estimates of physiological parameters (bustamante-marin and ostrowski, 2017), suggests that persistent motion could enhance the frequency of host-to-host transmission by orders of magnitude. key: cord-307813-elom30nx authors: yip, tsz-fung; selim, aisha sami mohammed; lian, ida; lee, suki man-yan title: advancements in host-based interventions for influenza treatment date: 2018-07-10 journal: front immunol doi: 10.3389/fimmu.2018.01547 sha: doc_id: 307813 cord_uid: elom30nx influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. keywords: host factors, influenza, cytokines, metabolism, immunomodulation introduction influenza remains a source of public health concern. influenza a virus (iav) has been the cause of historical noxious pandemics, such as the spanish flu 1918 h1n1, asian flu h2n2 1957, hong kong h3n2 flu 1968, and more recently the pandemic of h1n1 2009 (swine flu). influenza also causes seasonal epidemics and outbreaks with high morbidity and mortality rates such as the 2015 h1n1 outbreak in india (1, 2) . the error-prone nature of the viral rna polymerase (rdrp) and virus' capacity for genetic re-assortment (antigenic drift and shift) result in the viral components' susceptibility to mutations, allowing the viruses to evade the immune system and increases their resistance to control strategies. currently, influenza vaccination and two classes of antiviral drugs-m2 ion channel blockers (amantadine and rimantadine) and neuraminidase (na) inhibitor (oseltamivir, zanamivir, and peramivir)-and the novel treatment option using polymerase inhibitor (favipiravir) are considered as mainstays in influenza infection treatment and control. the use of influenza vaccinations remains challenging due to antigenic drifts and shifts, with seasonal variation of new circulating species. production of vaccine is time consuming with efficacy concerns, especially in the case of pandemic. variations in vaccine efficacy caused by age should be aware, with studies suggesting that vaccineconferred protection may not be optimal in certain age groups (3) . the disadvantages of using the conventional antiviral drugs have also been a concern. significant levels of resistance to both classes of drugs have been repeatedly reported (4, 5) . high level of resistance (up to 91%) to m2 blockers has been reported in h3n2 virus strain in american isolates (6) . resistance has also been reported in h5n1 virus (7) . iav resistance to na inhibitors has also become an increasingly prevalent concern, with the recent highly fatal outbreak of influenza a(h1n1)pdm09 in india 2015 associated with oseltamivir drug resistance (8, 9) . in addition, a large cluster of influenza a(h1n1)pdm09 viruses in japan was found to have increased oseltamivir and peramivir drug resistance (5) . there is an urgent need to search for alternative targets to treat influenza virus infections, including non-viral targets such as host cellular factors; which are promising as viruses rely on the host machinery for replication. while host immune response is intended to confer a degree of protection against the infection, an impaired or exaggerated host immune response could be detrimental-iav h5n1 and h7n9 virus infection was reported to exaggerate aberrant cytokine release, resulting in a cytokine storm that caused accelerated host death (10) (11) (12) . many recent studies have focused on the investigation of targeting host factors to control virus replication as well as modulate immune response, which we have previously evaluated (13) . in this review, we will discuss the latest studies (in the past 5 years) on the investigation of novel host-based approaches with potential for influenza treatment. the replication cycle of iav can be grossly divided into four different stages: (1) entry, (2) genome nuclear import, (3) replication and protein synthesis, and (4) genome nuclear export, apical transport, assembly, and budding. as an obligate intracellular pathogen, iavs are heavily dependent on host machinery for replication and propagation. to this extent, studies employing genome-wide rna interference (rnai) to screen for host factors involved in iav replication cycle have been performed (14, 15) and an increasing number of approaches targeting these host factors to control iav replication have been investigated. entry of iav into the host cell is divided into several steps (16, 17) . first, hemagglutinin (ha) on the surface of iav binds to the terminal α-sialic acid on the host cell receptor. this induces the internalization of the viral particle by clathrin-dependent, caveolin-, and clathrin-independent endocytosis (18) . macropinocytosis was revealed as an alternative entry pathway for iav (19) , which subsequently enters the canonical endocytic pathway (20, 21) . the vesicle-containing viral particle forms an early endosome (also known as sorting endosome), which matures into a late endosome as the endocytic pathway progresses. a gradual decrease in intraluminal ph from ph 6.5 to 5.0, mediated by v-atpase proton pump (22) , takes place as the endosome matures (23, 24) . this ph drop in the endosomal lumen induces a conformational change in ha, which is activated by proteolytic cleavage to generate ha1 and ha2 from precursor molecule ha0 (25, 26) . this conformational change triggers the fusion of the viral envelope with the endosomal membrane, releasing the viral genome into the cytoplasm. acidification of the endosome causes the subsequent acidi fication of viral lumen via the iav m2 proton channel (27) , which in turn promotes the dissociation of m1 layer from both the viral envelope (24) and the viral ribonucleoprotein (vrnp) complex (28) . interestingly, a sharp decrease in ph from neutral to an acidic ph of 5.0 as utilized by acid bypass has been observed to be sub-optimal for viral replication. it is hence proposed that a gradual decrease in endosomal ph is necessary for sequential reduction in viral stiffness, dissociation of m1 from the np in the vrnp complex, destabilization of m1 layer from the viral envelope, and the eventual conformational change of the ha for the release of viral genome and proteins to the cytoplasm from late endosome (24) . proteolytic cleavage of ha0 to ha1/ha2 is an important step in iav replication. this cleavage relocates ha2, converting previously uncleaved ha0 to a metastable conformation that induces membrane fusion at acidic ph (29) . inefficient cleavage and activation of ha leads to low infectivity (30) . as identified proteins encoded by the viral genome do not possess proteolytic properties, the virus is dependent on host protease for the cleavage of ha. this provides a potential target to control iav infection. ha is commonly cleaved by trypsin-like proteases at the single arginine residue at position 329. human airway epithelium serine proteases hat and tmprss2 were identified as the host factors for cleavage at this residue (31) . aprotinin, purified from bovine lung (32) , is a protease inhibitor with a long history of clinical use as an antifibrinolytic agent in cardiac surgery (33) . its potential as an anti-iav drug has been recognized for over a decade (34) and has been shown to reduce the infectivity of a broad spectrum of iav strains (34, 35) both in vitro (26) and in vivo (36) . once withdrawn from the western drug market due to its association with mortality (33), aprotinin has been approved as a locally administered, small-particle aerosol drug for the treatment of iav infection in russia (36) . however, side-effects associated with the systemic administration of aprotinin raises the need for an alternative protease inhibitor for use in treatment of iav infections. camostat, a serine protease inhibitor, was reported to demonstrate anti-iav potential in mice dating back to 1996 (37) , but little to no research has been conducted to develop it into an anti-iav treatment. it was revisited and proven to be one of the most efficient serine protease inhibitors for the inhibition of iav replication in primary human tracheal epithelial cells in vitro when tested compounds were used at similar molarities (35) . at present, camostat is widely administered for the treatment of liver fibrosis, chronic pancreatitis, and cancer (38, 39) , making it a highly promising candidate for drug repurposing. despite the lack of association between camostat and increased mortality (as with aprotinin), reports of camostat potentially inducing acute eosinophilic pneumonia (38) warrants the need for careful consideration and further research into the repositioning of drugs from the same class. highly pathogenic iav, such as the h5 and h7 subtypes, are reported to have ha cleavage sites rich in basic residues (30) . the polybasic nature of the cleavage sites provides multiple targets for a broad spectrum of proteases, including the more ubiquitously expressed intracellular proteases such as furin (40) . this increased protease spectrum could be utilized by these viruses for the activation of ha prior to viral budding, allowing for evasion of potential inhibition by exogenously administered serine protease inhibitors. furthermore, an in vivo study utilizing mice treated with a single protease inhibitor prior to infection with h7 virus bearing a polybasic cleavage site showed poor efficacy despite good results were obtained for infection with h1n1 virus bearing single cleavage site (41) , suggesting strain specificity in using serine protease inhibitors to treat iav infections. endosomal acidification is required for the release of iav genome (in the form of a vrnp complex) into the cytoplasm (24) . research has shown that an increase in endosomal ph during the early phases of infection could inhibit iav infection in vitro (42) , bringing to light the possibility of controlling iav infection through the prevention of endosomal acidification. the v-atpase inhibitor bafilomycin a1, when used at high concentrations (10-100 nm) has been proven to inhibit iav replication through the efficient suppression of v-atpase (43, 44) . however, prominent cytotoxicity to host cells was also observed at such concentrations (44) . interestingly, lower concentrations (0.1 nm) of bafilomycin a1 lack inhibitory effects on v-atpase attenuated iav replication due to disruption of endosomal trafficking. thus, bafilomycin a1 is suggested to exert its antiviral function via distinct mechanisms at differing concentrations. diphyllin, isolated from the plant cleistanthus collinus, is a natural compound able to induce a v-atpase inhibitory effect (45) . in contrast to bafilomycin a1, diphyllin is well-tolerated in vitro without inducing obvious cytotoxic effects (46) . most notably, diphyllin is found to effectively inhibit replication of viral strains resistant to amantadine and/or oseltamivir (46) . since drug resistance to these widely administered antivirals is of major public health concern (13), diphyllin is regarded as a promising antiviral against drug-resistant iav strains. the release of iav genomic material during replication requires the fusion of the endosomal membrane with the viral envelope. since cholesterol plays a major role in controlling the fluidity of the lipid bilayer in cells, it is hence suspected to have a role in the infection cycle of iav. interferon-induced transmembrane proteins (ifitms) are proteins expressed in many vertebrates (including humans) and are found on the plasma membrane, the membranes of early and late endosomes, as well as on lysosomes (47, 48) . while humans express ifitm1, ifitm2, ifitm3, ifitm5, and ifitm10, only ifitm 1, 2, and 3 are both immune-related as well as interferon (ifn)-inducible (48) , and have been observed to restrict the replication of different viruses, including iav (49) . studies suggest that ifitms limit viral infection by reducing membrane fluidity and hence restrict the hemifusion (the mixing of lipid bilayer without the release of viral content) of viral and endosomal membranes (50) , probably via the disruption of cholesterol homeostasis of late endosomes, where viral fusion and genome release conventionally take place (51) . a recent study using rnai also demonstrated that cholesterol homeostasis can be regulated via acid phosphatase 2 (acp2)-mediated niemann-pick c2 activity and impaired the membrane fusion of iav and influenza b virus (ibv) (52) , further suggesting the importance of controlling cholesterol homeostasis in the release of viral genome to cytoplasm. on the contrary, later studies suggest that ifitm3 exerts its antiviral activity in a cholesterol-independent manner, showing that an increase in cholesterol composition of late endosomal membranes fail to inhibit viral membrane fusion (53) . in addition, studies suggested the accumulation of cholesterol level in the late endosome does not inhibit the iav genome release into cytoplasm (54, 55) . with the modulation of cholesterol levels in host endosomal membrane as a mean to inhibit iav host cell entry is still under debate, further studies are required before clear conclusions can be drawn. by comparing the mirna profiles of the iav-permissive hek 293t cells and (less permissive) hela cells, mirna-33a has been identified as a negative regulator for iav infection via the inhibition of archain 1 (arcn1, also known as δ-copi) (56). arcn1 is a subunit of the copi complex that is required for intracellular trafficking and endosome function (57) , depletion of which has been reported to inhibit iav infection (14) . despite impaired iav internalization caused by arcn1 depletion via sirna (56, 58), it was not able to recapitulate through acute inhibition of copi complex by pharmaceutical means (58) . it is hypothesized that the long-term (lasting days) perturbation on arcn1 by rnai affected the general endosomal trafficking network, a phenomena which cannot be recapitulated by acute pharmaceutical inhibition to block iav infection (58) . the potential of targeting arcn1 for iav treatment deserves further investigation, despite the favorable results from rnai studies. nuclear import of vrnp complexes from the cytoplasm following fusion of the viral and the endosomal membrane is required for replication to take place (59) . an early study suggested that vrnp complexes could be transported to the periphery of the nucleus (60), while recent studies report that vrnp complexes utilize the importin-α-importin-β1 (impα-impβ1) system for nuclear import (59, 61) and lacking of importin-α7, in an importin-α7 knockout mouse model were found to be resistant to iav infection (62) . ivermectin has long been clinically administered for the treatment of parasitosis (63) , but has recently come to attention as a potential inhibitor of impα/β (64) . ivermectin inhibition of impα/β has shown to inhibit the replication of rna viruses such as dengue virus and hiv-1 (64) . ivermectin was recently tested for the inhibition of iav in vitro, with nuclear import of vrnp complex (of both wild-type and antiviral mxa escape mutant) efficiently inhibited (65) . given ivermectin's longstanding record of clinical applications and fda-approved status, repurposing of this drug for the treatment of iav should be considered, especially while under threat of pandemic iav outbreak. following the import of the vrnp complex into the nucleus of the host cell, rdrp uses the vrna as a template to synthesize mrna or crna. synthesized crna remains in the nucleus for new vrna generation, while mrna is exported out of the nucleus for translation. viral protein products are either transported to the cell surface via golgi (in case of ha and na) or imported back into the nucleus to bind with vrna, forming new vrnp complex (59) . numerous host factors are involved in this process and hence could be possible targets for therapeutic intervention. out of the eight genome segments of iav, the m and ns segments are well known for undergoing splicing to generate at least two different mrnas per individual segment (66, 67) . cdc2-like kinase 1 (clk1) is a kinase which regulates alternative splicing of pre-mrna (68) . inhibition of clk1 by the chemical tg003 or knockdown of clk1 is shown to cause a decrease in m2 mrna generation and disrupt downstream m2 protein expression, prominently reduced iav propagation (15) . clypearin and corilagin were both found to be potent anti-iav compounds, with a higher therapeutic index than tg003 in vitro (69) . clypearin is isolated from herbs used by chinese medicine practitioners for treating respiratory tract diseases during replication, viral mrna is exported from the nucleus to cytoplasm, where protein synthesis takes place. human rna polymerase ii activity is found to be correlated with iav replication through the inhibition of nuclear export of certain viral mrnas, such as m1 mrna (70) . cyclosporine a (csa) is a fda-approved drug with immunomodulatory functions (71) that has been found to have an anti-iav effect in both cyclophilin a (cypa)-dependent and -independent manners (72) . the cypa-dependent effect was found to correlate with nuclear export of vrnp complex (see targeting nuclear export complex). the cypa-independent effect caused inhibition of host rna polymerase ii. csa is a prospective drug candidate for treatment of iav infections with a relatively high barrier for development of intrinsic drug resistance, as opposed to commonly used antivirals (73) . nuclear rna export factor 1 (nxf1) is a host factor that has been identified to be involved in the nuclear export of iav mrna. the knockdown of nxf1 in hek 293t cells revealed prominent viral mrna nuclear retention in host cell nucleus (74) . protectin d1 (pd1), an endogenously produced lipid in the respiratory tract, has been identified to have potent anti-inflammatory and antiviral effects (75) . pd1 production was notably found to be reduced in the lungs of iav-infected mice. therapeutic administration of pd1 was shown to significantly reduce iav mrna expression, lower lung viral titer, as well as improve survival of iav-infected mice. mechanistic studies revealed attenuated cytoplasmic translocation of viral mrna with such treatment. a decrease in recruitment of viral transcripts to nxf1 was observed while nuclear export of host rna remained largely unaffected, suggesting a role of pd1 in regulating nxf1 in nuclear export of viral rna. natural pd1 expression in the human airway makes this an ideal candidate for novel therapeutics in the treatment of iav infection. the eukaryotic initiation factor-4a (eif4a) family plays an important role in protein translation (76, 77) . eif4a impairment has been proven to be related to antiviral activity in a broad spectrum of rna viruses in vitro (78) , with inhibition of iav mrna translation (79) . the eif4a inhibitors, silvestrol and pateamine a were demonstrated to arrest viral protein synthesis, thus blocking viral genome replication in vitro (80) . although both silvestrol and pateamine a caused high cytotoxicity at the concentration required effective for iav inhibition, drugs targeting mrna translation for various diseases have been approved by fda or are under active development (81) . as such, inhibition of iav infections by disrupting mrna translation may well be a therapeutic approach in the future. post-translational modifications during protein maturation ensure proper function of proteins, with proteins of iav no exception. nitazoxanide, a fda-licensed drug used to treat enteritis, was found to be effective in controlling iav infection by interfering with ha n-glycosylation as well as intracellular trafficking in host cell and eventually led to a reduction in viral budding (82) . despite the mechanism of nitazoxanide being presently unknown, its ability to inhibit replication of numerous viruses [iav, respiratory syncytial virus, coronavirus, hepatitis b virus, and many others (83) ] suggests that it may act on host machinery. the drug has also been proven in vitro to inhibit the propagation of many circulating strains of human iav, including those resistant to oseltamivir or zanamivir (84) . nitazoxanide has a high barrier of resistance to iav (83) and other viral strains resistance to neuraminidase inhibitors (85), making it a very promising therapeutic target for iav treatment. the drug is currently under phase iii clinical trials (83). in the later stage of viral replication, viral rnas of iav packed with rdrp and np (known as vrnp complexes) are exported from the nucleus (59), assembled (86) , and transported to the plasma membrane [apical in polarized cells (87) ] for budding. novel targets for influenza treatment frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1547 exportin 1 (xpo1, also known as crm1) is well known for its function in the nuclear export of protein (88) and rna, including viral rna (89) . similar to hiv (89, 90) , iav viral rna does not directly bind to xpo1 but is instead held together by several viral proteins. the viral nuclear export protein (nep, or previously known as ns2) and the vrnp complex have been proposed as the nuclear export complex (91) . cellular xpo1 has been proven to be crucial in the nuclear export of the vrnp complex, with early studies using leptomycin b (lmb), a potent xpo1 inhibitor, revealing that in vitro inhibition of xpo1 led to nuclear retention of vrnp complex (92, 93) . however, lmb was deemed unsuitable for development as a potential drug in the phase i clinical trial due to observed cytotoxic effects (94) . verdinexor (also known as kpt-355) is a new bioavailable selective inhibitor of xpo1. it has been shown to be effective against different strains of iavs both in vitro and in vivo as prophylactic and therapeutic treatments (95, 96) . it is worth mentioning that delayed administration of verdinexor at day 4 post-infection was still deemed beneficial, with reduced viral load in vivo (96) . this suggests a prolonged therapeutic time window when compared to the mainstay antiviral drugs such as oseltamivir, where recommended administration is at the early stage of infection (within 48 h of symptom onset) (97) . currently, verdinexor has passed the phase i clinical study trials, suggesting that it does not pose severe cytotoxic effects as lmb does. in addition, a recent report demonstrated that a new drug, dp2392-e10, which binds and inhibits the function of xpo1, can suppress iav replication in vitro (98) further strengthens the concept of iav intervention by targeting xpo1. viral m1 protein is crucial in assisting the nuclear export of vrnp complex. it was commonly suggested that m1 protein links vrnp complex to viral nuclear export protein nep which interacts with xpo1 for nuclear export (59) . thus, viral m1 protein may serve as a target to inhibit nuclear export of vrnp. as previously mentioned (see inhibition of mrna export), csa inhibits iav replication via both cypa-dependent and -independent mechanisms. a recent study using a transgenic mice overexpressing cypa showed greater resistance to iav challenge (99) . in the cypa-dependent mechanism, csa enhances the binding of cypa to m1 protein (72) , increases the self-association of m1, and hinders m1 nuclear import (100) . csa also promotes the cypa-dependent degradation of viral m1 protein (72, 101) . csa seems to be a promising drug to inhibit the nuclear export of vrnp complex by inhibiting viral m1 protein stability and function. recently, cd151, a tetraspanin (defined by four transmembrane domains with conserved residues) that is expressed abundantly in lungs and interacts with integrins has been implicated in the regulation of iav replication in vitro and in vivo (102) . knockdown of cd151 in primary human nasal epithelial cells resulted in the nuclear retention of host xpo1, viral np, nep, and m1 proteins, with an increased survival rate observed in iavinfected cd151 knockout mice. co-immunoprecipitation assays suggest that cd151 interacts with viral np, m1, and nep proteins (102) ; however, the exact domains involved in interaction and the mechanism of cd151 function in nuclear export remain unclear. given that a small molecule inhibitor for cd151 is now under development (103) , more data revealing the role of cd151 in iav infection and subsequent use in targeting cd151 as anti-iav therapy is anticipated. during iav infection, raf/mek/erk signaling cascade is activated, while the inhibition of mek by u0126, probably mediated via myosin (light chain) (104), a known motor protein, impairs the nuclear export of vrnp complexes (105) . suppressing iav replication by inhibition of raf/mek/erk signaling cascade has been illustrated both in vivo (106) and in vitro (105) . the replication of ibv (107) as well as borna disease virus (108) was shown to be inhibited by u0126, suggesting the versatility of this approach in controlling infection by different viruses. despite being effective when administered locally to lungs via aerosol, u0126 has little effect when administered orally (106) . another mek inhibitor, ci-1040 (also known as pd184352) was shown to have high potency against iav in vitro (106). ci-1040 has completed phase ii clinical trials as an anti-tumor drug, with the application of ci-1040 as a potential anti-iav drug candidate recently revisited. unlike u0126, ci-1040 is orally bioavailable and oral administration of ci-1040 at 48 h post-infection protected 60% of the iav-infected mice, while the oseltamivir-treated group experienced a 100% death rate (109) . oseltamivir is known to be effective only when administered in the early stages of iav infection. this suggests the potential use of ci-1040 as an agent used in iav treatment due to its potentially longer therapeutic time window than mainstay antivirals. formyl peptide receptor 2 (fpr2) located at the host cell surface was identified as an erk stimulator (110) . antagonizing fpr2 promoted the survival of iav-infected mice (110) . furthermore, fpr2 antagonists have been described to possess antiviral activity against not only iav but also ibv infection (111) , promoting the idea that antagonizing fpr2 to suppress raf/mek/erk signaling cascade could potentially be a novel approach for the treatment of a broad spectrum of influenza viruses. after the nuclear export of the vrnp complexes, host cell's intracellular transport mechanism is required to deliver vrnp complexes to the host plasma membrane for the assembly of viral rnas and proteins at the final stage of viral replication. among the various vesicular compartments found in a cell, the rab11a + endosomes are known to recycle endocytosed membrane proteins and lipids to the plasma membrane for membrane homeostasis (112) , a property utilized by many rna viruses, including iav (87, (113) (114) (115) . iav progeny virus production was found to be significantly reduced in rab11a + knockdown human cell lines (116) . furthermore, vrnp complex plasma membrane transport perturbation was observed in rab11a knockdown cells (114, 115) ; in cells expressing deletion mutant of rab11 family interacting proteins (87) ; as well as cells treated with chemicals to interfere microtubule (114) . direct interaction of vrnp complex with rab11a has also been verified (114, 115) , demonstrating the dependence of vrna complex transport on rab11a + vesicles and the microtubule network during viral replication. since rab11a proteins do not confer any mobile properties to the vesicle, molecular motors such as kinesins are required for the active transportation of vesicles through cytoskeletons. kif13a, a kinesin-3 family member, was recently identified as a molecular motor for plasma membrane transportation of vrnp-loaded rab11a + vesicles (117) . kif13a knockdown was found to reduce progeny virus production. overexpression of a mutant form of kif13a lacking in motor capacity resulted in disruption of the plasma membrane distribution of vrnp complex during later stages of infection. this data suggest that the apical transport of viral components via rab11a or kif13a could potentially serve as therapeutic targets against iav infection. further examination is merited. tubulin acetylation and deacetylation affects microtubule stability (118) . histone deacetylase 6 (hdac6) was found to deacetylate α-tubulin, one of the subunits of microtubule (119) . a study has demonstrated that hdac6 is involved in iav replication (120) . inhibition of hdac6 by tubacin or knockdown of hdac6 gene resulted in an increase of progeny virus production with vrnp complex redistributed toward the periphery of infected cells. in addition, transportation of ha to the plasma membrane for viral budding was also found to be inhibited by hdac6. this data suggests that activation of hdac6 by its stimulant could be a potential approach to anti-iav therapy, despite hdac6 stimulants still being under development. while several studies have suggested iav transmission between cells through apical membranes (121) and intercellular connections (122) , virus budding from cell membranes remains the major route for transmission of viruses to uninfected cells. na is responsible for the cleavage of sialic acid to prevent the interaction between ha and the host cell during viral budding. besides, viral na, viral ha, m1 as well as m2, are also suggested to play an important role in the initiation of the budding process (123, 124) . in section "controlling cholesterol homeostasis, " we discussed the involvement of host cholesterol in viral membrane fusion and viral genome release to cytoplasm. recent studies have demonstrated that host cholesterol may also play an important role in viral budding. it was demonstrated that overexpression of annexin a6 (anxa6), a phospholipid binding protein, could lead to a decrease in cholesterol levels within the golgi apparatus and plasma membrane (55), ultimately causing a reduction in egression of progeny virion from infected cells (54) . this reduction could be reversed by the addition of exogenous cholesterol (55) . similar to anxa6 overexpression, addition of a hydrophobic polyamine, u18666a, could reduce cholesterol level in plasma membr ane, also inhibited viral replication (55) . since iav is assumed to bud from lipid rafts (cholesterol-rich plasma membrane domains) (123) , it was demonstrated that anxa6 overexpression or u18666a treatment could hinder progeny virus production by lowering the cholesterol content in the plasma membrane. this hypothesis was strengthened through recent studies resolving the cholesterol-binding site of viral m2 protein, suggesting that iav m2 clustering (which provides membrane curvature for scission) is mediated by cholesterol (125) . a recent report utilizing two different fda-approved cholesterol-lowering drugs, gemfibrozil and lovastatin, stated that there was reduction in stability and infectivity of progeny virus compared to that replicating within cholesterol-sufficient host cells (126) . taken together, this data suggests that controlling cellular cholesterol content would be an effective alternative with drugs available for repurposing iav treatment. further in vivo works are needed to confirm this hypothesis. the gi-type g-protein coupled receptor α2-adrenergic receptors (α2-ars) have been recently identified as a key host factor involved in iav replication (127) . apical transport of the viral protein ha is inhibited by low intracellular camp level after stimulating the α2-ar-mediated signaling. in vitro stimulation of α2-ar by its agonist clonidine inhibits iav replication. therapeutic administration of clonidine reduced pulmonary edema and improved survival rate of iav-infected mice. development of a new antiviral targeting the α2-ar-mediated signaling seems promising and deserves further investigation. although targeting host factors for viral interventions generally provides a better resistance barrier, emergence of resistance may still arise (61) . therefore, combined use of interventions targeting both virus and host factors have been recommended to reduce opportunities for viral development of resistance. one such example would be the combined administration of na inhibitor (oseltamivir) alongside an anti-host factor [such as v-atpase inhibitor diphyllin (46) , ha maturation inhibitor nitazoxanide (85) , fpr2 antagonists (111) , and xpo1 inhibitor verdinexor (96) ]. while further direct assessment for the ease of emergence of escape mutants between single and combinatory use of drugs is required, the synergistic effects of a combined, multi-drug approach observed thus far highly suggest an increased effectiveness over a single-drug approach. table 1 summarizes novel host targets regulating iav replication. compared to rnai, small molecular chemicals remain the best choice as drug candidates due to their fast acting and easy-todeliver properties. although small molecular chemicals targeting certain host factors aforementioned have yet to be developed, their rnai-identified involvement in the iav replication cycle provide leads for the development of new iav interventions. the immune system aims to protect the host from infection and clear the pathogen once an infection occurs. in addition, the complex networks formed between the host physiology and the immune system co-operatively shape the disease outcome; modulations on the networks could alleviate disease severity in iav infections. the immunological responses elicited by iav infection has been reviewed in detail (128) (129) (130) . at the initial stage of iav infection, the respiratory epithelial cells are the primary target for infection. once the infection is initiated, the recognition of infection is accomplished via the detection of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) (see toll-like receptors), and lead to the expression and secretion of different cytokines and chemokines, such as il-6, il-8, tumor necrosis factor (tnf)-α, and ccl2 as well as type i and iii ifns. as sentinel cells, alveolar macrophages could also be infected, inducing cytokines and is the main source of type i ifns (128, 129) . type i ifns are known inducer for the upregulation of death receptor 5, which is the receptor for tnfrelated apoptosis-inducing ligand (trail), in lung pneumocytes (128) . il-8 and ccl2 produced by both epithelial cells and macrophages act as chemoattractants for neutrophils and monocytes, respectively. neutrophils are one of the earliest immune cells being recruited to the site of infection (131) with transmigration of neutrophils carry out by adhesion molecules, such as cd11a, cd11b, and cd18 (132) . in addition to the antiviral activity of neutrophil-released reactive oxygen species (ros), defensin and pentraxin (132) , uptaking iav by neutrophils could also help in controlling viral propagation as these cells do not support replication of iav (133) . besides controlling viral replication, neutrophils also play an important role in guiding the migration of iav-specific cd8 + t-cells in the infection site by secreting and leaving a trail of cxcl12 (134) . infiltrated monocytes will, however, differentiate into macrophages or dendritic cells (dcs). the monocytes-derived macrophages are reported to be a permissive host for iav production (135) , sustaining inflammation by producing cytokines in a magnitude larger than that of the resident alveolar macrophages. the monocyte-derived dc as well as the resident airway cd11c low b220 + plasmacytoid dc (pdc) and two types of conventional dcs (cd103 + cd11b low and cd103 − cd11b hi ) acquire the antigen of the invading pathogen through either direct infection or up-taking infected dead cells (129) . in the presence of type i ifns, dcs mature when encountering pamps from invading pathogen (129) . depending on the sub-cellular localization of the antigen, cytosolic and endosomal antigen will be loaded onto major histocompatibility complex (mhc) class i and ii molecules respectively (130) . once mature, dcs migrate from the infection site to the draining lymph nodes via the interaction of ccr7 and ccl19/ccl21 (130, 136) for antigen presentation via mhc class i and ii to naïve cd8 + and cd4 + t-cells, respectively (137) (138) (139) (140) . interestingly, monocytesderived dcs that engulfed the infected dead cells are poor antigen presenters for cd8 + t-cells and require the transfer of intact mhc class i/peptide complex to lymph node-resident cd8α + dcs which are the most efficient antigen-presenting cells to cd8 + t-cells (137) . in addition to antigen presentation, pdc are well known for their high ability in type i ifns production to limit viral propagation (141) . within the lymph node, naïve cd8 + t-cells are activated by the dcs, differentiate and clonal expand into cytotoxic t-lymphocytes (ctls) with the aid of various cytokines, including ifn-γ, il-12, type i ifns, and il2 (142, 143) , and the help from activated cd4 + t helper cells (144). differentiated ctls downregulate their lymph node homing receptor ccr7 and upregulate ccr4 and cxcr3 for the migration to the site of infection. within the site of infection, ctls control viral replication by targeting and inducing apoptosis of virus-infected cells via the secretion of perforin and granzymes as well as the ligation of death receptors on the infected cells by tnf, fas ligand, and trail. on the other hand, cd4 + t-cells are activated by the presentation of mhc class ii/ antigen complex by dcs, with co-stimulatory receptors such as cd28 expressed on the t-cells and the ligand for cd28 (cd80 and cd86) expressed on dcs playing an important role (144). activation of cd4 + t-cells lead to differentiation into different effector cells subsets, including the classical th1 and th2, and the more recently identified regulatory t cells, follicular t helper cells, th9, and th17 subsets (144). th1 cells regulate to the differentiation of ctls as mentioned whereas th2 cells contributes to the activation of b-cells through cd40l. within the pregerminal center of the lymph node, the follicular t helper cells interact with antigen-primed b-cells and promote their proliferation. antigen-primed b-cells differentiates into plasmablast and undergo antibody class-switching in the germinal center (145) . detailed functions of regulatory t cells, follicular t cells, th9, and th17 cells are discussed elsewhere (144, 145). plasmablasts enter the blood-stream, are recruited to the inflamed tissue, and terminally differentiate into plasma b cells which specialize in the production of antibody for pathogen neutralization, opsonization, and antibody-dependent cell-mediated cytotoxicity, etc. memory t-and b-cells are also developed during the maturation process, and has been discussed and reviewed elsewhere (146) (147) (148) (149) . a schematic diagram showing a summary of the immune response after iav infection has been illustrated in figure 1 . the yin and yang theory is always used to describe the importance in balancing the host immune response. in the light of this theory, the treatment strategy aims to suppress the overwhelming activation of the host immune response and in reverse to compensate any unfavorable suppression. although adaptive immune responses are important in viral clearance, the immediate innate immunity play an important role in the early control of an infection, and conversely, is a major factor for disease severity due to immunopathology. dysregulated immune responses caused by viral infections have been implicated in severe disease development (150, 151) , such as acute lung injury (ali). ali in its most severe form, known as acute respiratory distress syndrome (ards), is reported to be the most prevalent cause of mortality in iav-infected patients (152) . studies suggested that iav strains could be associated with either over-activating (human infection by avian h5n1 and h7n9) (153, 154) or suppressing (h1n1, h3n2) (155) immune response. recent history has seen the outbreak of iav pandemics of varying severity takes place at the cost of millions of lives. one such example would be the deadly spanish flu of 1918, which claimed the lives of 20-50 million of the 500 million people infected worldwide. the pathological examination of lung sections from mice infected with reconstituted 1918 iav virus revealed necrotizing bronchiolitis and severe alveolitis in tissue, with neutrophils observed as the predominant inflammatory cell type present (156) , suggesting neutrophil involvement in the pathogenesis of iav infection. the majority of immune cells in blood circulation are neutrophils; of which they are among the first innate immune cells recruited to the site of infection (131) . neutrophils characteristically control microbial infections by generating bactericidal (157) neutrophil extracellular traps (nets), consisting of granule proteins, histones, and decondensed chromatin (131) . both protective and destructive role of neutrophils in iav infections have been described. the contrasting role of neutrophils could be explained by factors such as viral strain and viral dose used in different experimental setup, etc. the protective role of neutrophils was observed when mice infected with a low, non-lethal dose of iav h3n2 strain hkx31 displayed neutrophil-mediated viral clearance via phagocytosis (132, 158) . depletion of neutrophils has found to enhance viral load in the iav-infected animals (158) . on the contrary, this protective nature is disputed due to the association of neutrophil-generated nets. extensive net formation was observed in mice infected with pr8, an iav strain highly pathogenic to mice (159) . histones and myeloperoxidase within the net induce cell death of lung epithelium and endothelium (157) , leading to the loss of integrity of the alveolarcapillary barrier, a characteristic of ali. yet, while histones have been shown to suppress iav replication in vitro (160) , in vivo study demonstrated that there was increase in lung inflammation and damage in iav-infected mice treated with histones (161) . interestingly, co-treatment of lethally infected mice with anti-histone antibody and oseltamivir resulted in an increase in animal survival when compared to infected mice groups treated solely with oseltamivir (161) . in agreement with the in vitro and in vivo data, it has been reported that net produced by cultured neutrophils from patient with h7n9 and severe h1n1 infection increased alveolar epithelial cell permeability (162) leading to ali. more importantly, plasma net level positively correlated with the disease severity index (including higher acute physiology and chronic health evaluation ii score) and multiple organ dysfunction syndrome (162) , further demonstrating the detrimental role of net in the pathogenesis of severe iav infections. studies have demonstrated the involvement of superoxide dismutase and myeloperoxidase in netosis, the formation of net (159) . the presence of anti-myeloperoxidase antibody as well as the superoxide dismutase inhibitor (detc) significantly reduced netosis. finally, tetrahydroisoquinolines (163) and a panpeptidylarginine deiminase (pad) inhibitor, named cl-amidine (164) have been suggested to inhibit netosis. despite it has been reported that during iav h1n1 infection, pad4 knockout mice displayed only slight improvement in weight loss and a slight prolonged but no end-point survival advantage was observed compared to wt mice (165) , based on the extensive findings presented above, targeting net to prevent ali in the severe case of iav infection, including the highly pathogenic avian iav, remain promising and may warrant further investigation. innate lymphoid cells are cells of lymphoid lineages that do not express antigen-specific b-or t-cell receptors (166) . similar to t-helper cells, they are classified into subsets by their ability to produce type 1 (th1), type 2 (th2), and type 3 (th17 and th22) cytokines. previous studies confirmed the involvement of ilcs of group 2 linage (ilc2) in iav infection and airway inflammation (166, 167) . on the positive side, during the recovery phase of iav infection, ilc2 expresses amphiregulin which promote airway epithelium repair (166, 168) , thus facilitating the recovery of the infected lung. on the other hand, in response to il-33 produced by macrophages, dcs, and nkt cells, ilc2 secretes il-5 and il-13 and induce airway hyper-responsiveness. recruitment of eosinophils by il-5 to the lung also mediates airway inflammation (166) . since eosinophilia is a characteristic of allergic asthma and influenza is a major cause for morbidity and mortality in asthma patients (166) , it will be of particular interest to investigate the role of ilc2 in iav infection, particularly in asthma patients. ilc1s have been initially described as immature nk cells residing in the liver and share many phenotypic similarities with nk cells (169) . it was recently appreciated that tissue-resident ilc1s other than the previously recognized nk cells are the major early source of the antiviral ifn-γ at the primary site of various viral infection, including iav (170) . interestingly, ifn-γ was found to suppress ilc2 activity and reduce il5 production which exacerbates disease severity during influenza a(h1n1) pdm09 infection (171) . this data may highlight a link between ilc1 and ilc2 and suggesting ilc1 can suppress ilc2 activity via ifn-γ production during iav infection. with ilcs finally identified, functions of these cells and their role in immune response to tumors and pathogen infections have been massively investigated in recent years. type i ifns, prostaglandin i2, corticosteroids, and testosterone have been reported to suppress ilc2 activity (172, 173) . in addition to il-33, the epithelial cytokines il-25, thymic stromal lymphopoietin, as well as the lipid mediator prostaglandin d2 were found to activate ilc2 (173) . the therapeutic potential of these ilc2 activators and suppressors is yet to be deduced. with more and more studies demonstrating the involvement of ilc in iav infection, the interplay between different ilc subtypes in iav infection would, therefore, be an interesting area to explore and modulate the ilc activity may be a future approach to combat iav infection. reactive oxygen species, generated by specialized enzymes such as nadph oxidases, are released during iav infection (174) . the nadph oxidase family consists of enzymes containing different catalytic subunit named nox1-5 and dual oxidase (duox) 1 and 2. ros have been reported to display both beneficial (limiting viral replication) and detrimental (promoting ali) effects in the course of iav infection. interestingly, the protective or destructive effect of ros is dependent on the enzyme of which the ros is generated (174) . dual oxidase1 and 2 are found to be host-protective (174, 175) . in vitro, ros generated by nuclear duox indirectly regulates the splicing of iav mrnas via the nuclear speckle-associated splicing complex (175) . in addition to altering viral mrna splicing, ros generated by doux2 has been attributed to the production of ifn-λ, an important anti-iav ifn. in response to iav infection, increased viral mrna replication was observed when duox2 was silenced in vitro (176) . increased viral replication was also observed in mice with doux silenced (175) , further depicting the protective role of doux in iav infection. unlike doux, nox2 activation could be harmful to host. iav infection was reported to induce nox2-dependent endosomal ros production (177) . ros could target the conserved cys98 on toll-like receptor (tlr) 7, and inhibit tlr7-mediated type i ifn expression during a mild iav h3n2 infection in vivo (177) . iav-infected mice treated with specific nox2 inhibitor, cholestanol-conjugated gp91ds-tat, were found to have reduction in endosomal ros production, restored tlr7 activity, and displayed a decreased viral load (177) . in addition to nox2, nox4dependent ros production has also been reported to activate mapk/erk signaling (178) , enhancing the export of vrnp complex, thus increasing viral replication (see targeting the raf/ mek/erk pathway). nox4 knockdown resulted in a reduction of viral replication in vitro (178) . targeting the different nadph oxidase isoforms, instead of scavenging ros should be considered as the therapeutic approach for iav infection, as doux-mediated ros production is beneficial (175, 176) , while nox2 and nox4 are harmful during iav infections (177, 178) . finally, ns1 (not to be confused with iav ns1 protein) has been demonstrated to be a nox inhibitor, which could inhibit the activity of nox1, nox2, and nox4. a study demonstrated that ns1 suppresses iav-induced nox2 and significantly inhibits iav virus replication (179) . besides cholestanolconjugated gp91ds-tat and ns1 aforementioned, apocynin, a phagocytic nox2 inhibitor as well as ros scavenger (180) (181) (182) , has been demonstrated to ameliorate hyper upregulation of cytokines induced by iav infection through socs1 and socs3 in vitro (154) and reduce peri-bronchial inflammation and viral titer in vivo (183) . interestingly, ebselen, another nox2 inhibitor and glutathione peroxidase mimetic, could reduce inflammatory status measured in bronchoalveolar lavage fluid (balf) of mice pre-exposed to cigarette smoke and subsequently infected with iav (184) . taken together, these reports highlight the potential use of nadph oxidases inhibitors and ros scavengers to treat iav infections. dysregulated cytokine production has been associated with the elevated mortality rate observed in severe iav infections (185, 186) . as such, the immunomodulation of cytokines are regarded as promising therapeutic tactics. recent advancements developed with this approach will be highlighted in the following section. tumor necrosis factor has two main functions during viral infection-it activates nf-κb, inducing the expression of cytokines responsible for the host immune response; and induces apoptosis through activation of a signaling cascade involving tradd, fadd, and caspase 3, 7, 8, and 10 (187) (188) (189) . tnf is known to be highly upregulated in iav-infected hosts, especially in hosts infected with highly pathogenic iav (153, 190) . however, it is both protective and counter-protective functions associated with tnf that makes it a target in the treatment of iav. the protective role of tnf is observed during infection by low pathogenic iav, where extrinsically derived tnf is responsible for attenuating tissue-damaging cd8 + t-cell response (191) . in addition to recruiting monocytic cells to the infection site, cd8 + t-cells response was observed to deteriorate lung pathology (192) and damage healthy, non-infected lung epithelial cells (193) upon iav infection. furthermore, tnf deficiency has been associated with an increased detection of il-15 and il-6 in balf (192) , which promote the survival of and proliferation of cd8 + t-cells (194, 195) and subsequent tissue damage. exacerbated lung pathology caused by the upregulation of the monocyte chemoattractant protein-1 was observed in tnf −/− mice infected with sub-lethal dose of iav (196) . in addition, decreased cd8 + t-cell contraction due to enhanced expression of the anti-apoptotic protein bcl-2 was observed in sub-lethally iav-infected tnfdeficient mice when compared to wt mice (192) . as a whole, there is substantial evidence supporting the protective role of tnf in iav infection. on the other hand, the correlation of tnf with pulmonary edema has been well-documented (197) . tnf has been observed to stimulate the expression of cxcl2 in alveolar epithelial cells in a transgenic mice model resembling extensive iav infection in lung tissue, causing alveolar damage, lung edema, and hemorrhage (198) . in addition to lung edema, tnf has also been reported to correlate with iav-associated encephalopathy (199, 200) . however, it is notable that despite iav-associated encephalopathy, direct invasion of the central nervous system is rare (201) , suggesting that iav-associated encephalopathy could instead be a result of peripheral infection. furthermore, tnf has been shown to increase the permeability of the blood-brain barrier (bbb) (202, 203) , contributing to neural damage (204) . these studies further support an anti-tnf approach as a potential therapy for severe iav infection. at present, etanercept, an anti-tnf drug administered in the treatment of rheumatoid arthritis, is the only tnf inhibitor (or even tnf directed treatment) tested for iav treatment. etanercept has been shown to protect against the in vivo lethal infection of mice with a highly virulent, mouse-adapted iav strain (205) , with observations made of an increased survival rate with decreased morbidity, expression of the proinflammatory cytokine il-6, lung injury, and edema (205) . the protective role of il-6 was demonstrated in mice challenged with sub-lethal iav infection. il-6-deficient mice displayed exacerbated pulmonary damage (206, 207) and lung injury due to an observed decline in the survival of alveolar type ii cells and alveolar epithelial cells (207) . iav suppresses the anti-apoptotic mcl-1 and bcl-xl expression, causing cell death of neutrophils which are critical in viral clearance (206) . addition of il-6 restored the expression of mcl-1 and bcl-xl in vitro and is considered as the underlying mechanism for the observed survival advantage of wt mice over il-6 knockout mice during mild iav infection. il-6 has also been shown to induce the proliferation of lung il-10 + regulatory t cells and il-27, which act to limit excessive proliferation of cd8 + t-cells and subsequent cd8 + -inflicted damage. this would hence prevent the tissue damage observed in lung immunopathology (208) . despite the apparent protective role of il-6, high levels of il-6 in serum or cerebrospinal fluid have been reported in severe neurologically complicated iav cases, with il-6 used as a marker for prognosis (199-201, 209, 210) . the role of il-6 in regulation of bbb permeability was reported (211) , with potentially detrimental neurological complications. as such, the suppression of hyper-induced il-6 as a form of therapy in severe iav infection should be considered. one such option is the anti-il6 antibodybased drug tocilizumab, which is currently administered clinically for the treatment of rheumatoid arthritis. however, study on the usage of this drug to treat hyper upregulation of il-6 due to severe iav infection has yet to be conducted. on the other hand, in a case of h1n1 virus-induced ards, the use of an extracorporeal cytokine hemoadsorption device to remove cytokines including tnf and il-6 from the bloodstream (212) has showed beneficial to the patient (213) . more research is required to confirm whether the removal or neutralization of il-6 could be a potential therapy for severe iav infections. the activation of cd8 + t-cell is crucial for viral clearance. it should, however, be tightly regulated to limit cd8 + t-cell inflicted host cell damage. il-6 mediates il-27 induction (208) . il-27 acts to suppress cd8 + t-cells and reduce morbidity through il-10 and regulatory t-cells (208) . much like other immunomodulatory approaches, the timing for applying il-27 should be carefully assessed. compared to placebo-treated iavinfected group, early administration of il-27 to iav-infected mice in fact led to poorer viral clearance, increased morbidity, and deteriorated lung histopathology, while il-27 administration during the recovery phase (5-10 days post-infection) accelerated recovery and improve lung immunopathology (214) . notably, il-27 could also suppress th17 responses and increases susceptibility to secondary s. aureus infection (215) . therefore, co-administration of antibiotics should be considered when utilizing il-27 as potential iav treatment. both type i and iii ifns have antiviral properties, with viruses counteract ifns to gain an advantage for their propagation. the iav viral protein ns1 inhibits the production of ifns by antagonizing irf-3, a key transcriptional factor for ifns. this prevents the processing of cellular pre-mrnas (including those for ifns) and directly interacts with retinoic acid-inducible gene (rig)-i receptors, which are critical in innate sensing, to suppress ifn production during infection (216, 217) . in addition to inhibiting ifn expression, the induction of socs3 inhibits ifns signaling by suppressing cytokine signaling has been documented (155) . the recognition of 5′ triphosphate on viral rna by rig-i receptor is shown to induce the expression of socs3, which in turn represses type i ifns expression (155) . due to ifns being a key contributor to antiviral immune response, an impairment of type i or iii ifn production may cause the escalation of otherwise mildly pathogenic iav infection into a life-threatening one (218) . while type i ifn has been demonstrated to inhibit iav replication in vitro (219) ; the in vivo administration of type i ifn in animal models only displayed effectiveness in a prophylactic capacity. a lowered viral titer was detected in the nasal wash of test animals. however, host susceptibility to iav infection remained unchanged (219) . notably, this protective effect is only conferred by an optimal dose of type i ifn of low to moderate amounts (10-100 units per mice daily); with higher dosages (1,000-10,000 units per mice daily) shown to increase morbidity (220) . in addition, clinical trials demonstrated that prophylac tic administration of type i ifn reduced disease severity and lowered susceptibility to iav in males and participants aged 50 or above (221) . despite relatively successful results seen in the prophylactic use of ifns, its therapeutic use is of greater clinical relevance. mice treated with type i ifn post-iav infection showed a successful reduction in lung iav titer but displayed increased morbidity and mortality in comparison to vehicle-treated mice (222) . a possible explanation for this phenomenon is the induction of excessive inflammatory response and trail-dr5-mediated epithelial cell death by type i ifn (223) , which accounts for the observed lung pathology in iav-infected animals treated with type i ifn (224) . in addition, downregulation of γδ t-cells by type i ifn has been correlated with increased susceptibility to secondary s. pneumoniae infection (225) , further arguing against the potential use of type i ifns for the treatment of iav infection. in comparison to type i ifns, the administration of type iii ifns may provide advantages in the control of iav replication (176, 222, 224) without the risk of previously reported type i ifns-mediated immunopathologic side-effects (222, 224, 226) . however, a recent study aiming to stimulate ifns signaling through the systematic administration of rig-i ligand post-iav infection demonstrated that type i, but not type iii ifns signaling is important in conferring protection during fatal iav infection in vivo (227) . though, this study did not measure the production of type i and iii ifns as well as any changes in viral load with respect to ifnar or ifnlr knockout. in addition, while human immune cells are not primary targets in iav infection, they could be susceptible to iav and become efficient host cells for virus replication. they are reported to possess a subpar response to type iii ifns (222) ; leading to the preliminary conclusion that solely using type iii ifn as treatment may not be feasible. as such, reports suggesting the use of type iii ifns over type i ifns as a front-line therapeutic agent to counter iav infections may require further investigation. the inhibition of cox-2 by selective inhibitors, nimesulide and celecoxib, was previously demonstrated to suppress the hyper upregulation of pro-inflammatory cytokines induced by highly pathogenic avian iav (228) (229) (230) . in addition, the use of zanamivir in tandem with a specific cox-2 inhibitor was shown to increase the survival rate of mice lethally infected with avian h7n9 iav, when compared to mice treated solely with zanamivir (229) . activated cox-2 regulates downstream prostaglandin production. one such example is pge2, a major type of prostaglandin recently demonstrated to play an important role during iav infection. pge2 was significantly upregulated in response to iav infection, leading to the inhibition of antiviral type i ifn production in macrophages and the subsequent increase in virus replication (231) . the use of chemicals ah6809 and gw627368x to antagonize pge2 downstream signaling molecules ep2 and ep4 respectively, was shown to induce antiviral type i ifn production. the in vivo treatment of mice lethally challenged iav with both ep2 and ep4 antagonists significantly improved the survival rate. a recent study demonstrated the ability of a modified tcm decoction to reduce peg2 production and subsequent morbidity in mice lethally challenged with iav. improved lung pathology was observed (232) . the long history of clinical tcm use supports the clinical feasibility of peg2 inhibition as an option to treat severe iav infections. pattern recognition receptors on host cells sense specific pamps present on the viral surface or generated during replication. prrs can be broadly divided into two classes by their function or location. when defined by location, prrs are classified into 3 groups-membrane-bound (tlrs and c-type lectin receptors), cytosolic (rig-i-like and nod-like receptors), and secreted (collectins and pentraxins) (233) . significant research has been conducted on prrs with regards to iav infection. tlrs and rig-i receptors have been extensively studied for their major roles in eliciting host immune responses (cytokine and ifn expression) during iav infection (234) (235) (236) . rig-i receptors have been investigated for their functional relevance to iav infection and targeting these receptors as a form of iav treatment has been extensively reviewed (237) (238) (239) . this section will cover recent research on tlrs and the targeting of different tlrs to treat iav infection. humans have been identified to express tlr1-10, while mice have been identified to express functional tlr1-9 as well as tlr11-13 (240) . most tlrs-with the exception of tlr3utilize myd88 as an adaptor protein during signal transduction. tlr3 utilizes trif as an adaptor. tlr4 is known for its ability to utilize either myd88 or trif, with the choice of adaptor dependent on its sub-cellular location (241) . different tlrs, such as tlr3, 7, and 8 (240) as well as tlr2, tlr4, and most recently tlr10 (235) , have been revealed to play a role in the orchestration of host immune responses contributing to iav pathogenesis. with tlr10 being an exception (242) (243) (244) , tlr activation largely causes the release of pro-inflammatory cytokines, with hypercytokinemia leading to ali as a major cause of mortality in severe iav infections. in addition to dysregulated cytokine release, excessive production of ros has been associated with ali development. in fact, lung injury during severe pulmonary infections, such as iav and sars, could be caused by oxidative stress (245) . iav infection activates nadph oxidase that subsequently produces oxidized papc, an endogenous phospholipid. the oxidized papc serves as an agonist for tlr4, activating a tlr4-trif-traf6-nf-κb signaling cascade to eventually trigger the release of il-6, ultimately inducing the onset of ali. in addition to oxidized papc, the induction of endogenous protein s100a9 upon intracellular prr ddx21 recognition of iav subsequently induces the activation of tlr4, further contributing to iav-induced mortality (246) . since tlr4 has been proven to be important in ali induction (and hence iav-related mortality), manipulating the stimulation and antagonism of tlr4 could potentially reduce the severity of iav infections. eritoran (e5564) is a specific tlr4 antagonist initially purposed for the treatment of sepsis, but a failed a phase iii clinical trial due to improved patient care in the placebo group prevented its eventual use in sepsis treatment (247) . in vivo administration of eritoran in mice lethally infected with iav resulted in improved clinical score, lung pathology results, and reduced viral titer. delayed administration of eritoran, at day 6 after infection beyond the recommended therapeutic time window (within 48 h after the first display of clinical symptom) for use of oseltamivir (248), also demonstrated a significant benefit to infected mice compared to non-treated group, suggesting a prolonged therapeutic time window for iav treatment when compared to mainstay antiviral drug treatment. a newer and structurally simpler specific tlr4 antagonist, fp7 (249), alongside a newly developed decoy peptide 2r9 that has been shown to disrupt tlr2, 4, 7, and 9 signaling via tirap, has been shown to protect mice from lethal iav infection (250) . these results support the potential use of tlr4 antagonism as a means to treat severe iav infection. the suppression of other tlr signaling pathways-such as blocking tlr2-mediated signaling through the use of an anti-tlr2 antibody, significantly protected against lethality when administered on day 2 and 4 post-iav infection (251) . a study also demonstrated that h5n1-infected tlr3 knockout mice had better survival than h5n1-infected wild-type mice, which is evident through the significantly faster regaining of body weight post-infection, lower viral titer in the lung, and fewer pathological changes in the lung (252) . an increasing number of tlr antagonists are now under development (253, 254) , alongside several other agents also shown to have effects on tlrs. polysaccharides isolated from r. isatidis, a traditional chinese medicinal herb used to treat iav infection, have recently been shown to inhibit pro-inflammatory cytokines such as il-6 and ccl-5 in vitro by down-regulating upstream tlr3 expression (255) . menk, an endogenous protein expressed in the adrenal medulla, was shown to both prophylactically and therapeutically increase the survival rate while reducing viral-caused lung pathology and viral titer in mice lethally challenged with iav (256) . this was determined to be caused by the downregulation of tlr7. these results suggest the potential of down-regulating tlr expression in the treatment of iav infection. the above-mentioned data suggest modulation of tlr signaling or expression as a promising approach in treating severe influenza disease and deserves immediate investigation. table 2 summarizes new immunomodulatory approaches to combat iav infections. it is well documented that patients with diabetes mellitus have a greater tendency to develop severe iav infection than healthy patients (257) . hyperglycemia increases susceptibility of the host to iav infection via viral uptake, through the promotion of v-atpase assembly (258) and immunosuppression (257) . in addition, viruses rely on host metabolism to perform essential functions during replication (259) (260) (261) (262) . these processes exert a large energy demand on the host within a very short period of time (263) ; energy of which is supplied by and is dependent on host metabolism. iav viruses have been reported to modify the metabolic state of the host. for example, increased c-myc-dependent glycolysis and glutaminolysis has been demonstrated in infected cells (264) . the changes in glucose and glutamine metabolism were reversed upon the addition of bez235, which inhibited the iav-mediated c-myc induction. administration of bez235 2 days prior to infection and up to 4 days post-infection was shown to decrease lung viral titer and improve the survival rate in iav-infected mice. small molecules such as clotrimazole and α-mangostin that target lipid metabolism have also been demonstrated to suppress iav replication in vitro (264) . in addition to being important for generating energy and biosynthesis, recent research demonstrates that cellular metabolism affects immune cell function. dysregulated immune responses observed in many diseases are associated with specific metabolic configurations. viruses, influenza inclusive (265) , were found to induce drastic alterations in metabolic levels and programs (263) . macrophages in infected hosts were observed to have marked differences in the krebs cycle, a key metabolic pathway. this is of significance due to the role of macrophages, which are immune cells critical in the pathogenesis of many inflammatory diseases (263, 265, 266) . in activated macrophages, succinate, a krebs cycle intermediate, was found to possess inflammatory signal. accumulation of succinate generates ros, leading to subsequent activation of hypoxia-inducible factor 1α and the induction of cytokines such as il-1β (267) . a recent study identified the ability of itaconate, another krebs cycle-derived metabolite, to block the production of inflammatory factors. this prevented inflammation, protecting mice from lethal levels of inflammation that can occur during infection (268) . this data suggest the critical roles of krebs cycle intermediates in regulating cytokine profiles and inflammation. metabolites generated by innate immune cells in distinct configurations could have different roles beyond that of bioenergetics, with functions in signaling regulation, transcription, and orchestrating innate immune responses. despite the lack of research conducted thus far on the application of immunometabolic approaches to influenza treatment, the prospect of manipulating immune responses by modulating immune cell metabolic state is promising. further research should focus on the identification of metabolites for modulation of immune cell function with substantial improvement of therapeutic strategies to treat iav disease. latest advancements in high-throughput technologies, e.g., meta bolomics is a useful approach to systematically investigate the changes of metabolic mechanisms during iav infections. identification of important metabolites involved during iav infection should be a new approach by modulating the host metabolism for interventions. multiple host-based intervention strategies against influenza have been developed or are under development. while approaches targeting host machinery required for virus replication seem to be promising thus far, additional research is needed to determine the effect of modulating host immune response on influenza treatment. this is increasingly important, since targeted host factors may play distinct roles in response to infection by different influenza viral strains (252) , making the management of influenza through solely targeting a single specific host factor is difficult. host-based interventions offer obvious advantages over conventional antivirals, such as a higher barrier to drug resistance (73, 83, 107) due to greater genetic stability of host factors than the mutation-prone nature of viral components. in addition, administration feasibility is a key factor to consider the usage of drugs. the mainstays of antivirals for iav infections, the na inhibitors, and m2 blockers, are recommended to be administered within 48 h of symptom onset for optimal antiviral activity. this short treatment window may not be fully fulfilled in a clinical setting. novel host-based interventions were reported to have therapeutic time windows longer than this conventional timeframe (96, 109, 214, 251) , even up to 6 days post-infection (248) , providing a clear clinical advantage over na inhibitors and m2 blockers. in addition, hypercytokinemia and ards could contribute to disease severity and mortality in instances of severe influenza infection, with virustargeting antivirals providing little to no alleviation of such complications. since host immune response is indispensable in host defense against invading pathogens, the use of immune-modulators to suppress detrimental effects while retaining beneficial protection of the host remains challenging. the timing and dosage of medication administration would be critical in determining the drug effectiveness in influenza treatment. targeting virus-induced metabolic changes to restore host normal metabolism may be a new direction to combat influenza disease. further research in the immunometabolism field, along side studies on modulating immune response to infectious disease by altering host metabolic processes; would create a new direction for future research and is expected to yield significant discoveries that may provide new therapeutic options in the treatment of iav infections. smyl conceptualized the work. il and asms drafted some review sections and tfy and smyl wrote the manuscript. influenza a(h1n1)pdm09 outbreak detected in inter-seasonal months during the surveillance of influenza-like illness in pune avian influenza a (h7n9) virus infections in humans across five epidemics in mainland china age and influenza-specific pre-vaccination antibodies 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very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny stealing the keys to the kitchen: viral manipulation of the host cell metabolic network viral activation of cellular metabolism targeting metabolic reprogramming by influenza infection for therapeutic intervention metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling krebs cycle rewired for macrophage and dendritic cell effector functions succinate is an inflammatory signal that induces il-1beta through hif-1alpha itaconate is an anti-inflammatory metabolite that activates nrf2 via alkylation of keap1 key: cord-102458-7sssm3zk authors: milanez-almeida, pedro; martins, andrew j.; torabi-parizi, parizad; franco, luis m.; tsang, john s.; germain, ronald n. title: blood gene expression-based prediction of lethality after respiratory infection by influenza a virus in mice date: 2020-10-27 journal: biorxiv doi: 10.1101/2020.10.27.357053 sha: doc_id: 102458 cord_uid: 7sssm3zk lethality after respiratory infection with influenza a virus (iav) is associated with potent immune activation and lung tissue damage. in a well-controlled animal model of infection, we sought to determine if one could predict lethality using transcriptional information obtained from whole blood early after influenza virus exposure. we started with publicly available transcriptomic data from the lung, which is the primary site of the infection and pathology, to derive a multigene transcriptional signature of death reflective of innate inflammation associated with tissue damage. we refined this affected tissue signature with data from infected mouse and human blood to develop and validate a machine learning model that can robustly predict survival in mice after iav challenge using data obtained from as little as 10 μl of blood from early time points post infection. furthermore, in genetically identical, cohoused mice infected with the same viral bolus, the same model can predict the lethality of individual animals but, intriguingly, only within a specific time window that overlapped with the early effector phase of adaptive immunity. these findings raise the possibility of predicting disease outcome in respiratory virus infections with blood transcriptional data and pave the way for translating such approaches to humans. influenza a virus (iav) infection of the respiratory tract can lead to severe immune activation and lung tissue damage in mice, ferrets, macaques and humans (1-6). intrinsic virulence, replication capacity and initial infectious dose, together with the host's genetic background, immune status and overall health, determine the extent to which host immunity is triggered (7, 8) . substantial evidence indicates that lethality is associated with an excessive innate immune response, with lung dysfunction arising from epithelial and endothelial damage induced by infiltrating leukocytes, in particular monocytes and neutrophils (9) (10) (11) (12) (13) . our previous work in a murine model of infection with iav uncovered clusters of co-regulated genes in the lung associated with lethal influenza infection; one of those lethal clusters was highly associated with an overwhelming neutrophil response and, consistently, early post-infection partial neutrophil depletion rescued animals from lethality, establishing a direct link between the innate response in the tissue and death (13) . while that earlier work focused on gene expression signatures in the infected pulmonary tissue, here we sought to identify a blood-based signature for prediction of lethality. previous attempts to develop gene expression signatures in blood in the context of iav-induced illness focused mostly on distinguishing iav from non-iav infections, symptomatic from asymptomatic iav carriers, or low from high influenza vaccine responders (14) (15) (16) (17) (18) (19) (20) . a notable exception was the recent description of blood transcriptomics data from a large cohort of subjects enrolled in the mechanisms of severe acute influenza consortium (mosaic) study (21) . in that report, severity of infectionmeasured in terms of need for mechanical ventilation -was associated with a weak transcriptional "viral response" signal and a strong transcriptional "bacterial response" (and activated-neutrophil) signal in blood in comparison with non-severe cases. however, these transcription patterns were also strongly associated with duration of illness and the authors emphasized the importance of timing in the interpretation of immune activity to iav infection (21), which, for obvious reasons, can be hard to control for in natural human infection studies. we aimed to develop a blood biomarker for early identification of individuals at risk of adverse disease outcome after influenza infection in a well-controlled mouse model of iav infection, where a precise delineation of the evolution of the response associated with severity could be achieved. we utilized transcriptomic data from the lungs, the focal point of infection, and also data from blood post infection, to derive a transcriptional signature of lethality across various iav and mouse strains. this early-response signature could distinguish mice at high risk of death with different influenza a virus strains. these results provide an impetus for seeking to translate this approach to human respiratory virus infection characterized by damaging inflammatory responses. the first question was how to select a panel of candidate genes whose expression in blood could be used to predict lethality after iav exposure. we focused on genes whose expression early after infection was associated with eventual lethal outcome, rather than with infection (14, 16) . we reasoned that the focal point of infection, the lungs, where lethal processes unfold, would contain the relevant biological signal. several whole-genome transcriptomics datasets from mouse lung tissues after infection with iav are publicly available, including from our own previous work (13, 22, 23) . we utilized these resources (i.e., transcriptomic data from the mouse lungs at several time points after infection) to derive a gene signature of lethality from the lung across several iav and mouse strains, followed by integration with blood data from influenza-infected mice and humans. briefly, to be considered for inclusion in the signature of lethality, on days 1 to 3 after infection genes had to be differentially expressed (de) , in comparison to pbs-treated animals, in the lungs of lethally infected mice but not de in the lungs of non-lethally infected animals (see fig. 1 and methods for more details) (13, 22, 23) . furthermore, genes were included in the signature only if they were in at least one of the gene clusters associated with lethal, but not with non-lethal, iav infections that we uncovered previously in the lungs (13). the above procedure (the lung signature of lethal influenza infection) yielded 1,069 genes, which were enriched for several biological processes, including regulation of epithelial cell proliferation, cell adhesion, morphogenesis of an epithelium, regulation of vasculature development and regulation of to refine this lung lethality signature with blood data, we only retained genes that were de in the blood of mice two days after infection with a lethal dose of the highly pathogenic h1n1/pr8 iav strain (fig. s1) , and excluded genes that were de in the blood of humans upon low pathogenicity infection (24). 81 genes passed these pre-selection filters. as a positive control for detection of iav infection itself, independent of lethal outcome, 10 genes previously used as classifiers of respiratory virus infection in blood were selected (14, 16) as well as 5 reference "housekeeping" genes for normalization (25). primers against 96 genes were designed for high throughput rt-qpcr (table s1 ). since (a) our past work used non-lethal infection data to develop the specific set of lethalityassociated gene clusters, (b) we excluded genes using human low pathogenicity data, and (c) we excluded genes de in the lungs of non-lethally infected mice, the hope was that the candidate genes selected into the integrated tissue and blood signature would have better specificity for association with lethality. to determine whether the expression of these candidate genes in blood could be used as a starting point to train a machine learning model of lethality in mice after challenge with iav, gene expression data were collected from rna isolated from 50 µl of blood drawn from 34 animals four days after treatment with pbs or a range of lethal and sublethal influenza strain a/puerto rico/8/1934 h1n1 (pr8) doses ( fig. 2a-b) . we used a statistical learning method known as elastic net and leave-one-out cross validation to assess whether predictive models could be built from our data (26-28). briefly, survival (fig. 2b ) and gene expression data (fig. s2) were fitted via elastic netregularized multinomial logistic regression to generate a model to classify mice into three categories: 1) not infected, 2) surviving upon infection, or 3) dying upon infection. during training (i.e., model selection via cross-validation), the algorithm learned that 23 genes had expression values in blood that could be linearly combined to determine the probability of an individual animal being in one of the training categories ( fig. 2c and fig. s3a ). some of the positive control genes were selected by the algorithm to help differentiate between infected and non-infected animals, and fewer to separate infected survivors from non-survivors (fig. s3a) , suggesting that the death-associated candidate genes were indeed enriched for detecting signals of lethality in blood. in the approach described above using logistic regression, the algorithm attempts to learn how the expression of each gene can be combined to discriminate mice in one category from another. in a hypothetical scenario of resource prioritization, however, one might also be interested in estimating the time to a relevant clinical event -death, in this case. this can be achieved with cox proportional hazards regression, where time to event is taken into consideration and the relative risk of death for each subject can be derived. hence, to examine whether the expression of lethal candidate genes in blood would distinguish mice at high risk of early death after challenge, we combined survival and gene expression data from the 81 lethal candidate genes in cox regression regularized via the elastic net. during training (i.e., model selection via cross-validation), the algorithm learned that 11 genes had expression values in blood that could be linearly combined to generate a scoring system of infected animals as a function of the day of death ( in both the multinomial and the lethal cox models, high levels of expression of genes associated with monocytes and neutrophils, together with low levels of transcripts associated with lymphocytes, indicated high risk of death (fig. s3 ), consistent with previously described analyses of the immune response to iav infection (10, 12, 13) and also recent data from covid-19 patients (29). art4, the gene most positively associated with lethality in both models, is highly expressed in hematopoietic stem cells and immature lymphocytes according to the immgen database (30), indicating a potential association of lethality with dysregulated hematopoiesis and release of immature cells into circulation. an important aspect of machine learning-derived models is whether their performance is generalizable, which means whether they perform well on unseen test data that has not been used in training. here, the models were tested for their ability to predict lethality based on gene expression data from independent cohorts of mice that were not available for training. performance was tested in three different ways: 1) on mice infected with high doses of either a low or a high pathogenicity influenza strain (i.e., non-lethal high dose strain a/texas/36/91 h1n1 (tx91) vs. lethal high dose pr8); 2) by training our models on this second dataset (low vs. high pathogenicity data) while testing on our first dataset described above from infection with different doses of pr8; and 3) by testing on the scenario of infection at one lethal dose 50 (ld50), where mice are challenged with virtually the same viral dose but only half of them survives the infection. in the first round of validation, an independent cohort of 14 mice bled on day two after treatment with pbs or with a high dose of either tx91 or pr8 provided the data ( fig. 3a-b) . although the test set was from an earlier time point of infection and included one virus strain and one dose not used in training, both the multinomial and the lethal cox models showed good predictive accuracy ( fig. 3c-d) . in the second round of validation, after reversing the roles of each dataset (i.e., training on the set with two different iav strains on day two of infection and testing on the set with five different doses of pr8 on day four of infection), the multinomial model did not perform as well as the lethal cox model, which showed good accuracy for predicting outcome ( fig. s4a-b) . considering only the lethal cox model, for our third round of validation we turned our attention to the challenging scenario of predicting lethality among genetically identical, sex and age matched, cohoused mice given the same infectious bolus at an ld50 of pr8 (n = 24 mice). in this setting, typically about half of the mice succumb while the other half survives viral exposure. importantly, it is not known mechanistically what drives this outcome dichotomy, since measuring putative candidate factors such as lung viral titer and immune infiltration requires sacrificing the mice and, thus, precludes assessment of the actual outcome of disease. to us, the most obvious candidate mechanism was that differences in initial infectious bolus effectively received by each animal during the infection procedure would determine the dichotomy. in that case, our lethal model should be able to predict the outcome of infection very early on, and thus help shed light on the biological mechanisms of life or death under these particular conditions. incubation period is the time that transpires from the moment of exposure to a pathogenic agent to when the host starts showing signs of infection. during this period, a virus needs to arrive at accessible tissues and bind to receptors to enter and replicate in susceptible cells. from infected cells, new infectious viral particles are released, and the cycle starts again, with viral titers temporarily following an exponential growth curve at least while virus replication is unperturbed by the host immune system. naturally, the time to trigger effective host immunity is influenced by viral virulence, replication capacity and initial infectious dose. s5c) . considering that only a very small number of pr8 virions is required to induce pathogenic lung disease, these results indicate that on day two of 1 ld50 pr8 infection the virus had not yet reached high enough levels in the respiratory tree for the early anti-viral response to become detectable in blood, and, thus, our model was not able to detect any signs of lethality in the blood of mice on day two post 1 ld50 pr8 infection. by day four, however, de of all 9 positive control of infection genes could be detected in the blood of animals infected with 1 ld50 pr8 (fig. s5c) , likely reflecting the spread of pr8 in the lungs and evolving host immunity. in addition, on day four, our lethal model correctly placed the animals at an intermediate level of risk of death -higher than mice infected with low pathogenicity tx91, where no mice were at any risk of death, and lower than high dose lethal pr8, where every animal would eventually succumb (fig. s5d) . however, on day four, our model was unable to predict accurately which of the individual mice within the 1 ld50 group would survive and which would die (fig. s5e ). retraining our model on 1 ld50 blood gene expression data also failed, as did predictions based on loss of body weight up to day four, suggesting that the fate of these mice might be indistinguishable at this early point after infection. in an attempt to further characterize the infection dynamics, we devised a longitudinal experiment in which ethically small samples of blood (~10 µl) were taken daily from the same animals in a different cohort of 16 mice during days four to eight of infection with 1 ld50 pr8 (fig. 4a) , followed by rna isolation, high-throughput rt-qpcr, and testing based on the existing lethality model (i.e., without retraining). the model had statistically significant power to distinguish within group, interindividual differences in relative risk of death after 1 ld50 challenge on days five and six, but not on days four, seven or eight (fig. 4b) . these results suggest a specific time window within which processes associated with ld50 lethality is reflected by our signature in blood. while the lack of accuracy later in the course of infection (i.e., days seven and eight) was likely due to the fact that the model was trained for early detection of lethality, before adaptive immune cells fully developed and reached the circulation, these data suggest that pamps and damps eventually reach different levels in the lungs of different mice, impacting their blood cell composition and lethal score, which can be used for prediction of lethality of individual animals even in the challenging scenario of experimental infection with an 1 ld50 iav dose. prediction tools for infectious disease outcomes would enable evidence-based treatment decisions and resource allocation, in particular during pandemics. we show that a transcriptional signature predictive of lethality can be developed via machine learning in a mouse model of influenza infection early after exposure, from as little as 10 µl of blood. this was achieved by integrating tissue and blood signatures of lethal influenza infection. the model also revealed a specific time window for prediction of lethality in the challenging scenario of 1 ld50 pr8 infection. earlier in the course of infection, our model tended to place mice infected with 1 ld50 pr8 at an intermediate risk level in comparison to mice infected with high dose pr8 or tx91, suggesting that our model can delineate effects associated with infection severity. in the scenario of ld50 pr8 infection of inbred and cohoused littermates with virtually the same infectious bolus, our model revealed a later split (~days 5 and 6) between animals who would eventually die or survive. these intriguing results raise the possibility that differences in initial infectious bolus were not the primary determinants of life or death of mice infected with 1 ld50. rather, these mice seem to behave like one group undergoing similar responses until they reach the boundaries of a tipping point, with survival or death being the result of biological variation around that tipping point (schematic model in fig. s5f) . the time when these differences were observed (i.e., days 5 and 6) overlaps with the early effector phase of adaptive immunity. since adaptive immunity, in particular cytotoxic t cells, plays an important role in reducing viral load and, hence, in stopping the feedforward stimulation of the damaging innate response (13, (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) , the timing when predictions can be made suggests that early inter-individual differences in the adaptive response may make a critical contribution to differences in the outcome of disease. although the average t cell response to infection is remarkably efficient and constant (44-48), formation of the naïve repertoire of antigen-specific t cells is a semi-stochastic process that varies in each host, resulting in a range of precursor frequencies (49-51) and, presumably, leading to a spread among the mice in the kinetics of reaching an adequate effector t cell number for effective viral clearance. therefore, the timing of the split between survivors and non-survivors in the 1 ld50 scenario potentially reflects differential interference with virus production during the early phase of the effector t cell response, which may be the determinant of life or death at the tipping point of infection. however, a cause/effect relationship is difficult to test directly due to the lack of tools to accurately examine, for example, virus titer, lung infiltration by antigen-specific cells, or the size of their precursor population in live mice, because these would require terminal experiments that would preclude determination of which animals would eventually die several days later due to the actual infection. nonetheless, preliminary experiments adding gene probes to our panel that report activated cd8+ t cell abundance in the blood samples suggest that there is a relationship between the strength of these lymphocyterelated signals in day 5 or 6 samples and survival after infection. further tests along these lines are planned to refine the signature and relate these blood findings in a large cohort of infected animals to effector function and viral abundance in the lungs as directly assessed by multiplex tissue imaging. our experiments have several limitations, such as the lack of validation of the models on human iav infection data. unfortunately, to our knowledge the only report to contain both whole blood wholegenome transcriptomics and disease severity data that includes severe human iav infection, contained data from only three severe cases beginning five days after onset of symptoms or earlier (21). beside the fact that such low number of early cases preclude attempts to validate our models, one also needs to take into consideration the incubation time -the time from exposure until development of symptoms -when considering the temporal evolution of infection. the models presented here were developed for prediction very early after exposure, and, while animal models of infection can be used to clearly delineate the evolution of the immune response along its temporal component, it remains to be determined which time window post exposure in mice most closely translates to time after onset of symptoms in human infection. experimental iav challenge in humans with relatively mild strains indicates a peak of symptoms (mostly runny nose and sore throat but no need for mechanical ventilation) around day 3-4 after exposure (52), but it is not clear how long it takes, on average, for a symptomatic carrier to seek medical treatment, in particular in case of severe disease with highly virulent and/or highly pathogenic strains. such questions would need to be addressed to enhance preparedness for the next pandemic and before predictive models developed in animal experiments could be realistically deployed to human populations, including with respect to the ongoing sars-cov2 pandemic. although mice and humans have substantial differences, including different distributions of influenza viral receptors in the respiratory tree, which is crucial to create an inter-species barrier for transmission, the immunobiology of highly pathogenic influenza infection, once transmission has been established, is quite similar across the several species that have been studied with regard to strong innate immune activation (7, 53, 54) . genes that are part of the lethal cox model are highlighted (fig. s3b) . data visualization and analysis was performed in r 4.0.2 and rstudio (60), using packages downloaded from cran and bioconductor 3.11 (61) . data were handled, plotted and visualized with the foreach (62), dplyr (63) , ggfortify (64), ggplot2 (65) and gridextra (66) packages. for rna-seq, blood was collected postmortem via heart puncture on day two after infection (n = 3 mice/treatment) and kept at -20 °c in rnalater (thermo fisher scientific a list of candidate genes for the affected lung tissue signature of lethal influenza infection, as well as respiratory virus infection positive controls and reference genes was created by checking for overlap between the gene lists that were either downloaded from the original publications or created using the geo data with the geo2r tool (bh-fdr = 0.1; see text and fig. 1 for details) (13, (22) (23) (24) . when entrez gene ids were not available, the mouse genome informatics database (jackson) was used to find current and old gene symbols as well as synonyms. a final list with selected genes and primers is shown in table s1 . bioanalyzer. only samples with rin score above 7 were further analyzed. primer design and gene expression analysis were performed with the 96.96 ifc using delta gene assays software (d3 assay design) and protocols for pre-amplified samples for use with biomark hd, following the manufacturer's instructions (fluidigm) as described previously (71) . to generate a standard curve for assessment of primer performance, samples from infected and non-infected animals were pooled and applied in a standard curve in duplicates (two no-sample controls were also included). initial quality control was performed on the biomark hd using real-time pcr analysis v4.1.3 (fluidigm), with automatic threshold generation. genes with primers unable to generate high quality results across the range of dilutions of the standard curve (r 2 > 0.85 in ct value vs. dilution plots) or generating more than one peak in the melting curve were excluded from further analysis. data was imported to r and normalized with htqpcr (72) . normalization was based on the standard delta ct method (subtraction of the mean of the reference "housekeeping" genes from all other values). in the machine learning algorithm, training was performed with glmnet (26-28) using leave-one-out cross-validation to tune the regularization parameter lambda (alpha = 0.5, family = "multinomial" for the test error of the scoring model was estimated both using leave-one-out cross-validation within the training cohort (also known as nested cross-validation, in which the leave-one-out procedure is used (a) in a sub-cohort of the training samples to tune the hyperparameter lambda, followed by predicting on the one sample not included in that sub-cohort, and (b) repeated for every sample in the training cohort) and on an independent cohort of mice. a cox proportional hazards regression model was fitted to the predicted score (i.e., the relative risk derived using the predict function of the glmnet package [type = response]) of each mouse using the survival package and checked that they did not violate the proportional hazards assumption at alpha = 0.05 using the cox.zph function also in the survival package (74). importantly, the low number of mice per category precluded the use of cross-validation to estimate the test error of the classification model, which was done only using the independent cohort of mice. fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus avian influenza virus (h5n1): a threat to human health aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses into the eye of the cytokine storm innate immunity to influenza virus infection ccr2+ monocyte-derived dendritic cells and exudate macrophages produce influenza-induced pulmonary immune pathology and mortality lung epithelial apoptosis in influenza virus pneumonia: the role of macrophageexpressed tnf-related apoptosis-inducing ligand innate immune responses to influenza a h5n1: friend or foe? tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection a systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection gene expression signatures diagnose influenza and other symptomatic respiratory viral infections in humans pathological findings of covid-19 associated with acute respiratory distress syndrome suppressive myeloid cells are a hallmark of severe covid-19 disease severity-specific neutrophil signatures in blood transcriptomes stratify covid-19 patients orchestrating high-throughput genomic analysis with bioconductor ggfortify: unified interface to visualize statistical results of ggplot2: elegant graphics for data analysis pathogen-related differences in the abundance of presented antigen are reflected in cd4+ t cell dynamic behavior and effector function in the lung fast gapped-read alignment with bowtie 2 moderated estimation of fold change and dispersion for rnaseq data with deseq2 panther in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees distinct nf-κb and mapk activation thresholds uncouple steady-state microbe sensing from anti-pathogen inflammatory responses htqpcr: high-throughput analysis and visualization of quantitative realtime pcr data in r predicted relative risk (log) for each mouse is shown for comparison, with the red dot and red line representing mean and standard deviation in each group. (f) schematic model of risk of lethal inflammation rising with time early after infection with different doses of and strains iav. n = 31 in a/b (all infected mice new expression profiling by high throughput sequencing data reported here are available on geo with accession number gse124404. we would like to thank emily condiff, dr. antonio p. table table s1 : selected genes whose expression was measured in the blood of mice upon treatment for training and testing model. class defines whether a gene is part of the lethal affected tissue signature (lethal), positive controls of infection (infection) or reference "housekeeping" genes (reference); fp: forward primer; rp: reverse primer. key: cord-286741-h3oix9zc authors: park, mee sook; kim, jin il; bae, joon-yong; park, man-seong title: animal models for the risk assessment of viral pandemic potential date: 2020-04-22 journal: lab anim res doi: 10.1186/s42826-020-00040-6 sha: doc_id: 286741 cord_uid: h3oix9zc pandemics affect human lives severely and globally. experience predicts that there will be a pandemic for sure although the time is unknown. when a viral epidemic breaks out, assessing its pandemic risk is an important part of the process that characterizes genomic property, viral pathogenicity, transmission in animal model, and so forth. in this review, we intend to figure out how a pandemic may occur by looking into the past influenza pandemic events. we discuss interpretations of the experimental evidences resulted from animal model studies and extend implications of viral pandemic potentials and ingredients to emerging viral epidemics. focusing on the pandemic potential of viral infectious diseases, we suggest what should be assessed to prevent global catastrophes from influenza virus, middle east respiratory syndrome coronavirus, dengue and zika viruses. of the four types of influenza viruses, influenza a virus (iav) and influenza b virus (ibv) cause major respiratory diseases to humans [1, 2] . the iavs can be classified into different subtypes by the antigenicity of surface glycoproteins, hemagglutinin(ha) and na(neuraminidase). so far, 18 and 11 subtypes have been identified from the ha and na proteins, respectively, and the last two subtypes (17 and 18 subtypes in ha and 10 and 11 subtypes in na) were recently discovered from bats [3, 4] . all other subtypes (h1 through h16 and n1 through n9) have been identified in aquatic birds, which are considered as the main reservoirs of iavs [5] . in contrast to the iavs, ibvs are classified into two antigenically distinct lineages, namely victoria and yamagata [1, 5, 6] . while the iavs infect diverse avian and mammalian hosts including humans, the ibvs are circulating mostly among human beings with a few exceptions of spillover cases reported in seals and swine [7] [8] [9] [10] . iav and ibv infections show similar clinical signs of 'influenza-like illness' and outcomes [11] [12] [13] [14] . there have been four major influenza pandemics since 1918 with some glimpses of pandemic-like events in history [15] [16] [17] . the h1n1 influenza pandemic of 1918 (pdm1918) is estimated to have caused up to 50 million human deaths across the globe [18] , symbolizing how devastating one pandemic outbreak can be. it is believed that influenza pandemics can be occurred by antigenic shift, which generally results from the introduction of certain gene segment(s) from nonhuman sources to human infecting iavs through a genetic reassortment process [5, 16] . the efficient human-to-human transmission and lack of immunity against the novel virus in humans can be driving forces to facilitate the dissemination of the virus and then to result in a pandemic. after a pandemic wave, the virus may lose momentum under increasing immune pressures among humans and persist as a seasonal virus. this seasonal virus will retain genetic mutations by circulating season by season, and its viral antigenicity may change, which is so-called antigenic drift, and it is the main reason that the vaccine viruses need updates every year. currently, the h1n1 and h3n2 subtypes of iavs, which are the descendants of 2009 and 1968 influenza pandemics, respectively, and the victoria and yamagata lineages of ibvs are circulating as seasonal viruses in humans. before the h1n1 pandemic in 2009 (pdm2009), an avian h5n1 iav had been remarked as a strong candidate that would cause a next pandemic given accumulating human infection cases with the virus [19, 20] . recently, an avian h7n9 virus has become the focus of attention concerning the increasing number of human infection cases in china [21, 22] . however, it is important to remember that pdm2009 was caused unexpectedly by a swine origin iav [16] , emphasizing the importance of the surveillance of swine iavs [23] . there are also other subtypes of avian ha and na isolated from human influenza cases sporadically [24, 25] . given their pandemic potential, we need to assess these humaninfecting zoonotic iavs in detail by comparing with the viruses that had caused past influenza pandemics. recently, middle east respiratory syndrome coronavirus (mers-cov) is dubbed 'camel-flu' virus [26] . seven years after its first human infection in 2012 [27] [28] [29] , more than 2400 human cases have been reported with approximately 35% case fatality rate [30] . mers-cov has a singlestranded positive-sense rna genome consisting of two partially overlapping large replicase open reading frames (orfs) and at least nine downstream orfs including the orfs encoding the four canonical structural proteins of coronaviruses, the envelope proteins s, e, and m and the n protein [31] . similarity of mers-cov with influenza viruses is not in its genome organization but probably in its respiratory symptoms, zoonotic potential, and the mode of respiratory transmission [32] [33] [34] . in addition to influenza viruses and mers-covs, arthropod-borne viruses, such as dengue and zika, may also pose pandemic threats even though persistent human-to-human transmissions have been rarely reported [35] [36] [37] [38] [39] . in this review, we intend to figure out the recipe and the ingredients of a pandemic by looking into the past pandemic events. iavs have eight segmented genomes of single-stranded, negative-sense rnas, which express similar major proteins, such as polymerase basic 2 (pb2), polymerase basic 1 (pb1), polymerase acidic (pa), ha, nucleoprotein (np), na, matrix 1 (m1) and matrix 2 (m2), nonstructural 1 (ns1), and nonstructural 2 (ns2/nep) [1] . studying the past influenza pandemics helps us to understand the mechanisms of such devastating outcomes. theoretically,144 different iav subtype viruses can be generated by the combinations of 16 ha and 9 na subtypes of avian iavs. however, only the h1n1, h2n2, and h3n2 subtypes have been identified as the causes of human influenza pandemics. of these, the h1n1 subtype caused the 1918 and 2009 pandemics and the 'abortive pandemic'-like swine influenza epidemic in 1976, which hundreds of soldiers at fort dix, new jersey, the united states were infected with [15, 40, 41] (fig. 1) . although diverse iav subtypes have been isolated from swine, h1, h2 and h3, and n1 and n2 subtypes have been mainly established [42] [43] [44] . as mentioned above, the pdm2009 and 1976 h1n1 viruses were swine-origin and readily transmissible among humans [15, 16, 45] . another pandemic h1n1 virus, pdm1918, however, appeared to be closely related with avian strains, which would be ultimately the ancestor of subsequent human and swine h1n1 iavs [46] . since human-infecting avian iavs were shown to be less transmissible among humans [47] [48] [49] , a 'spill-over' infection with avian iavs was not likely to be a fig. 1 timeline of influenza pandemics. each pandemic event is marked by a bold arrowhead on the timeline, which is not to the scale. the open arrowhead designates the 'aborted pandemic'. the animal symbols designate the host origins of the has of the pandemic strains. lines with the diamond heads designate the duration and the end of the circulation. the arrowed lines designate being circulated currently. the dotted lines designate unknown direct source of pdm1918 [50, 51] . as the receptor specificity is considered another prerequisite for avian iavs to infect and transmit to humans, there are some doubts about the avian-origin pdm1918 [52] . the h2 ha of the 1957 h2n2 pandemic and the h3 ha of the 1968 h3n2 pandemic were introduced from the avian reservoir to circulating human iavs [53, 54] , but whether the reassortment events occurred in humans or in other hosts, such as swine, immediately before transmitting to humans remains unanswered [16] . iavs can be largely divided into the human-like and avianlike types given the receptor specificity of their ha proteins. normally, human-like iavs bind to α-2,6 sialic acid (α-2,6 sa) receptor whereas avian-like iavs prefer α-2,3 sa receptor [55, 56] . it has been revealed that an aquatic bird mallard expresses more α-2,3 sa than α-2,6 sa in the respiratory tract but α-2,6 sa is barely expressed in the intestinal tracts [57] . the preference of avian iavs to α-2,3 sa might be related with fecal transmission of the viruses [58, 59] . regarding zoonotic transmission of iavs, swine is considered the intermediate host that can shuffle genetic segments between avian and human iavs to produce a novel strain [54] . because swine expresses both α-2,3 and α-2,6 sas in the upper respiratory tract, avian and human iavs all can infect swine [60] [61] [62] . it has been also challenged by additional studies that demonstrated similar sa distribution between human and swine [63] [64] [65] [66] . however, it has been shown that avian iavs could transmit between swine, and novel strains could be generated from contact swine by the genetic reassortment between avian and swine iavs [67] , which cannot be demonstrated in humans. hence, it appears that swine rather than humans may play a major role for the generation of novel strains at the interface of avian and human iavs [68] [69] [70] , and swine might be considered an adaptation host of iavs, as indicated in the cases of zoonosis [71] [72] [73] [74] [75] [76] and reverse zoonosis of iavs [43, 77] . then, it should be questioned whether avian iavs can be transformed into a pandemic virus by the adaptation only in humans. even though some reports indicated acquired transmissibility of avian iavs through multiple passages in ferrets [78] and transmissibility of avian iavs in swine [67] , it may be limited contact opportunities of the same avian iav to be repeatedly adapted in humans, as demonstrated in herfst et al. [78] . whether a rare adaptive mutant would grow out to dominate in the human host would be another issue. unlike severe symptoms observed in novel avian iav-infected patients, however, swine may be asymptomatic when infected with avian iavs [79, 80] . unless efficient transmissibility of an avian iav in humans was adaptively acquired during a single human infection, there would be only very limited close contact transmission from the patient to the care giver. close contact transmission of avian h7n9 iavs between patients and care givers have been recognized, but the contacted care givers have rarely shown the signs of infection [81] [82] [83] . this may demonstrate why avian iavs have acquired necessary adaptive mutations in swine rather than in humans to be pandemic viruses [69] . from the examples of the past influenza pandemics discussed so far, the recipe of a pandemic may be drawn up. the ingredients of influenza pandemics appear to be (1) non-human animal reservoir(s) that provides novel antigenic sources continuously, (2) adaptation host(s) where accumulated mutations result in host specificity changes or genetic reassortment occurs, (3) proper transmissibility between adaptation host(s) and humans back and forth, (4) efficient human-to-human transmission, and (5) pathogenicity in humans (fig. 2) . the second and third ingredients may work together to generate a virus with the human adapted genes with a new antigenic flavor. we have seen that the influenza pandemics came about with these ingredients and the human activity of socializing and traveling. determining whether a virus has these ingredients might be an important step in assessing its pandemic potential. the sudden reappearance of 1950s seasonal h1n1 strains in 1977 that was dubbed 'russian flu' right after the 1976 swine iav epidemic spurred the awareness of the need for pandemic planning [15, 40] . although the virus had rapidly spread among people under 25 years of age and had been 'drifting' as seasonal strains until the appearance of the pdm2009 [18] , the 1977 h1n1 virus may not be considered a pandemic virus. the virus was only a reappearance of previous human iav most likely by an accidental release and met an immunity gap among young people. the virus was not of a nonhuman reservoir origin, according to the pandemic ingredients summarized above. in case of the h5n1 and h7n9 avian iavs, they may lack efficient transmissibility to and between humans. however, as shown in ferret studies, they should be under close surveillance for their pandemic potential in advance. in addition to these avian iavs, swine iavs should be also monitored because novel strains can be generated by genetic reassortment in swine between avian and human iavs, as presented in the genesis of pdm2009. in contrast to iavs, ibvs do not have animal reservoirs, so they might be considered a virus of less pandemic potential. mers-cov appeared to originate from a bat coronavirus [28, [84] [85] [86] [87] and has become enzootic since a certain time point among dromedary camels [32, [88] [89] [90] , which is readily transmissible to and between humans [34] . unlike other human virus, such as measles [91] , mers-cov might evolve constantly in the dromedary camels [92] , which show a high rate of seroconversion and carry the virus mostly asymptomatically [90] . continuous back and forth transmission between humans and the dromedary camels constitutes very similar situations with iavs. frequent recombination during mers-cov replication in the reservoir host [93] [94] [95] might be used as a tool of adaptation by antigenically novel mers-cov strains or closely related bat coronaviruses, similarly as genetic reassortments of iavs in swine [96] . if there might be a mers-cov pandemic, subsequent mers-cov epidemics by antigenically 'drifted' strains might follow the pattern of seasonal influenza viruses until antigenically shifted (recombined) mers-cov strains hit humans again. mosquitos are a vector and non-human reservoir of dengue and zika viruses. back and forth transmission of these viruses between mosquitos and humans and the 'antibody dependent enhancement' of infection to dengue and zika viruses might potentially support the expansion of susceptible human pools [97] [98] [99] . however, currently there are very limited cases of human-tohuman transmission of these viruses through body fluid contacts [35] [36] [37] [38] [39] , and it is unlikely to result in rapid global spreading of the viruses like iavs, especially since mosquito distribution is geographically limited [100] . hence, possible human-to-human transmission of the dengue and zika viruses is inevitably limited by the requirement of intimate contacts or blood transfusion [101] . even though this ecological limitedness, solid control measures against mosquitos should be implemented to prevent dissemination of these arthropod-borne viral diseases in a global scale. small laboratory animals are surrogate models used in the experiments of human infecting viruses. viral behaviors in a natural host are often different from those in humans. viruses causing serious diseases in humans are often asymptomatic in their natural hosts. this is why natural hosts have a limitation as animal models. in case fig. 2 the ingredients and recipe of influenza virus pandemic. the workings of the five ingredients of iav pandemics are depicted. the ingredients are numbered (1) through (5) . avian iav, swine iav, and human iav are given as aviav, swiav and huiav, respectively. the viruses generated through (1), (2), and (3) may have a diverse range of transmissibility. when a virus with a nonhuman origin ha and an efficient human transmissibility gets transmitted from the adaptation host swine to human (4), a pandemic might ensue (5) of iavs, avian and swine species should be considered the natural reservoir animals, and in case of mers-covs, bats and dromedary camels [32, 87, 90] . viruses causing severe diseases and deaths in humans are the targets of prevention and control. researches using animal models for such viruses are carried out in two main directions: (1) investigation of viral characteristics in hosts, such as replication capacity, cellular tropisms, pathogenicity, and transmission, and (2) development of antivirals and vaccines. no animal models can be a perfect replicate of humans. certain animal models can have advantages in representing viral infection and, at the same time, disadvantages in other aspects. therefore, experimental questions may determine the best animal model, and experiments should be conducted such a way that the results obtained using animal models can be translated to humans. non-reservoir animals used for influenza virus infection experiments include ferrets, mice, guinea pigs, syrian hamsters, and non-human primates [102] [103] [104] [105] [106] [107] [108] [109] . historically, the discovery of the first human influenza virus was made by infecting ferrets with throat washings of influenza patients [110] . ferrets could also be readily infected with swine influenza virus. the different species of guinea pigs, mice, rabbits, hamsters, hedgehogs, and monkeys did not develop flu-like symptoms [103] . however, these asymptomatic animals have become useful for special purposes of influenza virus infection experiments now because virus detection and titration methods have become sophisticated (table 1) . especially, mice and guinea pigs are the most accessible animal models cost-and space-wise. in the case of mice, human infecting influenza viruses do not usually infect mice well, which is overcome by adapting the virus in mice through serial passages [103, 119, 120] . mice express the avian type α-2,3 sa in the lower respiratory tract, similarly as humans, but not the human type α-2,6 sa [121] . this is in line with the tendency of experimentally inoculated avian iavs, regardless of lowpathogenic (lpaiv) or highly pathogenic avian iavs (hpaiv), being mouse lethal with a relatively low 50% mouse lethal dose (mld 50 ) [104, 122] . as far as seasonal isolates of human iavs are concerned, dba/2 mice have been shown to be highly susceptible to diverse strains of un-adapted iavs [123, 124] . ferrets, guinea pigs, and syrian hamsters could be infected with most of iavs without adaptation, but only guinea pigs could be infected with ibv without adaptation and supported the airborne transmission [108, 111] . syrian hamster has been tried to replace mice because iavs could infect it without adaptation and there were airborne transmissions among syrian hamsters [108] . however, since guinea pigs can be infected with un-adapted ibvs as well as iavs and support airborne transmissions, which is readily detectable by the nasal wash titer, any advantage of syrian hamsters over guinea pigs awaits further reports of the use of animal models in influenza virus research. nonhuman primates (nhps) are genetically closest to humans. although nhps are not a readily accessible animal model, nhps are indispensable in the cases of vaccine and antiviral tests, where data relevant to humans in terms of pharmacokinetics and physiology are critical [125] . 'animal efficacy rule' of the united states food and drug administration (fda) requires for the therapeutics to demonstrate efficacy in two animal models manifesting human-like symptoms including at least one non-rodent model [126] . although the needs of testing in nhp models are clearly present [113] [114] [115] [116] [117] , most laboratories cannot afford nhps, and there is other ethical uneasiness about using nhps. how best to do without nhps may be a persisting issue in search of appropriate animal models. in the case of influenza virus research, among the frequently used animal models, ferrets appear to be the only non-rodent model other than nhps [127] . animal models of mers-cov are restricted by the availability of the receptor dpp4 that contains distinct amino acid sequence motif. besides the reservoir host dromedary camels and bats, nhps, rabbits, and other livestock animals, such as goat, cow, sheep, and pig, have been shown to express dpp4 that can bind to mers-cov [85, [128] [129] [130] . however, dpp4 of frequently used small the designation of "o" means that there are many studies using the animal for the purposed study the designation of "?" means that there are no or not many studies using the animal for the purposed study animal models like mouse, hamster, and ferret did not bind to mers-cov [128] . in nhps, mers-cov infection showed similar clinical signs as in humans, ranging from mild to severe, depending on the species of nhps. although dpp4 expressions were similar between rhesus macaques and common marmosets, the disease severity was from mild to moderate and from moderate to severe, respectively [131, 132] . lack of replication of mers-cov in small animal models poses problems of cost and space, especially since experiments using mers-cov must be carried out in an animal biosafety level 3 facility [29, [133] [134] [135] . mouse engineering technology has been deployed in diverse ways to generate mice expressing human type dpp4 (hdpp4) [136] [137] [138] [139] [140] . mers-cov infection in mice having hdpp4 exhibited only moderate signs of respiratory pathology, most likely due to the low level expression of hdpp4 in the mouse lung [135] , but mers-cov could be adapted in these mice to a more pathogenic virus [139, 141] . in addition to nhp and hdpp4-mouse models, rabbits might be a good candidate for mers-cov transmission experiments due to its camel-like receptor distribution in the upper respiratory tract (table 2 ) [142, 150] . however, while dromedary camels and new world camelids could transmit mers-cov upon contact, rabbits could hardly transmit the virus [130, 143] . mers-cov has been shown to use α-2,3 sa as a receptor assistant, which dromedary camels but not rabbits express in the nasal epithelium [130, 151, 152] . humans do not express the primary receptor dpp4 in the upper respiratory tract but transmits mers-cov well [153] . despite the controversies, humans have been reported to express α-2,3 sa in the upper respiratory tract [61, 64] . contribution of the 'pre-attachment' receptor α-2,3 sa or any other 'assistant' receptors to mers-cov transmission might be worth further investigation [151] . as far as the animal efficacy rule of fda is concerned, there appears no other choices but hdpp4-mouse and nhp models in the case of mers-cov studies. human-like symptoms of mers-cov infection have not been reproduced in other animals than hdpp4-mice and nhps. starting from the distinct receptor specificities of the ha proteins between avian and human iavs, host restriction determinants of iavs have been documented [56] . receptor specificity and amino acid signatures at pb2 residue 627 are well established host determinants critical for the interhost transmission of iavs [154] . however, iavs with avian or human receptor specificity can infect swine, as mentioned above. furthermore, the pb2 protein of the triple reassortant swine iav lineage, which comprises a majority of north american swine iavs [155] , retains the avian type e627 (glutamate in the pb2 residue 627) [156] . this was also a part of the molecular signatures of pdm2009 [16] . some human infecting avian iav isolates have shown acquisition of the human type k627 (lysine in the pb2 residue 627) but not acquisition of the human type receptor specificity determinants, and some acquisition of both [80, 157, 158] . iavs were shown to bind cells lacking sialic acid, and replicated efficiently [159] . acquisition of pb2 k627 might be more advantageous than acquisition of human type receptor specificity for avian iavs to replicate in the upper respiratory tract of humans, which is not an optimal temperature for pb2 e627 [157] . it has been also shown that the pb2 e627k mutation can emerge in a human case infected with an avian iav [160] . of note is that, although there have been avian-to-human transmission cases of avian h5n1 and h7n9 iavs, there have been no sustained human-to-human transmission of avian iavs. indeed, it has been reported that an h5n1 hpaiv harboring the human-type pb2 e672k mutation (change from glutamate to lysine in the position 627) and human-type ha q226l and g228s mutations (change from glutamine to leucine and from glycine to serine in positions 226 and 228, respectively, by h3 numbering) by itself could not transmit via airborne droplets between ferrets [78] . this may suggest that airborne transmissibility of avian iavs in humans might not be determined only by the presence or absence of molecular determinants. several studies using reassortant viruses have shown that the competence of reassortant viruses may not be predicted simply by the presence of the specific molecular determinants [161] [162] [163] [164] . an experiment testing a swine 'mixing vessel' hypothesis by co-housing pigs infected with an avian h1n1 strain or with a swine h3n2 strain and naive pigs revealed 40 and 60% transmission efficiency, respectively [67] . in that experiment, the reassortant viruses the designations of "o" and "?" are the same as in table 1 appeared to be well replicated (59/63) in the middle or lower respiratory tract, regardless of the presence of swine pb2 or avian pb2, although all but one reassortants (62/63) contained the swine ha. four out of the 63 reassortants did replicate in the upper respiratory tract and three out of four of those were the swine pb2containing reassortants [67] . what we can learn from this experiment is that the reassortant with an avian ha is not frequently selected in pigs and that the reassortant with swine pb2 is selected for the replication in the upper respiratory tract of pigs. the proportion of iav infection in farmed pigs is relatively low [68] , and the likelihood of co-existing of pigs infected with avian iavs and/or with swine iavs in the same pen may be even lower. however, avian and swine iav reassortants have been established and isolated in pigs [155] , which is the evidence of ongoing 'genetic mixing' of iavs. surveillance of newly emerging iavs may be approached in two ways; isolating and sequencing a virus using nextgeneration sequencing (ngs). as we discussed above, it is not enough to look for the molecular determinants by sequencing. even though full genome sequences are recovered, and their evolutionary relationships are reconstructed, further studies using the viruses should be carried out [165, 166] . the classical method of growing viruses in madin-darby canine kidney (mdck) and human airway epithelial a549 cells might be the first step after a genetic sequence analysis. the mdck cells express both α-2,3 and α-2,6 sas and support replication of influenza viruses ubiquitously due to the lack of the mx protein anti-influenza signaling [167, 168] . therefore, viral growth in the mdck cells is considered to evaluate the inherent growth potential of the viruses under such a condition where no innate and adaptive immune responses of the host are counted in [157] . on the other hand, the a549 cells, expressing more α-2,6 sa than α-2,3 sa like in the human upper respiratory tract, may indicate the growth potential of the viruses in the human upper respiratory tract [169, 170] . any swine isolatesavian, swine endemic, or reassortant originshowing equivalent to or better growth rates in mdck and a549 cells than the swine-origin pdm2009 virus may be further studied for their pathogenicity and transmissibility in animal models, to determine their pandemic potential. viral pathogenicity is related to host cell tropism but may be separate from the susceptibility of hosts to the virus. iavs are pathogenic to humans but not guinea pigs, although both are permissive to the virus. the degree of pathogenicity, the virulence of an iav, is inevitably associated with how well the virus replicates in a tissue or an organ, impairment of which results in a serious disease. in cases of respiratory viruses, those replicating in the lower respiratory tract tend to be more pathogenic than those in the upper respiratory tract [171, 172] . therefore, viral pathogenicity is closely related to inherent replication ability and receptor specificity of the virus. an avian iav with pb2 k627 has been shown to replicate better in both upper and lower respiratory tracts in mice and more pathogenic to the infected mice than with pb2 e627 [157] . however, in case of the pdm2009 isolate a/california/04/09 virus (ca04), the virus lacked previously identified molecular markers of iav virulence or transmissibility [173] , although was more pathogenic to mice than the seasonal h1n1 virus [174] [175] [176] . droplet transmissibility of pdm2009 in ferrets was shown to be slightly lower than the seasonal h1n1 virus whereas their contact transmissibilities were equally efficient [174] . this suggests that antigenic novelty might play a more important role for the pandemic potential of a certain iav than viral transmissibility. ferrets have been known to have human-like glycan distributions in their respiratory tract and may develop respiratory symptom after iav infection [104, 127] . however, although most human iavs including swine iavs are not pathogenic to mice, mice could be a good initial testing model in terms of cost and handling easiness compared with ferrets, especially for an isolate containing avian origin has. since mice have α-2,3 sa in the lower respiratory tract [121] , avian iavs tend to be pathogenic to mice without a prior adaptation [177] . therefore, viral pathogenicity in mice could be an initial pathogenicity indicator of certain iavs. the first pdm2009 isolate ca04 was more pathogenic in balb/c mice than the later pdm2009 isolates [176] . the pdm2009 isolates from fatal cases were also more pathogenic in mice than from the mild cases, and replacing the ha of the mild case isolate with that from the fatal case could make a more pathogenic virus in mice [178] . these experiments show that iav pathogenicity in mice may reflect inherent lung pathogenicity of iavs in humans. however, the results of mouse experiments should be interpreted in comparison with a pathologically well-characterized control. human iavs adapted in mice vs. avian iavs adapted for human-to-human transmission through a serial adaptation process, more pathogenic viral isolates can be recovered. for an iav with the receptor specificity to α-2,6 sa to replicate in mice, where there is hardly any or small amount of α-2,6 sa in the respiratory tract [121, 179, 180] , the virus must have used surrogate receptors such as c-type lectins or else [181] [182] [183] . iavs have been shown to bind to and replicate in sa-free or sialidase treated cells, although to a lower degree than in the untreated cells [159, 183] . abolishing na activity has been shown to be another mechanism of adapting to the host expressing a low level of the specific receptor [184] . indeed, the ha protein of a mouse-adapted pdm2009 has been shown to have acquired higher affinity to α-2,3 sa and lower affinity to α-2,6 sa compared to the wild type [185] . the difference between a human iav adapted in mouse and an avian iav infecting humans may be determined by the usage and availability of appropriate sa or equivalent molecules. to be pathogenic to mice, human iavs with α-2,6 sa specificity must adapt to use non-sa receptors or α-2,3 sa abundant in mice [180] , but avian iavs with α-2,3 sa specificity may have a possibility to replicate in the lower respiratory tract of humans without a prior adaptation. it has been known that humans express α-2,3 and α-2,6 sas in the lower and upper respiratory tracts, respectively [61] . but, it has been also reported that humans express both α-2, 3 and α-2,6 sas at a similar level in the upper respiratory tract [64] . given the availability of animal models that reflect human respiratory diseases, we conceptually suggest human, swine, and ferret respiratory tracts in fig. 3 , based on the study of de graaf et al. [63] . human or swine iavs with α-2, 6 sa specificity would be trapped in the upper respiratory tract of the respective host, where α-2,6 sa is abundant (fig. 3a) . some replicating viruses may overflow down to the lower respiratory tract, where both α-2,3 and α-2,6 sas are expressed. avian iavs with α-2,3 sa specificity would be also trapped in the upper respiratory tract by α-2,3 sa, but replicate poorly due to the unfavorable temperature. under such circumstances, only a high dose of avian iavs allows to escape the trapping in the upper respiratory tract and reach the lower respiratory tract, where the temperature is more favorable for avian iavs. even though avian iavs might overflow from the lower respiratory tract up to the upper respiratory tract, the virus might not replicate there due to the unfavorable temperature, unless there was the pb2 e627k mutation. this may be the reason that avian iavs are not easily transmissible between humans. in terms of viral adaptation, a rare appearance of avian iav mutants with α-2,6 sa specificity may not have special selective growth advantages in the lower respiratory tract of humans due to the overwhelming dominance of α-2,3 sa. only when avian iavs replicating in the upper respiratory tract, although poorly, acquires the pb2 e627k mutation or a reassortment, with or without acquisition of α-2,6 sa specificity at the same time, the variants may grow out well [67] . serial passaging of a wild-type h5n1 hpaiv in ferrets could not make the virus airborne transmissible between ferrets, but only those containing the mutations conferring the human type α-2,6 sa specificity and pb2 e627k could acquire airborne transmissibility after several passages in ferrets [78] . basically, these experiments suggest that, even if a rare mutant retaining the human-type receptor specificity and pb2 determinants might appear during replication of avian iavs, the mutant might not be easily selected to a domination over multiple passages, at least in ferrets. the reason may be that avian iavs have their niche of efficient replication in the lower respiratory tract of human, swine, or ferret (fig. 3) . the issue of avian iav adaptation in humans may not be whether adaptive mutations appears but whether there is a selective force enough for a virus to possess adaptive mutations to grow out to dominance. the ha protein of avian h5n1 iavs has been reported to require both human-type q226l and g228s mutations to bind to both α-2,3 and α-2,6 sas [186] , and that of avian h7n9 iavs only needs the q226l mutation [122, 187] . interestingly, a h7n9 human isolate was contact transmissible in pigs, regardless of pb2 e627 or k627, but only in the acquisition of human-type ha q226l determinant, which indicates the importance of the acquisition of human-type receptor specificity for the avian iav transmission in pigs, potentially also in humans [188] . however, as demonstrated in fig. 3b and c, the acquisition of human-type pb2 k627 would have been more critical due to the presence of saα-2,3 glycans in the upper respiratory tract of pigs. avian iavs with the pb2 e627k mutation should be able to replicate in the upper respiratory tract of humans and may be transformed to be transmissible between humans (fig. 3c) . however, they were poorly transmissible in ferrets (fig. 3d ). there have been no reports that investigated the transmissiblity of avian iavs with the pb2 e627k mutation between humans. all the first three human isolates of avian h7n9 iavs, which contained the human-type pb2 e627k and ha q226l mutations, showed approximately 30% airborne transmissibility in ferrets [48, 80, 122] . these appear to match with the conceptualized transmission in ferrets of fig. 3d . the h7n9 isolates with pb2 k627 and ha l226 would have replicated in the upper respiratory tract of ferrets, but their replication might have been inefficient since the receptor binding of the h7n9 q226l ha was still weaker to α-2,6 sa than to α-2,3 sa [187] . airborne transmission of the h7n9 virus in ferrets lacking α-2,6 sa might be explained by the overflow of the virus replicating in the lower respiratory tract, since ferrets poorly express α-2,3 sa in the upper respiratory tract [189] . this model also indicates similar level of transmission of avian iavs in ferrets without any human-type determinants. therefore, the conceptual model of iav transmission based on the receptor distribution appears to agree only with the ferret experiments, and probably only with h7n9 transmission in ferrets, since avian h5n1 iavs were not transmissible in ferrets [78, 190] . this conceptual model is apparently an oversimplification. there may be more factors involved in the transmission of iavs, such as the functional balance between the ha and na proteins and inherent replicability of iavs [191, 192] . the problem is, due to the discrepancy between what is expected in humans by the conceptual model and the reality, how to translate the results of transmission experiments evaluated in animal models to the natural transmission environments between humans. we may compare the transmissibility of certain viral isolates to that of ca04 in ferrets, but it is difficult to determine what the level of the transmissibility of these viruses indicate in terms of the pandemic potential of the viruses. the reports on the α-2,3 sa expression status in the upper respiratory tract of ferrets, pigs, and humans appear to be inconclusive [63] . determination of iav receptor distributions in humans and animal models appears critical for the interpretation of pathogenicity and transmission experiments assessed in animal models. we have discussed how to approach to the determination of the pandemic potential of iavs. similar principles may apply to mers-covs. previously, mers-cov replication was noted in nhp-derived cell lines, vero, and llc-mk2 cells [28] . it is now known that dpp4 is a functional cellular receptor for mers-covs and that vero cells express dpp4 [193] . vero cells also express α-2,3 sa, which has been shown to assist receptor binding of mers-covs [130, 151, 152, 167] . vero cells are well known for an impairment in the type i interferon pathways [194] . . avian iav with e627 or k627 pb2 might behave similarly in ferrets. an avian iav, without the specific receptor in the upper respiratory tract of ferret, may replicate in the lower respiratory tract but relatively poorly due to relatively low expression of saα-2,3 glycans. the overflowing avian iav, although with a low likelihood, may not be 'cleaned up' due to lack of saα-2,3 glycans in the upper respiratory tract of ferret, and may occasionally get transmitted to nearby ferrets covs in the upper respiratory tract is likely to contribute to their better transmission [143] . therefore, growth characteristics of camel mers-covs in vero cells may give us initial clues about how efficient the transmission of novel strains might be. the pathogenicity of mers-covs is closely associated with dpp4 expression in the lower respiratory tract of humans [153] . like iavs, the pathogenicity of mers-cov in hdpp4 mice may be an initial indicator. while hdpp4 mice produced severe symptoms upon mers-cov infection [140] , mice whose mdpp4 replaced with hdpp4 or modified to contain mers-cov binding hdpp4 motif (m-hdpp4-mice) showed little clinical signs [138, 139] . hdpp4 mice might be good for the evaluation of antiviral candidates, but not as a pathogenicity indicator, due to the ectopic expression of hdpp4. hdpp4 mice might be better to observe increases of growths and clinical signs after mers-cov infection. comparison of viral titers in the lungs and the lung pathology of mers-cov infection with those of the first mers-cov isolate hcov-emc [28] would give clues concerning the replication potential of mers-covs. there are no small animal models for the evaluation of mers-cov transmission yet. even though avian iavs transmit extremely poorly between humans, mers-covs appears highly transmissible between humans. in case of mers-cov outbreaks in korea, 2015, a superspreader individual resulted in 28 infection cases [34] . however, it is difficult to appropriately interpret the transmissibility difference between iavs and mers-covs in ferrets. due to the distribution differences of dpp4 and α-2,3 sa in the lower and upper respiratory tracts in humans and dromedary camels, it might be a major problem in translating the results of transmission experiments using small animal models. hcov-emc exhibited no transmission in rabbits [143] , and a kind of transmission threshold has not been defined, so it would be a problem to determine the pandemic potential of mers-cov isolates. the dpp4 motif of nhps have the same sequence with that of humans [132, 147] . however, no mers-cov transmission has been reported between nhps. hence, some kinds of surrogate measures might be necessary to evaluate the transmissibility of mers-covs. mice are not capable of contact or aerosol transmitting iav but can be infected with aerosolized live-attenuated influenza vaccine virus using a nebulizer [195] . since it has been reported that the lower respiratory tract of hdpp4 mice is very similar to that of humans, replication kinetics of a mers-cov isolate in the lungs of hdpp4 mice would provide a clue concerning transmissibility and virulence of mers-covs. this suggests the feasibility and importance of hdpp4 mice for the evaluation of replication or transmissibility of mers-covs. we have discussed what may be required to be a pandemic virus by analyzing the past influenza pandemics. the uniqueness of the past influenza pandemics is in that the three pools of reservoirs or hosts (avian, swine, and human) keep the persistent potential of generating novel iavs and that no other pathogens are known to bring about pandemics recurrently. moreover, the ingredients of the influenza pandemics and the modes of transmission may apply to other pathogens exhibiting pandemic potential. unlike iavs, mers-covs have not swept global communities and appeared to need a persistent human reservoir. the ultimate goal of mers-cov researches may find a way to prevent a mers-cov pandemic. studying the pandemic viruses, such as pdm1918 and pdm2009, may provide scientific information regarding molecular and viral requirements of potential pandemic viruses. our conceptual interpretation of animal models also underlines the value and importance of preclinical experiments in terms of the translational purposes and insights of the pandemic potential of influenza and other rna viruses. this study was supported by a grant from the national research foundation of korea (nrf) funded by the ministry of science and ict, republic of korea (grant no. nrf-2017r1a2b2003773). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. availability of data and materials not 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compatibility of polymerase reassortant viruses derived from the pandemic (h1n1) 2009 influenza virus and circulating influenza a viruses rescue of influenza a virus from recombinant dna a dna transfection system for generation of influenza a virus from eight plasmids african green monkey kidney (vero) cells provide an alternative host cell system for influenza a and b viruses high yields of influenza a virus in madin-darby canine kidney cells are promoted by an insufficient interferon-induced antiviral state receptor binding specificity of recent human h3n2 influenza viruses differential susceptibility of different cell lines to swine-origin influenza a h1n1, seasonal human influenza a h1n1, and avian influenza a h5n1 viruses pathogenesis of influenza virus infections: the good, the bad and the ugly molecular pathology of emerging coronavirus infections introduction of virulence markers in pb2 of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses replication and pathogenic potential of influenza a virus subtypes h3, h7, and h15 from free-range ducks in bangladesh in mammals virulence determinants of pandemic a(h1n1)2009 influenza virus in a mouse model sialic acid recognition is a key determinant of influenza a virus tropism in murine trachea epithelial cell cultures effective replication of human influenza viruses in mice lacking a major alpha2,6 sialyltransferase the c-type lectin langerin functions as a receptor for attachment and infectious entry of influenza a virus nlinked glycosylation facilitates sialic acid-independent attachment and entry of influenza a viruses into cells expressing dc-sign or l-sign evolving complexities of influenza virus and its receptors adaptation of influenza a viruses to cells expressing low levels of sialic acid leads to loss of neuraminidase activity adaptation of pandemic h1n1 influenza viruses in mice structure and receptor specificity of the hemagglutinin from an h5n1 influenza virus three mutations switch h7n9 influenza to human-type receptor specificity analysis of recombinant h7n9 wild-type and mutant viruses in pigs shows that the q226l mutation in ha is important for transmission influenza h1n1 a/solomon island/ 3/06 virus receptor binding specificity correlates with virus pathogenicity, antigenicity, and immunogenicity in ferrets experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 h1n1 influenza pandemic effects of ha and na glycosylation pattern changes on the transmission of avian influenza a(h7n9) virus in guinea pigs dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc influenza a virus lacking the ns1 gene replicates in interferon-deficient systems aerosol vaccination induces robust protective immunity to homologous and heterologous influenza infection in mice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank sang woo kim, juho do, dahae hong, and eunji choi for technical assistance with the animal experiments.authors' contributions m-sp outlined this review. msp, jik, j-yb, and m-sp wrote this review. the authors read and approved the final manuscript. the authors declare no competing interests.received: 28 january 2020 accepted: 10 march 2020 key: cord-011438-imbpgsub authors: zhang, yun; xu, zhichao; cao, yongchang title: host–virus interaction: how host cells defend against influenza a virus infection date: 2020-03-29 journal: viruses doi: 10.3390/v12040376 sha: doc_id: 11438 cord_uid: imbpgsub influenza a viruses (iavs) are highly contagious pathogens infecting human and numerous animals. the viruses cause millions of infection cases and thousands of deaths every year, thus making iavs a continual threat to global health. upon iav infection, host innate immune system is triggered and activated to restrict virus replication and clear pathogens. subsequently, host adaptive immunity is involved in specific virus clearance. on the other hand, to achieve a successful infection, iavs also apply multiple strategies to avoid be detected and eliminated by the host immunity. in the current review, we present a general description on recent work regarding different host cells and molecules facilitating antiviral defenses against iav infection and how iavs antagonize host immune responses. influenza a virus (iav) can infect a wide range of warm-blooded animals, including birds, pigs, horses, and humans. in humans, the viruses cause respiratory disease and be transmitted by inhalation of virus-containing dust particles or aerosols [1] . severe iav infection can cause lung inflammation and acute respiratory distress syndrome (ards), which may lead to mortality. thus, causing many influenza epidemics and pandemics, iav has been a threat to public health for decades [2] . the virus is an enveloped, segmented, negative-strand rna virus, belonging to the orthomyxoviriae family. the eight viral gene segments encode as many as 18 proteins. besides polymerase basic 1 (pb1), pb1-n40, pb1-f2, pb2, polymerase acid (pa), hemagglutinin (ha), nucleoprotein (np), neuraminidase (na), matrix 1 (m1), matrix 2 (m2), nonstructural protein 1 (ns1) and ns2 (also known as nuclear export protein, nep), new viral proteins were recently uncovered, such as pb2-s1 [3] , pa-x (product of ribosomal frameshifting) [4] , pa-related proteins pa-n155 and pa-n182 [5] , m42 [6] , and ns3 [7] . ha, na, and m2 proteins constitute surface of the iav virion, where ha is the most abundant surface protein. according to the genetic and antigenic diversity of the ha and na proteins, iavs were divided into 18 ha and 11 na subtypes. h17n10 and h18n11 subtypes were recently identified in bats [8, 9] . ha is a type i glycosylated protein, which is responsible for virus entry to host cell. functional ha protein is a homotrimer structurally composed of a stem region and a globular head region in each monomer. the head region bearing n-acetylneuraminic acid (sialic acid, sa) binding pocket is critical for receptor attachment, and contains most antigenic determinants. the stem region undergoing conformational changes is responsible for low ph-triggered membrane fusion [10] , and plays an important role in cross protection against heterosubtypic iav infection [11] . n38 glycan at this region iavs can infect a broad spectrum of host species, including both wild and domestic birds, as well as many mammalian species. the virus is capable of interspecies transmission to new species. however, no interspecies transmission of the bat iavs has been reported so far [54] . furthermore, the high frequency of mutations and recombination increases the risk of iav adaptation in humans. besides three pandemic subtypes (h1n1, h2n2, and h3n2), other subtypes, including h5n1, h5n6, h7n7, h7n9, n9n2, and h10n8 could cross the species barrier and cause human infections [55] [56] [57] [58] . several effect factors are essential in iav host switch events, including the receptor-binding properties of ha [16] , as well as cellular receptors [59] [60] [61] [62] . long and his colleagues summarized the role of host factors in iavs adaption to humans, and the review is recommended here for further reading [63] . noteworthy is the fact that most phylogenetically diverse iavs with different origins could successfully replicate in swine [64] . since pigs have both sa α-2,6 and sa α-2,3 galactose receptors [65] , they can serve as a suitable mixing reservoir for both human and avian iavs, thus raising global concern on periodic zoonotic infections. take the emergence of influenza a (h1n1) pdm09 (ph1n1) and influenza a (h3n2) a/canada/1158/2006 strains for instance, both strains are swine-origin iavs and were the consequence of adaption and reassortment of several swine lineages [66, 67] . furthermore, some genes of these strains originated from avian iavs [68] . with the development of gene sequencing technology, machine learning (ml) facilitated with large genomic datasets are used in prediction about sequence changes in newly invaded viruses from other animal hosts, take the "batch-learning self-organizing map (blsom)" method for instance [69] . ml is also applied in characterization of distinct host tropism protein signatures [70] , and prediction of amino acid changes for interspecies transmission [71] . these studies provided measures in identification of potential high-risk strains. in addition, the nucleotides and dinucleotide compositions of viruses play important roles in prediction of viral host species [72] . combining gene sequencing technology viruses 2020, 12, 376 4 of 23 and ml methods, researchers applied large iav genomic datasets to analyze species selection bias of iav mono-/dinucleotide composition and predict human-adaptive swine or avian iavs [73, 74] . the application of multi-disciplinary subjects would provide useful information for prediction of pandemic influenza. in general, the life cycle of the iav is generally divided into four steps: virus entry into the host cell, transcription and replication of the viral genome, assembly, and virus budding. though alveolar epithelial cell is the primary target cell for iavs, different iav subtypes have different patterns of viral attachment (pva). for human iavs, alveolar type ii epithelial cells, as well as immune cells such as alveolar macrophages and dendritic cells, are major target cells for an established infection [75, 76] . two seasonal iavs and pandemic h1n1 virus, preferred to attach to ciliated epithelial cells and goblet cells in the upper respiratory tract (urt), and avian iavs, take h5n1 for instance, attached seldom to these cells [77] . in the lower respiratory tract (lrt), human iav h1n1 and h3n2 attached to more cell types than avian iav h5n1, a highly pathogenic avian iav (hpaiv) strain. however, h5n1 could bind to type ii pneumocytes [78] . considering the fact that metabolism in the type ii pneumocytes is quite active, infection of hpaivs is more likely to cause severe pneumonia [78] . other research on low pathogenic avian iavs (lpaivs), which generally do not cause severe pneumonia, showed that these viruses usually attach to human submucosal gland cells, thus can be cleared by the mucus [79] . iav infection starts from recognition of sa by ha protein, though in vitro research claimed that these n-linked glycans were not essential for virus entry [80] . the cleavage of ha precursor protein ha0 into ha1 (containing receptor binding domain) and ha2 (containing fusion peptide) in low ph environment during ha transport is critical for virion internalization [81] . some research showed that type ii transmembrane serine protease such as transmembrane protease serine 2 (tmprss2), human airway trypsin-like protease (hat), transmembrane protease serine 4 (tmprss4), homo sapiens serine protease desc1 and homo sapiens transmembrane protease, serine 13 (mspl) can cleave human and avian iav ha proteins at an arginine residue [82] . in addition, for avian iavs, ha0 of hpaivs can be cleaved by subtilisin-like protease, while that of lpaivs is cleaved by trypsin-like proteases [83] or thrombin [84] . therefore, in avian iavs, the cleavage sites are considered to be the major determinants for virus virulence [85] , and rna folding in the cleavage region could be an important factor for virulence determination [86, 87] . proteins in the vrnp complex contain different nuclear localization signals (nlss), thus helping the vrnp complex to enter the host cell nucleus via active transport, take the crm1-dependent pathway for instance [88] . the acidic environment of the endosome also activates m2 ion channel, hence acidifies the viral core, resulting in entrance of vrnp complex into the host cell [34] . replication of viral genome does not require a primer but a full-length complementary rna (crna), which is essential for the newly formed vrnp complex. the viral rna polymerases first bind to the 3 end and the 5 end of the segmented viral rna and crna, respectively, then start replication with the help of the 5 cap of host pre-mrnas via a pb1-pb2-mediated "cap snatching" mechanism [27, 89] . the conserved segment-specific nucleotides at the 3 and 5 ends of the viral genome could modulate genome expression and replication during infection [90] . in addition, dephosphorylation at a specific position of the h1n1 ns1 protein results in attenuated virus replication [91] . mature viral mrnas are transported to the cytoplasm by a "daisy-chain" complex and translated subsequently [88, 92] . new synthesis of ha occurs on the rough endoplasmic reticulum (er). glycosylation and palmitoylation of the protein are completed later in the golgi [93, 94] . after synthesis and maturation of na and m2 proteins, the trans-golgi network (tgn), together with coat protein i (copi) complex and gtpase rab proteins, transport the newly synthesized ha, na, and m2 proteins to the apical plasma membrane (pm). these proteins then assemble with viral genomic segments. the virions are finally closed and m1 and m2 proteins mediate virion budding from the apical side of the viruses 2020, 12, 376 5 of 23 cells [28, [95] [96] [97] [98] . na protein cleavages the sa residues, which allows the virions to be released from the plasma membrane [99] . since iav has a relatively small genome, host machinery is required in order to accomplish the viral life cycle. to uncover host dependency factors that are necessary for iav replication, numerous large-scale rna interference (rnai) screens and genome-wide crispr/cas 9 screen were performed [29, [100] [101] [102] [103] . for instance, son dna binding protein was important for iav virion trafficking in an early infection stage and cdc-like kinase 1 facilitated aiv replication [29] . usp47 facilitated viral entry, whereas tnfsf12 (april) and tnfsf12-tnfsf13 (twepril) helped with viral replication [101] . using genome-wide crispr/cas9 screen, several genes of sialic acid biosynthesis and related glycosylation pathways were involved with h5n1 infection [102] , and wdr7, ccdc115, and tmem199 were essential for viral entry and regulation of v-type atpase assembly [103] . furthermore, single-cell transcriptome sequencing (rna-seq) was applied to explore host-virus interactions, revealing a correlation between defective viral genomes and virus-induced host transcriptional programs [104] . these data provide valuable information for developing host-targeted therapeutics. host immune system functions immediately after detection of the virus. host mucosal immune system (mis), induced after virus invasion, serves as the first line to prevent iav from adhering to the susceptible cells. in the urt, mucosal response is induced in the naso-associated lymphoid tissues (nalt), while in the lrt, it occurs in bronchus-associated lymphoid tissues (balt). host innate immunity, including phagocytic cells, interferons (ifns), proinflammatory cytokines, etc., applies multiple mechanisms in defending iav infection [105] . host adaptive immunity, mediated by b lymphocytes and t lymphocytes, together with other immune mechanisms, reacts specifically to neutralize and eliminate the virus. on the other hand, to establish a successful infection, iavs also employ a plethora of strategies to avoid being detected or being cleared by the host immunity. notable strategies include regulation of ifn signaling [106] , inhibition of cytokine expressions [107, 108] , modulation of apoptosis [109] [110] [111] , interference of autophagy [112] , and effects on antibody production [113] . the iav-host immunity interaction was summarized by several reviews [114, 115] . upon detection of infection, innate effector cells, including natural killer (nk) cells, neutrophils, and dendritic cells (dcs), etc., are recruited to the infected sites. nk cells are large granular lymphocytes, making up 10% of the resident lymphocytes in the lung. after recruitment from the blood, nk cells interact with dcs and macrophages to secret various cytokines and restrict infection via lysis of the iav-infected cells. the lysis process is mediated by interaction between nk receptors p46 (most nkp46) and iav ha protein expressed by the infected cell [116, 117] . interestingly, liver nk cells other than lung nk cells possessed a memory phenotype to protect mice against subsequent iav infection, though the lung nk cells are important in control of primary iav infection [118] . however, nk cells are also shown to exacerbate iav pathology, since depletion of nk cells led to increased resistance to high dose h1n1 infection in mice [119, 120] . the contribution of nk cells to anti-iav defense in mouse models was later shown to be strain and dose dependent. in addition, the host genetic background also played an important role [121] . neutrophils are key innate immune cells recruited to infection sites by cellular migration through vascular endothelium. they function in clearance of pathogens via phagocytosis, producing extracellular traps, and degranulation [122] . in addition, they also regulate adaptive immunity via guiding influenza specific cd8 + t cells to the infection sites [123] . the function of dendritic cells (dcs) is to monitor invading pathogens. after iav infection, the conventional dcs migrate from lung to lymph nodes through interaction between ccr7 and its ligand, and present antigens to t cells [124, 125] . one study based on a mouse model showed that, during iav infection, immature and mature dcs were specialized in iav ha processing, since both types of dcs could present one epitope of h1n1 ha (ha amino acids 107-119), whereas another epitope (ha amino acids 302-313) could only be processed by mature dcs [126] . the complex role of dcs in initiation of robust immunity against iav infection is reviewed by waithman and mintern [127] . t cells and b cells are critical components in adaptive immunity against iav infection. cd8 + t cells differentiate into cytotoxic t lymphocytes (ctls) and defend iav infection via producing cytokines and effector molecules, and cytotoxic effects (i.e., lysis) of infected cells mediated by mhc class i. cd4 + t cells target iav-infected epithelial cells through binding with mhc class ii molecules and contribute to b cell activation thus consequently promote antibody production. the activation of t cells and b cells in iav infection will be exposited in section 3.5. the reaction of innate immunity is nonspecific. it is triggered by recognition of pathogen associated molecular patterns (pamps) via host pathogen recognition receptors (prrs). toll-like receptors (tlrs), retinoic acid-inducible gene-i proteins (rig-i), and nod-like receptors are common prrs, the activation of which leads to activation of innate immune signaling and further production of cytokines as well as other antiviral molecules. toll-like receptors are responsible for sensing pathogens at cell membranes, endosomes, and lysosome [128] . tlr3 and tlr7 are shown to be involved in iav detection at endosomes [105] . tlr3 recognizes double stranded rna (dsrna) which may be released by cellular stress and cell death [105] and unidentified rna structures in phagocytosed cells infected with iavs [129] . in macrophages and dendritic cells, tlr3 interacted with tir-domain-containing adapter, then activated the serine-threonine kinase iκkε (ikkε) and tank binding kinase 1 (tbk1) to phosphorylate interferon regulatory factor 3 (irf3), the process of which further led to expression of ifn-β [130] . in addition, an over-reacting tlr3 activation promoted iav pathogenesis, which could be reduced by a single-stranded oligonucleotide (sson) functioning as a tlr3 inhibitor, resulting in restrained viral loads both in vitro and in vivo [131] . tlr7 recognizes single stranded rna (ssrna). in plasmacytoid dendritic cells (pdcs), after activation of tlr7 during iav infection, irf7 or nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) were activated via myeloid differentiation factor 88 (myd88) to induce type i ifns [132] . in avian macrophages, activation of tlr7 produced pro-inflammatory molecules such as interleukin (il)-1β [133] . in addition, in mouse models, tlr7 played an important role in activation of nk cells [134] . it was also shown to be involved in development of adaptive immunity to prevent iav infection [135, 136] . rig-i recognizes ssrnas and transcriptional products of iavs, which triggers activation of the caspase activation and recruitment domains (cards) via dephosphorylation or ubiquitination by e3 ligases, resulting in activation of transcription factors including irfs and nf-κb [137] . otub1 played an essential role in regulation of rig-i [138] . in addition, melanoma differentiation-associated gene 5 (mda5) was also involved in sensing transcriptional products of iavs in the cytoplasm [139] . for nod-like receptor family, pyrin domain containing 3 (nlrp3) and nlr apoptosis inhibitory protein 5 were activated after iav infection [140] . iav m2 ion channel and pb1-f2 were involved in activation of nlrp3 inflammasome and stimulate il-1β secretion subsequently [141, 142] . the role of the nlrp3 inflammasome in regulation of anti-iav responses is discussed in detail by sarvestani and his colleagues [143] . delayed oseltamivir and sirolimus combined treatment could suppress nlrp3 inflammasome mediated secretion of il-1β and il-18, resulting in attenuation of h1n1-induced lung injury [144] . after detecting viral components, transcription factors including nf-κb and irfs are activated, leading to transcription of ifns and pro-inflammatory cytokines. ifns bind to receptors, resulting in upregulation of multiple interferon-stimulated genes (isgs) [145] . it is well known that type i ifns viruses 2020, 12, 376 7 of 23 (ifn-α and ifn-β) and type iii ifns (ifn-λ 1-4) play critical roles in antiviral responses. mice failed to restrict non-pathogenic iav when both type i and type iii ifn receptors were knocked out [146] . the expressed ifns consequentially bind to different receptors. type i ifns interact with ifn-α/β receptors (ifnar), whereas type iii ifns interact with ifn-λ receptors (ifnlr). janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [147, 148] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [149] . furthermore, ifn-λs served an important role in programming dcs to direct effective t cell immunity against iav infection [150] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [151, 152] . cholesterol 25-hydroxylase (ch25h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [153] . guanylate-binding protein 3 (gbp3) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [154] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim14 could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [155] . trim22 degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [156] . trim 25 regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [157] . trim32 recognized iav pb1 protein and reduced its polymerase activity [158] . trim41 targeted np for ubiquitination and degradation in vitro [159] . for further reading on other isgs, several reviews regarding ifn responses during iav infection are recommended here [160, 161] . a general description of activation of innate immunity and ifn signaling pathway after iav infection is illustrated in figure 1 . signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [147, 148] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [149] . furthermore, ifnλs served an important role in programming dcs to direct effective t cell immunity against iav infection [150] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [151, 152] . cholesterol 25-hydroxylase (ch25h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [153] . guanylate-binding protein 3 (gbp3) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [154] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim14 could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [155] . trim22 degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [156] . trim 25 regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [157] . trim32 recognized iav pb1 protein and reduced its polymerase activity [158] . trim in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [162] . the follow-up work showed that poly (adp-ribose) polymerase 1 (parp1) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [163] . ns1 is the most important ifns antagonist protein via mechanisms including inhibition of the trim25-mediated rig-i ubiquitination, suppression of protein kinase r (pkr), in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [162] . the follow-up work showed that poly (adp-ribose) polymerase 1 (parp1) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [163] . ns1 is the most important ifns antagonist protein via mechanisms including inhibition of the trim25-mediated rig-i ubiquitination, suppression of protein kinase r (pkr), phosphorylation of iκb kinases (ikk) α and β in the nf-κb pathway, interruption of the phosphorylation of stat1, stat2, and stat3 [39, 115] , and degradation of otub1 [138] . phosphorylation of ns1 is crucial for its function of antagonizing ifn-β expression, since dephosphorylation at position 73 and 83 of the protein induced a high level of ifn-β [91] . non-structural protein pb1-f2, identified from a+1 open reading frame (orf) of pb1 gene segment [164] , is multifunctional in deregulation of type i interferon [165, 166] . it counteracted rlr-mediated activation of ifn pathway not only by targeting mitochondrial mavs [115, 165, 167] , but also by binding to the dead-box helicase ddx3 to induce proteasome-dependent degradation [166] . furthermore, pb1-f2 interacted with mitochondrial tu translation elongation factor (tufm) to mediate formation of autophagosome, thus inducing complete mitophagy, which is critical for mavs degradation [167] . novel pa-x protein could also modulate innate immune responses. a review regarding the function of ns1 and pa-x proteins in antagonizing host innate immunity is recommended here [114] . though autophagy is essential for cellular metabolism and homeostasis, it also plays important roles in innate immune responses against pathogen infection. for cellular homeostasis, the mtor pathway is one of the most conserved autophagic pathways. the mtor complex 1 (mtorc1) negatively regulates the ulk1 kinase activity, thus affecting the autophagy induction [168] . c-jun n-terminal protein kinase 1 (jnk1) disrupts the bcl-2/beclin-1 complex through phosphorylation, thus regulating the autophagy induction [169, 170] . jnk1 is also reported to upregulate beclin-1 expression through phosphorylation of transcription factor c-jun in vitro [171] . in contrast to the autophagic pathways for cellular metabolism and homeostasis, less is known about autophagosome formation after iav infection [172] . to restrict infection of multiple viruses including iavs, trim23 is essential to mediate autophagy via its ring e3 ligase and adp-ribosylation factor (arf) gtpase activity [173] . beclin-1 and tufm-regulated autophagy also inhibited iav replication [112] . in hela cells and a549 cells, iav infection activated jnk1 to induce autophagosome formation and tgf-β-activated kinase 1 might contribute to the process [174, 175] . furthermore, autophagy was involved in maintaining memory b cells to counteract iav infection [176] . iav also utilizes autophagy to complete its life cycle. ns1 protein is proposed to suppress jnk1-mediated autophagy induction [174] . m2 could also block autophagosome maturation and mediate microtubule-associated protein 1 light chain 3 (lc3)-bound membrane redistribution, thus allowing filamentous budding of iav [177] [178] [179] . circ-gatad2a (gata zinc finger domain containing 2a), induced by iav infection, could inhibit autophagy and promote iav replication [180] . for a comprehensive reading on iav-induced apoptosis, a review is recommended here [181] . upon detection of iavs, dcs trigger production of ifns and cytokines, which in turns assist maturation of the dcs into antigen presenting cells (apcs), and initiate t cell immune responses. through the activation of ag-bearing dcs, naïve cd4 + t cells differentiate into th1, th2, th7, regulatory t cells (treg cells), follicular helper t cells, and killer cells. th1 and follicular helper t cells are the most abundant cd4 + t helper cells. they can secret antiviral cytokines, regulate cd8 + t cell differentiation, promote b cell activation, and maintain immunological memory [182, 183] . th17 cells induced pulmonary pathogenesis and could decrease mortality of iav-infected mice [184, 185] . in addition, γδ t cells, expanding in the late stage of iav infection with a t cell receptor (tcr)-independent viruses 2020, 12, 376 9 of 23 manner, could efficient eliminate iav-infected airway epithelial cells, resulting in lower viral titers [186] . new surrogate markers cd49d and cd11a were used to explore the kinetics of iav-specific cd4 t cells responses, revealing endogenous cd4 t cell response to primary iav infection is predominantly composed of t-bet+ cells [187] . cd8 + t cells are major components for virus clearance in adaptive immunity. after activated by dcs, cd8 + t cells undergo rapid expansion, differentiation, and migration to the infected sites. in general, to establish effective primary cytotoxic t lymphocyte (ctl) responses, cd4 + t cells play an essential role, with a mouse model as an exception [188] . ctls produce cytotoxic granules containing perforin and granzymes (gra and grb) to induce apoptosis and interrupt iav replication [189] . in addition, ctls produce cytokines, such as tnf, fasl, and trail, which recruit death receptors to induce apoptosis [190] . in addition, il16 deficiency enhanced the th1 and ctl responses upon iav infection [191] . furthermore, as cd8 + cells could last for two years in murine models, iav-specific memory ctls reacted specific to epitopes in conserved iav proteins [192] . in the nasal epithelia, they could prevent the spread of the virus from the urt to the lung [193] . to establish memory cd8 + t cells, autophagy plays an important role [194] , while the function of cd4 + t cells in memory ctl responses is "context-dependent". a recent study showed that cd4+ t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [195] . grant and her colleagues summarized and discussed the importance of cd8 + t cell immunity against iavs [192] , and this review is recommended for further reading. with the help of cd40 ligand (cd40l), cd4 + cells contribute to b cell activation [183] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [196] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blocking iav transmission [197] . in addition, iav-specific antibody-dependent cell-mediated cytotoxicity (cdcc) also plays a role in cross-protection against iav infection. a general description of adaptive immunity against primary iav infection is illustrated in figure 2 . antigenic shift and drift, resulting in reassorted and mutated ha and/or na, are responsible for aiv escaping from host immunity [50] [51] [52] . furthermore, additional glycosylation on h5 ha could also induce virus escape from neutralizing antibodies [198] . apoptosis represents programmed single cell death that occurs in cell physiological remodeling, cell proliferation, or immune response to invading pathogens [199] . besides prototypical changes, cells undergoing apoptosis can be detected through dna and biochemical assays, take the tunel and in situ end-labeling (isel) techniques for instance. two primary pathways are involved in activation of apoptosis: the intrinsic or mitochondrial pathway, and the extrinsic or death receptor pathway. the intrinsic pathway is also known as "the mitochondrial pathway", which operates in response to various intracellular stress. several factors such as nitric oxide (no), cytochrome c, and second mitochondria-derived activator of caspases (smac) can activate this pathway, and the key player of this pathway is proteins in the bcl-2 family, which are activated by stress signals and then release apoptotic factors via destabilizing the mitochondrial membrane [200, 201] , resulting in release of mitochondrial cytochrome c. cytochrome c then binds to apoptosis protease activating factor-1 (apaf-1) and forms a complex with pro-caspase 9 (then cleaved into caspase 9), the function of which is to cleave its effector pro-caspase 3 [202] . in addition, smac, localizing in the cytosol, could initiate activation of caspase 9 via blocking the activity of iap [203] . the extrinsic pathway is regulated by extracellular ligands acting on transmembrane "death receptors": the first apoptosis signal (fas) receptor-fas ligand (fasr/fasl) and the tnf-αtnf receptor 1 (tnfα/tnfr1) [199] . in the fasr/fasl model, fas ligand binds to its receptor fasr [204] , forming the death-inducing signaling complex (disc) with pro-caspase 8, resulting in activation of caspase 8 and downstream activation of other caspases (caspase-3, caspase-6, and caspase-7) [199] . in the tnfα/tnfr1 pathway, tnfr1-associated death domain protein (tradd) is activated after binding of tnfα to tnfr1, leading to recruitment of fadd and receptor interacting protein (rip) [205] . fadd then associates with pro-caspase 8 to form the disc, resulting in activation of caspase 8 and apoptosis. could prevent the spread of the virus from the urt to the lung [193] . to establish memory cd8 + t cells, autophagy plays an important role [194] , while the function of cd4 + t cells in memory ctl responses is "context-dependent". a recent study showed that cd4+ t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [195] . grant and her colleagues summarized and discussed the importance of cd8 + t cell immunity against iavs [192] , and this review is recommended for further reading. with the help of cd40 ligand (cd40l), cd4 + cells contribute to b cell activation [183] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [196] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blockin during iav infection, viruses modulate host apoptotic responses in a time-dependent manner [206] . for instance, in order to earn enough time for replication and virion formation, iav inhibited apoptosis via upregulating the anti-apoptotic phophoinositide-3-kinase-protein kinase b (pi3k-akt) pathway at the beginning of infection. however, in the later phase of infection, the virus suppressed this pathway to upregulate the pro-apoptotic p53 pathway, thus allowing successful release of virions [207] . several viral proteins are involved in regulation of host apoptosis. np protein induces host apoptosis to favor viral replication through interaction with ring finger 43 (rnf43) [208] , apoptotic inhibitor 5 (api5) [209] , or clusterin [111] . pb1-f2 also induced apoptosis and promoted viral replication through dysregulating mitochondrial potential [206] . furthermore, m1 promoted apoptosis by binding to heat shock protein 70, thus activating caspase and the subsequent apoptosis [210] . in addition, ns1 expression was reported to induce apoptosis in mdck and hela cells [211] . however, mutant iav lacking the ns1 gene could induce apoptosis in cultured cells [212] . the function of ns1 in inhibiting apoptosis may be explained by its ability to inhibit type i ifn [213, 214] . these data demonstrate sophisticated mechanisms of iav in regulating host apoptosis. furthermore, the role of these viral proteins in apoptosis suggests that these proteins may present suitable targets for anti-iav therapies. a comprehensive review on influenza a virus-induced apoptosis discussed by ampomah and lim is recommended here [181] . in addition, recent in vitro research found that apoptosis was induced at early iav infection stage, while later the cell death pathway was shifted to pyroptosis. the switch process was promoted by the type i ifn-mediated jak-stat signaling pathway through expression of the bcl-xl gene [215] . during iav infection, multiple immune systems coordinate together to protect the host. accordingly, viruses antagonize the immune system through multiple measures to establish a successful infection. considering the high frequencies in genome mutations and recombination, vaccination is the most effective way to defend against the viruses via inducing cross-protective antibodies and/or enhancing immune responses. several studies in vaccine development have tried to enhance host immune responses. for instance, vaccine candidate containing ha targeted to chemokine receptor (porcine mip1α) was shown to enhance t cell responses, resulting in a strong and cross-reactive cellular immunity in vaccinated pigs [216] . another example is an attempt to intranasally administer a polyanhydride nano vaccine (iav-nanovax), which could promote robust lung-resident germinal center (gc) b cells with lung-localized iav-specific antibody responses as well as lung-resident memory cd4 + and cd8 + t cell responses [217] . for anti-iav drugs, currently, na inhibitors (relenza tm and tamiflu tm ) are applied clinically as anti-influenza drugs [218] . these drugs inhibit the activity of na by preventing viral budding [21] . in addition, cap-dependent endonuclease inhibitor (baloxavir marboxil) targeting pa is also applied against influenza a and b virus infection [219] . our progressing understanding of the iav life cycle of the virus and iav-host interaction could contribute to anti-influenza drug design. since the recognition of ha protein to sa linked glycoproteins is the first step in iav infection, effective blocking of the interaction between viral ha and sa receptor serves as a favorable target in drug design [220, 221] . favipiravir, a nucleotide analogue that selectively inhibits the rna-dependent rna polymerase, is licensed in japan to be applied against emerging influenza viruses resistant to other antivirals [222, 223] . oleanolic acid (oa), a kind of pentacyclic triterpene natural product, and its analogues, as well as its derivatives, were shown to bind to ha, thus blocking the attachment of iavs to mdck cells [224] [225] [226] . pvf-tet is a peptide-based ha inhibitor, which was shown to sequester ha into amphisome (fusion of late endosome with autophagosome) and protected mice from the lethal iav infection [227] . new effective drugs targeting the polymerase would be a promising strategy against iav infection, since they would directly reduce or eliminate viral replication. numerous sites, including the cap-binding site [228] , the endonuclease [229, 230] , and pa-pb1 inter-subunit interface [231] can serve as potential targeting sites for new drug design. coumarin compounds, including eleutheroside b 1 , isofraxidin, fraxin, esculetin, fraxetin, and scoparone, were investigated for their antiviral and anti-inflammatory activities against influenza virus in vitro [31] . other candidates, such as naproxen, a non-steroidal anti-inflammatory drug, was shown to target np protein at residues f209 and y148, thus antagonizes the crm1-mediated nuclear export of np. it is suggested to have a broad-spectrum anti-influenza activity [232] . verdinexor (kpt-335), a novel orally bioavailable drug, blocks crm1-mediated nuclear export of np and repress nf-κb activation, thus reducing cytokine production and eliminating virus-associated immunopathology [233] . for further reading on candidate anti-iv therapeutics, a review summarized by davidson is recommended here [234] . with the increasing knowledge obtained through massive investigations on host immunity against iav infection, promoting host immune responses not limited to antibody enhancement would have good prospects not only for vaccine design, but also for development of novel antiviral agents. author contributions: manuscript preparation, y.z.; revision, z.x.; supervision, y.c.; funding acquisition, y.z. all authors read and approved the final version of the manuscript. funding: this study was supported by the "zhujiang talent program" overseas youth talent introduction program (post-doctoral program) and doctoral initiative project of natural science foundation of guangdong province (18zxxt49). the authors declare that they have no financial and personal relationships with other people or organizations that can influence the work. there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in this review. they do not have any commercial or associative interest that represents conflicts of interest in 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market emerging antiviral strategies to interfere with influenza virus entry discovery of the first series of small molecule h5n1 entry inhibitors favipiravir (t-705), a novel viral rna polymerase inhibitor favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase synthesis, structure activity relationship and anti-influenza a virus evaluation of oleanolic acid-linear amino derivatives design, synthesis of oleanolic acid-saccharide conjugates using click chemistry methodology and study of their anti-influenza activity design, synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors the inducible amphisome isolates viral hemagglutinin and defends against influenza a virus infection discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (vx-787) of influenza pb2 a novel endonuclease inhibitor exhibits broad-spectrum anti-influenza virus activityin vitro identification and characterization of influenza variants resistant to a viral endonuclease inhibitor polymerase acidic protein-basic protein 1 (pa-pb1) protein-protein interaction as a target for next-generation anti-influenza therapeutics naproxen exhibits broad anti-influenza virus activity in mice by impeding viral nucleoprotein nuclear export verdinexor targeting of crm1 is a promising therapeutic approach against rsv and influenza viruses treating influenza infection, from now and into the future key: cord-296890-08kqtw8s authors: toh, teck-hock; hii, king-ching; fieldhouse, jane k; ting, jakie; berita, antoinette; nguyen, tham thi; wong, see-chang; wong, toh-mee; lim, wei-honn; ha, siaw-jing; lau, chuet-zou; kong, sing-ling; bailey, emily s; warkentien, tyler e; husain, tupur s; gray, gregory c title: high prevalence of viral infections among hospitalized pneumonia patients in equatorial sarawak, malaysia date: 2019-02-13 journal: open forum infect dis doi: 10.1093/ofid/ofz074 sha: doc_id: 296890 cord_uid: 08kqtw8s background: although pneumonia is a known cause of morbidity and mortality in sarawak, malaysia, the etiology and epidemiology of pneumonia are not well described in this equatorial region. routine clinical diagnostics for pneumonia etiology at government hospitals in sarawak had historically involved only bacterial diagnostics. viral diagnostics were only obtained through outside consultations. methods: from june 15, 2017 to may 14, 2018, we collected nasopharyngeal swabs from 600 patients of all ages older than 1 month hospitalized with pneumonia at sibu and kapit hospitals. specimens were examined at our collaborating institutions with a panel of molecular assays for viral pathogens including influenza a (iav), ibv, icv, and idv, human adenovirus (adv), human enterovirus (ev), human coronavirus (cov), respiratory syncytial virus subtype a (rsv-a) or rsv-b, and parainfluenza virus (piv) types 1–4. results: of 599 samples examined, 288 (48%) had molecular evidence of 1 or more respiratory viruses. overall, the most prevalent virus detected was rsv-a (14.2%) followed by adv (10.4%) and iav (10.4%), then rsv-b (6.2%), ev (4.2%), ibv (2.2%), piv-3 (1.7%), cov (1.0%), piv-1 (1.0%), piv-4 (0.7%), and piv-2 (0.2%). no specimens were confirmed positive for icv or idv. conclusions: the high prevalence of viruses detected in this study suggest that respiratory viruses may be responsible for considerable morbidity in equatorial regions such as sarawak. access to viral diagnostics are very necessary for medical staff to determine appropriate pneumonia treatments. as viral respiratory tract infectious diseases such as avian influenzas and severe acute respiratory syndrome coronavirus have emerged as increasing threats to global health security, international stakeholders such as the world health organization and the united nations international children's emergency fund (unicef) are calling for increased global surveillance for emerging and re-emerging respiratory viruses [1] [2] [3] . locallevel surveillance systems are critical to understanding and monitoring the epidemiology of severe respiratory disease. understanding the etiology and epidemiology of pneumonia will guide antiviral interventions as well as enhance global emerging infectious disease preparedness. pneumonia is the second leading cause of death among children under the age of 5, resulting in more childhood deaths than diarrhea and malaria combined, with more than 95% of those cases occurring in low-and middle-income countries [4, 5] . a 2015 meta-analysis of the global burden of childhood pneumonia found the greatest proportion (39%) of severe pneumonia cases occurred in southeast asia [6] . however, because streptoccocus pneumoniae is the most common cause of vaccine-preventable severe pneumonia, much of the literature has focused on bacterial causes of pneumonia or antibiotic resistance, whereas fewer studies have prospectively investigated the viral etiology of pneumonia in southeast asia [7] [8] [9] . one study of respiratory samples collected from children living in kuala lumpur under 5 years of age between 1982 and 2008 found that 26.4% of the samples were positive by immunofluorescence assays and viral cultures for viral pathogens, with a prevalence of 18.6% for respiratory syncytial virus (rsv), 3.5% for parainfluenza viruses (pivs), 2.9% for influenza viruses, and 1.37% for adenovirus [10] . during the past 5 years, the directors of 2 government hospitals (sibu hospital and kapit hospital) in their namesake cities in sarawak, malaysia, have seen increasing pneumonia admissions (see supplementary figure 1 kapit hospital serves a smaller population of approximately 130 800 residents living along the rejang river [11] , 120 kilometers east and inland from sibu. the hospital has similarly experienced a recent increase in pneumonia admissions, estimated at more than 300 per year. before this study, routine clinical diagnostics for pneumonia etiology at both sibu and kapit hospitals involved chiefly blood culture and gram stains of endotracheal secretion, if intubated, to detect bacteria. molecular and immunofluorescence laboratory diagnostics were only ordered for severe cases, for which clinicians had to send specimens to a specialized virology laboratory in kuala lumpur, malaysia or the university centre in kuching, sarawak. the overall objective of this study was to examine the viral etiology of and risk factors for pneumonia among patients admitted to sibu and kapit hospitals between june 2017 and may 2018 and, in doing so, to assist malaysian collaborators with setting up sustainable real-time molecular assays for viral respiratory pathogens. study enrollment took place between june 2017 and may 2018 at sibu and kapit hospitals. the major ethnic groups in sarawak are iban, chinese, malay, melanau, bidayuh, and orang ulu. as of 2016, approximately 37.8% of the population in sibu was iban and 60.7% of the population in kapit was iban [11] . as previously described [12] , we adapted inclusion and exclusion criteria from 2 united states, large and comprehensive, community-based pneumonia studies published in 2015 [13, 14] . all patients older than 30 days admitted to sibu or kapit hospitals and diagnosed with pneumonia by an attending physician were considered for study eligibility. a medical officer (mo) evaluated eligible subjects for inclusion and exclusion criteria, including confirmation by chest radiography within 72 hours of hospitalization (see supplementary table 1 ). adults 18 years of age or older provided written consent, whereas children ages 7 to 18 provided written assent along with written parental or guardian consent. written consent was obtained from all parents or guardians of children under the age of 7. the study received a scientific review, and all procedures followed were in accordance with the ethical standards of the although this pilot study had sparse sarawak baseline virus prevalence data from which to calculate sample size, we approximated sample size based upon an estimated prevalence of influenza a (iav) causing 30% of pneumonias. hence, if we enrolled 600 pneumonia patients, we could be confident to calculate an estimated prevalence within 3.8% of the true prevalence of iav. from june 15 to july 27, 2017, the study team relied on convenience sampling to enroll as many patients as possible. for the remaining 10 months of the study, mos enrolled patients on 2 of 3 randomly selected days of the week, which were communicated to them by a study coordinator. after obtaining written assent and/or consent, mos administered a brief questionnaire. the questionnaire captured demographic information (age, gender, ethnicity) along with household size (number of cohabitants) and contact with animals within the last 30 days. in addition, all pre-existing health conditions and current medications were self-reported by the subject or their parent/guardian during the questionnaire, then confirmed by the mo through a review of the patient's medical records. the mo then used a flocked swab to collect 1 nasopharyngeal (np) swab from the subject's nose, which was quickly placed into a viral transport tube containing 3 ml sterile viral transport medium (bd universal viral transport; becton, dickinson and company, franklin lakes, nj). all specimens were stored at −80°c at the sibu hospital clinical research center (shcrc) until ribonucleic acid or deoxyribonucleic acid extraction was performed using the qiamp cador pathogen mini kit (qiagen, hilden, germany). specimens collected in kapit were stored at −80°c until they could be transported to sibu for processing. real-time reverse-transcription polymerase chain reaction (rrt-pcr) or real-time pcr (rpcr) was conducted on similar biorad cfx96 c1000 touch thermal cycler real-time systems at shcrc, duke-nus in singapore and at duke university in durham, north carolina. all np swabs were examined for molecular evidence of influenza a (iav), ibv, icv, and idv, human adenovirus (adv), human enterovirus (ev), human coronavirus (cov), rsv subtype a (rsv-a) and rsv-b, and piv types 1-4 (piv-1, -2, -3, and -4). primer and probes sequences for targeted pathogens are recorded in supplementary table 2. a positive control and a no-template control were included in each run. all specimens were first run at shcrc. one-milliliter aliquots of these specimens were later shipped on dry ice to either duke-nus or duke university for validation. laboratory staff at duke-nus and duke were blind to the results of the assays at shcrc. cycle threshold (ct) values <38 were considered positive, and ct values 38 to 40 were considered suspect to acknowledge that positivity could be the result of cross-reactivity or nonspecific amplification. cycle threshold values >40 were considered negative. all discrepant assays were noted and repeated at both shcrc and either duke or duke-nus, using the same original assays, and discrepancies were thus resolved. partial genome sequencing was also performed for several specimens to validate discrepant results. questionnaire data and molecular results were entered into redcap version 7.0 and verified at shcrc and at duke. data were imported into stata version 15.0 (statacorp, college station, tx), cleaned, and categorized for statistical analyses. we categorized continuous variables based on the distribution of the counts for household size (quartiles) and for age (quartiles). after examining age quartiles, we rounded age categories into approximate age quartiles so that age was treated as a whole number. demographic and clinical risk factors were examined for bivariate associations with the most prevalent positive molecular assays. pearson's χ 2 test or fishers exact test were used for bivariate work. risk factors with a bivariate test statistic p ≤ .1 were included in stepwise, manual, backward-elimination, unconditional logistic regression models. risk factors with p < .05 were retained in final models, and odds ratios (ors) and 95% confidence intervals (cis) were calculated. a total of 600 hospitalized pneumonia patients were enrolled at sibu and kapit hospitals between june 15, 2017 and may 15, 2018, with 389 of the subjects enrolled at sibu hospital (64.8%) and 211 enrolled at kapit hospital (35.2%). of the enrolled subjects, 325 (54.2%) were male. a total of 385 (64.2%) enrolled subjects were children 5 years of age or younger and 439 (73.2%) were of age 18 years and younger. the majority of participants identified as iban, with 253 (65.0%) iban subjects enrolled at sibu hospital and 188 (89.1%) iban subjects enrolled at kapit hospital (table 1) . of the 600 np swabs collected, 599 were run using rrt-pcr/ rpcr; 1 np swab collected from a pediatric patient at sibu hospital was accidentally destroyed before molecular screening. one or more viruses were detected by rrt-pcr/rpcr in 48.1% of the 599 samples testing positive or suspect positive for either iav, ibv, icv, idv, adv, ev, cov, rsv-a, rsv-b, piv-1, piv-2, piv-3, or piv-4. the prevalence of each pathogen out of the total number of patients enrolled by month is shown in figure 1 . we detected coinfections in 42 of the 275 patients with evidence of viral infection ( table 2) . of the coinfected specimens, adv was detected in approximately 65% (n = 27) with the most common coinfection combination being adv and rsv (9 adv/ rsv-b coinfections and 6 adv/rsv-a coinfections) followed by 7 adv/ev coinfections. respiratory syncytial virus subtype b was the second most prevalent virus among coinfections (n = 15); in addition to coinfections with adv-positive specimens, rsv-b was also detected in specimens found positive by rrt-pcr for rsv-a, piv-1, ev, iav, and ibv. one pediatric patient's specimen was positive for 3 viruses (rsv-b, adv, and ev). overall, the most prevalent virus detected was rsv (table 3) , with 122 samples testing positive for either rsv-a or rsv-b (overall prevalence of 20.4%; prevalence of 26.9% among children <18 years). a total of 85 specimens tested positive by rrt-pcr for rsv-a for an overall prevalence of 14.2%. an additional 7 specimens were suspect-positive for rsv-a (ct values ranged 38.3-39.7); however, suspect-positive specimens were not included in the final analyses. eighty-two of the rsv-a-positive samples were from children ≤5 years age 5, totaling a prevalence of 21.4%. a total of 37 (6.2% prevalence) specimens were positive for rsv-b by rrt-pcr, with 2 additional suspect-positive specimens (ct values ranged 38.9-39.6). thirtyfive of those specimens were collected from children <18 years (8.0% prevalence). after rsv, adv and iav were the most prevalent, each with 62 positive specimens detected by rpcr and rrt-pcr, respectively, for an overall prevalence of 10.4%. fifty-seven of the adv specimens were collected from children <18 years (13% prevalence), and 46 of the iav specimens were collected from children <18 years (10.5% prevalence). a lower prevalence (2.2%) of ibv was detected, with 13 ibv-positive specimens, 9 of which were collected from children <18 years of age. twenty-five specimens were positive for ev with a 4.2% prevalence overall and 5.3% prevalence among children <18 years. six specimens were positive by rrt-pcr for cov. among the piv types, piv-3 was the most prevalent, with 10 specimens testing positive for piv-3, followed by 6 piv-1 detections, 4 piv-4 detections, and 1 piv-2 detection. no specimens were determined to be positive for icv or idv; however, there were 2 suspect-positive idv samples (ct values ranged 38.2-39.3), which we were unsuccessful at isolating at duke university. one patient of the 600 enrolled was pregnant and tested positive for iav. three severe adverse events were reported to the malaysian ethics board for participants who died after enrollment. all 3 deaths were pediatric patients under 1 year of age with negative blood cultures. human adenovirus was detected in the np swab specimens of 2 of those patients, 1 of whom had an endotracheal tube culture that grew enterobacter aerogenes before death. respiratory syncytial virus subtype a was detected in the third patient's np swab. no coinfections were detected among these patients. due to low counts, cov, icv, idv, and piv1-4 disease outcomes were not included in the risk factor analysis. across the 6 disease outcomes examined (rsv-a, rsv-b, iav, ibv, ev, and adv), gender was not found as a statistically significant risk factor in the initial bivariate screenings. additional potential risk factors such as pre-existing diseases, treatment history, and animal contact defined as touched or come within 1 meter in the last 30 days. c other animals include the following: cow, rats, rabbits, snake, and monkey. ethnicity were eliminated in the stepwise, backward-elimination multivariate modeling. the month of enrollment was a statistically significant risk factor for most virus outcomes, including rsv-a, rsv-b, iav, and adv (tables 4 and 5 and supplementary tables 3 and 4 ). respiratory syncytial virus subtype a was most prevalent during the first month of enrollment, june-july 2017 with an adjusted or of 7.2 (95% ci, 1.6-33.5) compared with rsv-a detection by rrt-pcr in november-december 2017 (table 4 ). in contrast, there was a very low detection of rsv-b in june-july 2017 (see supplementary figure 2 ). compared to june-july 2017, rsv-b had an increased adjusted or beginning mid-february, with an adjusted or of 23.1 (95% ci, 2.9-185.6) between april 15 and may 14, 2018 (table 5) . we detected adv in over 30% of the 72 specimens collected from april 15 to may 14, 2018. compared to the month of october-november 2017, there was an increased adjusted or of 16.9 (95% ci, 2.1-133.4) of adv infection during the month of april-may. the only disease outcome for which location of hospitalization (kapit versus sibu) was statistically significant was ev; patients with specimens found positive for ev had a 2.4 higher or (95% ci, 1.1-5.5) of being enrolled at kapit hospital compared with sibu hospital (see supplementary table 5) . age was a statistically significant risk factor for rsv-a, rsv-b, and adv, with pediatric patients ages 1 month to 1 year and 1 to 2 years having increased adjusted or compared with patients >18 years of age. detection of rsv-a and rsv-b was associated with quartiles of household size (number of additional cohabitants) (tables 4 and 5 ). the only animal exposure that was statistically associated with a disease outcome was cat exposure for ibv, with those patients reporting coming into contact with a cat within 1 meter in the last 30 days having an or 5.4 times higher (95% ci, 1.8-16.9) than those patients who did not have contact with a cat. sarawak, located on the northwest side of the island of borneo, has an equatorial climate with a high relative humidity that rarely drops below 80% and temperatures ranging from 23°c to 32°c (see supplementary figure 2 ). both sibu (2.2873°n, 111.8305°e) and kapit (1.9951°n, 112.9331°e) are approximately 2 degrees north of the equator (supplementary figure 1) . the rainy season in this region typically falls between november and february, with december to march seeing the most rain. a drier season typically falls between may and september, with june to august seeing the least rain. our previous studies have investigated the relationship between relative humidity or absolute humidity and influenza virus transmission, suggesting that increased levels of humidity are linked with decreased transmission efficiency; however, the prevalence of iav in sibu and kapit hospitals suggest the high humidity associated with the equatorial climate in this region may not constrain iav transmission [15, 16] . our findings regarding the prevalence of rsv support the existing literature that the 2 antigenic subgroups a and b can cocirculate but typically differ by season [17] . unlike iav, we saw seasonal variation of rsv-a and rsv-b during the drier months (see supplementary figure 3) . the year-long data found a lower prevalence of rsv overall than an earlier analysis of the data collected during the first 2 months of the study, june and july 2017; the logistic regression model from the pilot study also found that hospital location was a risk factor for rsv-a infection, with patients at kapit hospital having a higher adjusted or (adjusted or = 3.2; 95% ci, 1.3-7.8) of testing positive for rsv-a compared with patients hospitalized at sibu hospital [12] . the finding that age was a statistically significant risk factor for rsv infection is consistent with the epidemiology of this virus. in the year-long study, we found a slightly elevated odds ratio of rsv-a and rsv-b infection among children ages 1 to 2 years (rsv-a adjusted or = 33. 8 after the initial statistical analysis, pediatric data were stratified and analyzed for the outcome of rsv-a (see supplementary table 6 ). in the stratified analysis, the same risk factors, including age quartile, household size, and month of enrollment, were found to be risk factors for rsv-a infection. we speculate that the high or (5.4; 95% ci, 1.8-16.9) observed for the association between cat exposure and ibv (see supplementary table 7) may have occurred by chance. children <18 years adults ≥18 years denominator is total number of specimens examined by rrt-pcr and rpcr for viruses, n = 599. when considering the number of cohabitants in the household of a subject testing positive for rsv-a, q4 (≥8) had a slightly increased or compared with q3 (6 to 7 cohabitants). the same was true when the analysis was run on the stratified data for pediatric subgroups (see supplementary table 5 ). in contrast, for rsv-b infection, q1 (0 to 3 cohabitants) and q2 (4 to 5 cohabitants) had slightly increased or compared with q3 (4.2 [95% ci, 1.1-16.7] and 4.1 [95% ci, 1.1-15.2], respectively). the mean household size among all enrolled subjects was 5.7 people with a range of 0 to 30 persons. household size was self-reported and did not capture the type of household (ie, single-family home or traditional longhouse). the study had a number of limitations. although the study offered the questionnaire in 3 different languages to ensure comprehension, it is possible that questions about behavior were not accurately understood. after the first 2 months of study, patients were supposedly recruited on 2 randomly selected days of the week; however, we cannot eliminate the possibility that mos selected the sicker patients in the ward for this study. in addition, this study was limited to hospitalized patients and therefore excluded milder cases of pneumonia not requiring hospitalization. although we used rigorously validated real-time pcr assays as the gold standard for virus detection, the study was limited to a panel of 13 viruses and we therefore likely missed other important pathogens present in specimens, including human metapneumovirus, rhinovirus, and bacteria. specifically, this study would benefit from an understanding of the prevalence of bacterial mono-or coinfections within the patient population. this study provides data on 1 year of pneumonia admissions at 2 district government hospitals in sarawak, malaysia; however, prevalence of the viruses investigated can vary between years. nevertheless, this study is one of the largest viral pneumonia etiology studies of its kind conducted in southeast asia. the findings led investigators to extend the study for an additional year, and a subset of 400 samples will be analyzed at duke university for rhinoviruses a, b, and c. through this study we found a viral etiology for a relatively high proportion (48.1%) of pneumonia cases, demonstrating the need for these hospitals to gain clinical diagnostics for viral pathogens. this will help hospital medical staff appropriately administer antiviral treatment for illnesses, such as iav and ibv, and avoid inappropriate use of antibiotics for pneumonia with a viral etiology. the findings from this study may assist the hospitals in their preparedness for future pneumonia admissions when new antivirals and vaccines are more readily available. whereas current therapies for rsv infection are largely supportive, as of 2018 there are more than 50 ongoing rsv vaccine trials and 45 rsv antiviral drug trials currently being evaluated, suggesting that new prevention and treatment mechanisms may be on the horizon [2] . the risk factor analysis of age categories and rsv-a detection also provides evidence to support and encourage breastfeeding among newborns. in a 2018 commentary on pneumonia published in the lancet, watkins and sridhar [18] describe the multifold factors limiting global action on pneumonia, including the challenges that arise from the complexity of pneumonia due to its multiple etiologies and consequentially diverse treatments. accurate diagnosis of pneumonia will not only allow clinicians to prescribe better treatment for patients, but it will also contribute to knowledge regarding the respiratory viruses most likely to cause illness in various age groups and communities. given the high prevalence of viruses detected among the patients enrolled in this study, routine surveillance and more sensitive molecular-based assays for respiratory viruses are recommended for hospitals in sarawak, malaysia and similar equatorial climates. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. potential conflicts of interest. t one pediatric patient specimen destroyed, assay results out of n = 599. surveillance for emerging respiratory viruses global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance addressing the public health burden of respiratory viruses: the battle against respiratory viruses (brave) initiative estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis global burden of childhood pneumonia and diarrhoea clinical and economic burden of community-acquired pneumonia amongst adults in the asia-pacific region the bacterial aetiology of adult community-acquired pneumonia in asia: a systematic review assessing the burden of pneumonia using administrative data from malaysia, indonesia, and the philippines epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years surveillance for respiratory syncytial virus and parainfluenza virus among patients hospitalized with pneumonia in sarawak community-acquired pneumonia requiring hospitalization among u.s. children community-acquired pneumonia requiring hospitalization among u.s. adults humidity as a non-pharmaceutical intervention for influenza a absolute humidity modulates influenza survival, transmission, and seasonality respiratory syncytial virus-a comprehensive review pneumonia: a global cause without champions we thank the medical officers of sibu and kapit hospitals (namely, nga-hung ngu, khai-fatt chao, cheng-ing kong, zhen-hao chin, edmund kwang-yuen wong, tiana ti, and hilary hon-yun kueh) for enrolling patients. we thank the laboratory staff at kapit hospital (namely, cornelius jambol, goh hieng hua, and velarie bill) and tiing-tiing chua at sibu hospital for processing specimens. we thank dr. cheesan lee for helping us to begin this collaboration. we thank jessica choi, sarah philo, kerry mallinson, sarah paust, calvin wang, and christine wang for laboratory support. we thank dr. larry park for his support in the statistical analysis. this work was conducted in partnership with duke university, the duke global health institute, sibu hospital clinical research center, and segi university sibu clinical campus. we thank the director general of health, malaysia for his permission to publish this paper.disclaimer. the views expressed in this article are those of the author(s) and do not necessarily reflect the official policy or position of the department of the navy, department of defense, or the united states government.financial support. this work funded by the us naval medical research center-asia and vysnova partners (sc-2016-saber-003-002, sc-2017-saber-010-001) and professor gregory gray's discretionary funds from duke university's global health institute. key: cord-275821-yu39aw54 authors: ciminski, kevin; thamamongood, thiprampai; zimmer, gert; schwemmle, martin title: novel insights into bat influenza a viruses date: 2017-09-14 journal: j gen virol doi: 10.1099/jgv.0.000927 sha: doc_id: 275821 cord_uid: yu39aw54 in 2012 and 2013, influenza virus genome sequences of two new influenza a virus (iav) subtypes were discovered in bat specimens, but further characterization was largely impeded by the lack of infectious virus. with the identification of highly susceptible cell lines, reconstitution of infectious bat iav by reverse genetics recently succeeded and allowed a first insight into the life cycle of these viruses. although there is a certain degree of functional compatibility between bat and conventional influenza a virus proteins, there are striking differences, including receptor usage, polarity of infection and reassortment potential. influenza a viruses (iavs) are the causative pathogens of annual epidemics and sporadically occurring pandemics responsible for substantial morbidity and mortality in the human population [1] . the main antigenic determinants of iav are the surface glycoproteins haemagglutinin (ha) and neuraminidase (na). based on the gene homology and antigenic properties of ha and na, iavs have been classically subdivided into 16 different ha (h1 to h16) and nine different na subtypes (n1 to n9) [2] . all known iav subtypes have been found in aquatic waterfowl, which is therefore thought to represent the natural iav reservoir [3] . nevertheless, avian iavs (aivs) have recurrently crossed species barriers thereby establishing new lineages in a wide variety of hosts, including sea mammals, domestic animals such as pigs and horses, domestic poultry, and humans [3] . here, migrating aquatic birds are of particular importance for the emergence and evolution of novel influenza viruses with zoonotic potential, since they carry all known iav subtypes and can disseminate aiv over long distances [3, 4] . currently, the iav subtypes h1n1 and h3n2 are circulating within the human population, causing annual epidemics. however, there is some concern that aiv subtypes such as h5n1 and h7n9 may adapt to the human species [5, 6] . since the human population is immunologically naïve with respect to these 'foreign' ha subtypes, successful establishment of a new lineage in the human population may eventually result in a pandemic associated with high mortality and morbidity. furthermore, as iav is a segmented negative-strand rna virus, co-infection of pigs with avian, human and/or porcine iav could lead to the emergence of new iavs harbouring a novel constellation of the eight genome segments [7] [8] [9] [10] . these so-called reassortant viruses have a high zoonotic potential, as recently exemplified by the 2009 pandemic h1n1 virus [7] . the previous understanding of the iav host range and diversity has recently been challenged by the discovery of two novel influenza subtypes in central and south american bat species. in 2012, a new influenza virus genomic sequence was identified in frugivorous yellowshouldered bats (sturnira lilium) in guatemala and provisionally designated as h17n10 [11] . only one year later, a distinct influenza genome was isolated from the flatfaced fruit bat (artibeus planirostris) in peru and initially classified as h18n11 [12] . serological surveys in central and south american bat populations revealed a broad prevalence of h17/h18-specific antibodies [12] , suggesting a widespread circulation of these viruses in different bat species. unfortunately, isolation of infectious virus from various bat tissues failed, greatly impeding further characterization of these new iav subtypes. however, phylogenetic analysis of these bat-derived influenza virus genomes indicated that most genes were most closely related to those of conventional iav [11, 12] . notable exceptions were the putative bat-derived ha and na genes, which showed only low homology with their counterparts from other iavs. as outlined below in detail, biochemical data have convincingly demonstrated that the surface glycoproteins of both h17n10 and h18n11 are strikingly different from conventional iav since they lack the canonical receptor-binding and receptor-destroying activities of conventional ha and na, respectively [12] [13] [14] [15] [16] . based on these marked differences, we suggested renaming the bat iav surface glycoproteins as ha-like (hl) and na-like (nl) [17] . the identification of bat iav expanded the host reservoir of iav and immediately raised the question of their zoonotic potential. only recently, it became possible to reconstruct infectious bat iav from synthetic dna using reverse genetic approaches [18] . in this review, we recapitulate recent findings on cell tropism, entry processes and the ability of these bat iavs to exchange genetic information with conventional iav. the iav genome comprises eight single-stranded viral rna (vrna) segments in negative polarity, each encapsidated by multiple copies of the viral nucleoprotein (np) and terminally bound by the viral rna polymerase, forming the viral ribonucleoprotein (vrnp) complex [19] [20] [21] [22] . all vrna segments share a similar structure in that they encompass a central coding region, flanked on both sides by short non-coding regions (ncr). located at the extreme 3¢ and 5¢ genome ends, 12 and 13 nucleotide-long complementary sequences are highly conserved between different iav segments and serve as promoters for the viral polymerase [23] [24] [25] [26] . in cells co-infected with different parental iavs, exchange of viral genome segments can give rise to iav with a new genetic composition, a process called reassortment [7, 9, 10, 27] . the exchange of genome segments between iavs allows rapid adaption to new host environments and is known to be responsible for the emergence of several pandemic iav strains in the past [7, 8] . an important prerequisite for genetic reassortment is the functional compatibility of the parental viral proteins [28] . however, segmentation of the viral genome concurrently complicates vrnp packaging as it demands a highly sophisticated mechanism that coordinates the selective packaging of the eight distinct vrnps into viral progeny particles [23] . although the precise mechanism of iav genome packaging into progeny virions has not been conclusively clarified yet, there is considerable evidence that each vrna segment bears so-called packaging signals within the 3¢ and 5¢ ncr together with adjacent parts of the vrna coding region [29] . it is believed that the packaging signals mediate the formation of a bundle of eight different vrnps by forming rna-rna or rna-protein interaction networks [30] [31] [32] [33] . bat iavs exhibit the characteristic genome structure of conventional iavs. they are composed of eight vrna segments in anti-sense orientation including terminal ncrs. likewise, the highly conserved complementary promoter sequences comprising 12 and 13 nucleotides at the extreme 3¢ and 5¢ ends of each bat genome segment are almost identical to those found in conventional iavs [11, 12] . moreover, with the exception of the envelope glycoprotein homologues ha, na and m2, the amino acid sequences of the remaining bat virus proteins are akin to known iav proteins reaching 50-80 % identity, depending on the protein sequence analysed. to determine the reassortment potential between bat and conventional iav, chimeric bat viruses were engineered containing six bat gene segments from either hl17nl10 or hl18nl11 virus, along with the iav ha and na genes from the prototypic iavs a/puerto rico/8/1934 (h1n1) (pr8), a/swine/texas/4199-2/1998 (h3n2) or a/sc35m (h7n7) [34, 35] . these chimeric viruses were shown to efficiently replicate in various cell culture models and in mice. of note, generation of these bat chimeric viruses by reverse genetics was only possible if the ha and na coding regions were flanked with the authentic bat iav ncr and parts of the coding region of the corresponding segment, already suggesting genome-packaging incompatibilities between bat influenza rna segments and those of conventional iav [34, 35] . indeed, co-infection experiments confirmed that bat chimeric viruses failed to reassort with the conventional iav subtypes h1n1, h3n2 and h7n7. however, gene reassortment was observed after co-infection with both hl17nl10-and hl18nl11-based bat chimeric viruses (fig. 1a ) [35] . in contrast to co-infection experiments, reverse genetics rescue experiments using the eight genome segments resulted in a significant higher number of reassortment events, since competition of homologous vrna segments from different viruses for genome packaging was avoided. this might also explain the successful generation of recombinant hl17nl10-based bat chimeric reassortant viruses harbouring the ha and na surface protein genes of pr8 plus an unmodified m segment from either pr8, a/swine/texas/4199-2/98 (h3n2) or a/ swine/kansas/11-107824/2011 (h3n2) [36] . these reassortant viruses replicated efficiently in various cell lines and in the lungs of infected mice. based on these findings, it is likely that the m segment packaging signals are at least partially compatible between bat and conventional iav. however, the failure of bat chimeric viruses to reassort with conventional iav was also partly due to an incompatibility between the internal proteins. whereas np of bat virus origin, and to a certain degree also the polymerase subunit pb2, supported the polymerase activity of the h1n1, h2n2, h3n2, h5n1 and h7n9 subtypes in polymerase reconstitution assays, pb1 and pa did not [34, 35, 37] . in addition, as the bat iav non-structural protein 1 (ns1) was previously shown to share the dsrna-binding property and ifn-suppression characteristics of conventional iav ns1s [38] , an infectious recombinant pr8 virus encoding the ns1 but not nep gene of hl17nl10 could be generated [39] . thus, the combination of both incompatible packaging sequences and incompatible viral proteins has likely contributed to the failure to exchange genetic information between bat and conventional iavs. surprisingly, although np of bat iav fully supported the polymerase activity of conventional iav of various subtypes, including h7n7, in a polymerase reconstitution assay, the generation of recombinant h7n7 viruses encoding bat iav np repeatedly failed, despite flanking the bat iav np coding region with authentic packaging sequences. however, partial substitution of h7n7 np amino acid residues with the corresponding ones from hl17nl10 np was tolerated and allowed the generation of viable h7n7 np mutant viruses. unexpectedly, these np amino acid substitutions caused impaired viral infectivity due to an uncoordinated packaging of genomic segments into viral particles [40] . mutational studies further revealed that as few as seven amino acid substitutions are sufficient to cause these packaging defects. depending on whether the highly conserved h7n7 np amino acids of the head or the body domain had been substituted with bat iav amino acids (fig. 1b) , the incorporation of specific rna genome segment subsets was differentially affected (fig. 1c) [40] . this is the first indication that a viral protein can specifically recognize vrna packaging signals, suggesting a complex interplay between np and the vrna packaging signals. therefore, the lack of reassortment between bat and conventional iav can be ascribed to the incompatibility of the vrna packaging signals and np. the vrna packaging signals and the np proteins of the exchange of genomic segments is known to take place in cells co-infected with different conventional iav subtypes. likewise, based on experiments with chimeric bat influenza viruses, genomic reassortment is believed to occur in cells co-infected with the known bat iav subtypes. however, reassortment between conventional and bat iavs is blocked, probably due to incompatibility of the vrna packaging signals and np. (b and c) highly conserved amino acids located in either the head (grey) or body (black) domain of a conventional iav np of the h7n7 subtype (a/sc35m) were partially substituted with the corresponding amino acids present in the hl17nl10 np (b). bat-specific amino acids introduced in the head or body domain are highlighted in pink or yellow, respectively. h7n7 viruses encoding these np mutants demonstrated a genome packaging deficiency (c). the incorporation of different rna segment subsets into viral particles was differentially affected, depending on whether the mutations were introduced into the head or body domain. the relative ratio of genome subsets identified in viral particle preparations is shown (raw data from moreira et al. [40] ). genome levels of wild-type (wt) h7n7 were set to 1. column colours correspond to the amino acid substitutions in either the head or body domain. bat and conventional iav might have evolved differentially in their respective hosts and probably have undergone co-evolutionary adaptation resulting in an optimal interaction of the eight distinct vrnps during the genome packaging process. hence, reassortment of the newly discovered hl17nl10 and hl18nl11 subtypes with conventional iav and emergence of pandemic reassortant viruses seems rather unlikely. the initial failure to generate infectious bat iavs from synthetic dna by reverse genetics was suspected to be related to the use of non-susceptible cell lines. this problem has recently been overcome by using vesicular stomatitis virus (vsv) encoding either hl-17 or hl-18 (vsv-hl) in place of the vsv-g protein [18] . screening of more than 30 cell lines from various species using these chimeric viruses led to the identification of madin-darby canine kidney type ii (mdck ii) cells as being most susceptible, whereas the related mdck i cell line turned out to be resistant to infection. in agreement with this observation, both recombinant hl17nl10 and hl18nl11 could be successfully propagated in mdck ii but not mdck i cells. however, both mdck cell types, which originated from the kidney of the same dog but differed in their passage history, are well known to support viral growth of conventional iav. subsequent studies with both chimeric vsv and recombinant hl18nl11 revealed differences in the entry mechanism compared to conventional iav. in polarized mdck ii cells, infection by hl18nl11 was initiated most efficiently from the basolateral site (fig. 2) , a property that is shared by other viruses including vsv, hepatitis b virus, hepatitis c virus, adenovirus types 2 and 5, vaccinia virus and measles virus [41] [42] [43] [44] [45] [46] . in contrast, conventional iavs are known to enter polarized epithelial cells via the apical site of the plasma membrane [47] . the preferential basolateral infection of polarized epithelial cells by bat iav suggests that the putative cellular receptor(s) for these viruses are preferentially expressed at the basolateral membrane but absent from the apical plasma membrane of mdck ii cells. at least two human cell lines, u-87 mg glioblastoma and sk-mel-28 malignant melanoma cells, were found to be susceptible to infection with vsv-hl17 and vsv-hl18 as well as with authentic hl17nl10 and hl18nl11 [18] . however, whether this already indicates a zoonotic potential of bat iavs remains to be shown. surprisingly, recombinant bat iavs were unable to infect cell lines from various bat species, including sturnira lilium and artibeus planirostris, which are believed to be naturally infected by these viruses (unpublished data). the reason for this unexpected resistance is unknown, but might be caused by the downregulation of the virus entry receptor(s) upon in vitro culturing of the bat cells. this was previously described for bat coronaviruses, where susceptibility or resistance of bat cell lines correlated with the presence or absence of the cell surfaceexpressed receptor cd26, also known as dipeptidyl peptidase 4 (dpp4) [48] . conventional iavs initiate the infection at the apical site of epithelial cells by receptor-mediated endocytosis after the binding of ha to sialic acid containing oligosaccharides exposed on the host cell surface. following completion of the iav replication cycle, progeny virions are then released from the apical plasma membrane where na facilitates the cleavage of sialic acid residues. in contrast, bat iavs enter polarized epithelial cells preferentially from the basolateral site via endocytosis upon binding of hl to a yet unknown receptor. budding of progeny bat iav virions occurs similarly to conventional iav at the apical plasma membrane. based on the analysis of the primary sequences, classical ha molecules are categorized into two phylogenetic groups, with group 1 comprising subtypes h1, h2, h5, h6, h8, h9, h11, h12, h13 and h16 and group 2 comprising subtypes h3, h4, h7, h10, h14 and h15 [2, 49, 50] . the number of identical amino acids in different ha subtypes varies from 40 to 70 %, and within the same subtype from 80 to 100 %. in contrast, the amino acid sequences of the bat iav hls are highly divergent from all known ha subtypes derived from conventional iavs. while both hl17 and hl18 have only 45 % amino acid identity to the 16 other known ha subtypes, they share 62 % amino acid sequence identity among each other. the primary amino acid sequences and structure analysis of these proteins revealed that these glycoproteins are more closely related to group 1 than to group 2 [11, 12] . despite the low amino acid sequence identity with conventional ha, the overall structures of hls and conventional has are highly similar. the hls also form a homotrimer containing a membrane-proximal stem region and a membrane-distal globular head, which contains the putative receptor-binding site (rbs) [12, 14, 16] . similar to most conventional ha subtypes, hl17 and hl18 harbour a monobasic cleavage site upstream of a highly conserved stretch of hydrophobic amino acids [12, 14, 16] . at this site, the hl proteins are cleaved by serine proteases such as trypsin or human tmprss2, resulting in the formation of two disulfide-linked subunits, hl1 and hl2 [51] . the proteolytic cleavage of hl proteins is essential for bat iav to be infectious. only following proteolytic cleavage were hl proteins able to exhibit low ph-triggered membrane fusion activity. this feature is shared by bat and conventional iavs and suggests that all of these viruses enter host cells by receptor-mediated endocytosis. the hl proteins of bat iavs were found to mediate autonomous propagation of hl-pseudotyped vsv without the need of support by nl proteins [18] . these chimeric viruses were able to propagate in susceptible cell lines such as canine mdck ii and bat indfspt cells, whereas recombinant vsv expressing exclusively the nl protein in place of vsv-g did not replicate autonomously. interestingly, propagation of chimeric vsv was not enhanced when the corresponding nl protein was expressed along with hl. in contrast, chimeric vsv expressing conventional ha was able to propagate only if na was expressed from the same virus genome, in agreement with the function of na as a receptor-destroying enzyme [52] . the important role of hl proteins in the entry of bat iav was subsequently confirmed by neutralizing hl17nl10 and hl18nl11 infection with antibodies specifically directed to hl17 and hl18, respectively [18] . on the contrary, the role of nl in the bat iav replication cycle remains a mystery. the function of this envelope protein obviously deviates from the classical role of conventional nas in virus release and dissemination. the receptor-binding specificity of conventional has has been studied in great detail. ha proteins from avian iavs preferentially bind to a2,3-linked sialic acids, whereas has of human iavs have a preference for sialic acids in a2,6linkage [53] [54] [55] . in principle, all the classical ha rbss consist of two parts, designated as edge and base. the edge portion is formed by three a-helices (the 130-loop, the 190helix and the 220-loop) while the base portion comprises only four conserved residues (y98, w153, h183 and y195), which mediate the binding specificity of ha [56] . intriguingly, the crystal structure of hl17 and hl18 revealed that, in contrast to conventional ha, there is no cavity present which could accommodate sialylated glycans. in conventional iav ha proteins, hydrogen bonds and salt bridge networks formed by the residues d136, q190, h226 and d228 enable strong interactions between the a-helices of the edge [12, 14, 16] . according to studies on the electrostatic surface potential, the putative rbs of hl is highly acidic, making binding to negatively charged sialic acid residues unlikely. consistently, glycan micro-array analysis revealed no detectable binding of recombinant hl17 and hl18 to more than 600 different glycan structures [12, 14] . all available data indicate that hl proteins recognize cellular receptor(s) that are dissimilar from sialic acid, the receptor determinant for conventional iav. interestingly, removal of sialic acids from cell surface glycoconjugates by sialidase treatment somewhat enhanced the infection of mdck ii cells by chimeric vsv-hl18, as well as by recombinant hl17nl10 or hl18nl11 [18] . the precise explanation for this phenomenon is not known, but removal of negatively charged sialic acids from the host cell surface probably facilitated binding of hl proteins to the cell surface by abrogating repulsion between sialic acid residues linked to cellular receptor glycoproteins and the negatively charged amino acids of the putative hl receptor-binding domain. in line with this proposed mechanism, a previous study showed that treatment of susceptible cells with tunicamycin, an inhibitor of n-glycosylation, reduced infection with hl-pseudotyped vsv [57] , suggesting that the unknown cellular receptor is a glycosylated protein. the extraordinary restricted cell tropism of chimeric vsv-hl and recombinant bat iav suggested that the putative cellular receptor(s) that are recognized by hl17 and hl18 are not ubiquitously expressed. nevertheless, they seem to be expressed by cells from different species and tissues [18, 52, 57] . recombinant hl17nl10 and hl18nl11 showed a similar cell tropism suggesting that hl17 and hl18 bind to the same or a very similar receptor. in order to gain comprehensive insight into the entry mechanism of bat iavs and to understand their extraordinary restricted cell tropism, it is imperative to identify the putative receptor(s) that mediate bat iav binding to the host cell surface. the knowledge of susceptible cell lines such as mdck ii and the availability of infectious recombinant bat iav and chimeric vsv-hl will undoubtedly facilitate the identification of receptor molecules. this in turn will advance our understanding of the potential host range and zoonotic potential of bat iavs. na of conventional iav exhibits sialidase (neuraminidase) activity and catalyses the hydrolysis of the glycosidic linkage between a sialic acid residue and the penultimate sugar of oligosaccharides. in this way, na destroys the receptor determinant of conventional iav and facilitates the release of progeny virus. in addition, removal of sialic acid residues from the viral glycoproteins prevents virus aggregation. finally, na may help conventional iav penetrate the sialic acid-rich mucin layers covering respiratory epithelial cells [58] . based on phylogenetic analysis, nine subtypes of na are distinguished and are subdivided into two groups. n1, n4, n5 and n8 belong to group 1 while n2, n3, n6, n7 and n9 are members of group 2 [59] . the na homologues of bat iav are only distantly related to the nas of conventional iav. therefore, these nas were primarily defined as nl10 and nl11 and were proposed to form a new group 3 [60] . conventional nas are type ii homotetrameric membrane proteins with a mushroom-like conformation. the head domain of each na monomer consists of six topologically b-sheets arranged in a propeller-like structure [61] . likewise, the overall three-dimensional structures of nl10 and nl11 share striking similarity with those of classical nas [12, 13, 15] . in addition, a calcium-binding site, which is normally required for the stabilization of the active centrum of conventional nas, is highly conserved in both nl10 and nl11. however, the crystal structures of nl10 and nl11 revealed that the conserved amino acids normally involved in sialic acid binding are not present. most of the amino acid residues required for na activity are substituted and the putative active site is wider because the 150-and the 430-loops have been displaced. in agreement with these observations, sialidase activity is not associated with nl10 or nl11 [12, 13, 15] and no evidence was found for binding of nl10 or nl11 to any carbohydrate structure [12, 15] . consequently, nl proteins might have a unique function distinct from the known sialidase activity of canonical nas. the recent identification of susceptible cell lines and the availability of reverse genetics systems for bat iav will certainly help in identifying the function of nl proteins in the viral life cycle. bats represent a natural reservoir for several pathogens including ebola virus, hendra virus, rabies virus and sars and mers coronaviruses. the identification of two new influenza a-like viruses, hl17nl10 and hl18nl11, has expanded the number of potentially zoonotic iavs. this raises important questions with respect to the origin and evolution of iav and poses public health concerns. recent findings demonstrated that the likelihood of reassortment between conventional iavs and bat iavs is limited. the inability to exchange viral rna segments is restricted by rna packaging signals and a matching np. although the risk of reassortment is low, the potential of adaptation to other mammalian hosts cannot be excluded at present. the reconstitution of synthetic hl17nl10 and hl18nl11 viruses marked a milestone in bat iav research and allowed for the first time the characterization of the mechanisms of virus entry and cell tropism. even though hl molecules play crucial roles in bat iav entry, the replication cycle of bat iavs may not follow the canonical life cycle of conventional iavs. moreover, the ability to infect human cell lines suggests that transmission of these viruses to the human population is still a possibility. the use of recombinant bat iav for experimental infection of bats will provide deeper insight into their tropism and transmission model in the natural host. importantly, identification and characterization of cellular receptors mediating virus entry will undoubtedly lead to clarification of the tissue tropism and zoonotic potential of these viruses. this study was supported by a grant from the deutsche forschungsgemeinschaft to m. s. (sch632/17-1) and the excellence initiative of the german research foundation (gsc-4, spemann graduate school) to t. t. the global impact of influenza on morbidity and mortality influenza hemagglutinin and neuraminidase membrane glycoproteins evolution and ecology of influenza a viruses global patterns of influenza a virus in wild birds biological features of novel avian influenza a (h7n9) virus human infection with a novel avian-origin influenza a (h7n9) virus antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans avian-to-human transmission of the pb1 gene of influenza a viruses in the 1957 and 1968 pandemics on the origin of the human influenza virus subtypes h2n2 and h3n2 potential for transmission of avian influenza viruses to pigs a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses structural and functional characterization of neuraminidase-like molecule n10 derived from bat 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efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide hepatocyte polarization is essential for the productive entry of the hepatitis b virus vaccinia virus entry, exit, and interaction with differentiated human airway epithelia basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellular transport in doubly infected madin-darby canine kidney cells cd26/ dpp4 cell-surface expression in bat cells correlates with bat cell susceptibility to middle east respiratory syndrome coronavirus (mers-cov) infection and evolution of persistent infection comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza a viruses sequence relationships among the hemagglutinin genes of 12 subtypes of influenza a virus the hemagglutinin of bat-associated influenza viruses is activated by tmprss2 for ph-dependent entry into bat but not human cells pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses 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) hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-mhz proton nuclear magnetic resonance study a surface plasmon resonance assay for the binding of influenza virus hemagglutinin to its sialic acid receptor structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution characterization of the glycoproteins of bat-derived influenza viruses influenza a penetrates host mucus by cleaving sialic acids with neuraminidase the neuraminidase of influenza virus bat-derived influenza-like viruses h17n10 and h18n11 structure of the catalytic and antigenic sites in influenza virus neuraminidase we thank quinnlan david for critical reading of the manuscript and hardin bolte for the generation of the figures. the authors declare that there are no conflicts of interest. five reasons to publish your next article with a microbiology society journal 1. the microbiology society is a not-for-profit organization. 2. we offer fast and rigorous peer reviewaverage time to first decision is 4-6 weeks. 3. our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as 'excellent' or 'very good'. 5. your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord-013174-whg64w0w authors: bhatta, tarka raj; ryt-hansen, pia; nielsen, jens peter; larsen, lars erik; larsen, inge; chamings, anthony; goecke, nicole b.; alexandersen, soren title: infection dynamics of swine influenza virus in a danish pig herd reveals recurrent infections with different variants of the h1n2 swine influenza a virus subtype date: 2020-09-10 journal: viruses doi: 10.3390/v12091013 sha: doc_id: 13174 cord_uid: whg64w0w influenza a virus (iav) in swine, so-called swine influenza a virus (swiav), causes respiratory illness in pigs around the globe. in danish pig herds, a h1n2 subtype named h1n2dk is one of the main circulating swiav. in this cohort study, the infection dynamic of swiav was evaluated in a danish pig herd by sampling and pcr testing of pigs from two weeks of age until slaughter at 22 weeks of age. in addition, next generation sequencing (ngs) was used to identify and characterize the complete genome of swiav circulating in the herd, and to examine the antigenic variability in the antigenic sites of the virus hemagglutinin (ha) and neuraminidase (na) proteins. overall, 76.6% of the pigs became pcr positive for swiav during the study, with the highest prevalence at four weeks of age. detailed analysis of the virus sequences obtained showed that the majority of mutations occurred at antigenic sites in the ha and na proteins of the virus. at least two different h1n2 variants were found to be circulating in the herd; one h1n2 variant was circulating at the sow and nursery sites, while another h1n2 variant was circulating at the finisher site. furthermore, it was demonstrated that individual pigs had recurrent swiav infections with the two different h1n2 variants, but re-infection with the same h1n2 variant was also observed. better understandings of the epidemiology, genetic and antigenic diversity of swiav may help to design better health interventions for the prevention and control of swiav infections in the herds. the influenza a virus (iav) is a negative-sense, single-stranded, eight-segmented rna virus belonging to the family orthomyxoviridae [1] . the main antigenic proteins are encoded by the surface gene segments hemagglutinin (ha) and neuraminidase (na). the six internal gene segments encode for polymerase b2 (pb2), polymerase b1 (pb1), polymerase a (pa), nucleoprotein (np), matrix (m1 and m2), and non-structural protein (nep-ns1) respectively [2, 3] . there are 18 different ha (h1 to h18) and 11 different na (n1 to n11) subtypes. most of these subtypes can be found in aquatic birds (h1-h16 and n1-n9), whereas only a few subtypes are found in mammals [1, 4] . in pigs, circulation of iav, so-called swine influenza a virus (swiav), is currently mainly limited to three different subtypes including h1n1, h1n2 and h3n2 [5] [6] [7] . avian-like swine h1n1 swiav, was first detected in european pig herds in the late 1970s [8, 9] and caused epizootic disease outbreaks that resolved shortly within a few weeks [10, 11] . however, several recent studies have shown that the dynamics of swiav have changed to a more enzootic form where the virus persists in the herds for months or even years [12] [13] [14] [15] [16] [17] . the altered dynamics of swiav from a short-term epizootic disease to continuous circulation in the herds, is probably a consequence of increased herd sizes and the continuous supply of naïve individuals that maintain the infection [18, 19] . in european pig herds, an average prevalence of 31% has been estimated for swiav infection [6] . the most prevalent subtypes identified in europe in recent years were the eurasian avian-like swine h1n1 (53.6%), the pandemic a/h1n1 2009 (h1n1pdm09) (10.3%), the human-like reassortant swine h1n2 (13%), and the human-like reassortant swine h3n2 (9.1%) [6] . in addition, a number of studies have also shown evidence of reassortants with internal gene segments of h1n1pdm09 and the surface gene segments of predominant enzootic swiav [20] [21] [22] [23] . in denmark, the human-like reassortant h1n2 has never been detected. however, another h1n2 reassortant is widespread among danish pig herds. this subtype is termed "h1n2dk" and has the ha gene of the eurasian avian-like h1n1 subtype and the na gene of the h3n2 human-like swiav [24] . in addition to the subtypes mentioned above, introductions of human seasonal iav occurs regularly, increasing the risk of novel swiav reassortants, and making the disease more difficult to control in the herds [23] . in addition, similar to other rna viruses, swiav has a high mutation rate, which drives the viral evolution and helps the virus evade the immune system by creating novel variants with modified antigenicity [25] [26] [27] . mutations in the antigenic sites (cb, sa, sb, ca1 and ca2) of the ha protein can lead to the virus being able to escape the binding of neutralizing antibodies [28, 29] . moreover, mutations in b-cell epitopes and t-cell epitopes of other iav proteins might also impact the host immunity [30] [31] [32] [33] . finally, mutations favouring altered n-linked glycosylation (nlg) sites near/within ha and na antigenic sites can also affect the binding of antibodies [34] . according to the danish agriculture and food council, denmark produces approximately 30 million pigs annually from around three thousand herds of which approximately 10 million are exported as weaners to other countries such as poland and germany [35, 36] . in contrast, denmark imports a very limited number of live pigs. in denmark and other countries worldwide swiav is one of the causes of respiratory infection in pigs [37] . swiav infections lead to destruction of the epithelial cells and impairs the immune system, thereby making the host more susceptible to infection by other viruses and bacteria. co-infection of pigs with distinct variants of swiav and other respiratory pathogens (like pasteurella multocida, mycoplasma hyopneumoniae, haemophilus parasuis, actinobacillus pleuropneumoniae, porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus) are known to cause enhanced disease compared to single pathogen infections, and are all part of the porcine respiratory disease complex (prdc), which can lead to massive economic losses [11, [38] [39] [40] . the aim of the present study was to monitor the molecular epidemiology of swiav circulating in pigs between two weeks of age to 22 weeks of age in a danish pig herd. a newly developed high-throughput real-time pcr (rtpcr) system (fluidigm, south san francisco, usa), which consists of 21 rtpcr assays targeting 18 selected respiratory and enteric viral and bacterial pathogens [41] was applied for initial detection of swiav in nasal swab samples. moreover, next generation sequencing (ngs) was used to characterize the complete genome of swiav during natural infections, and to examine the genetic variability in the antigenic sites of the virus ha and na proteins. in this study, only nasal swabs were collected and thereby the study did not include the introduction of a needle, which according to the danish law on animal experimentation (lbk number 474 of 15 may 2014) is the minimum intervention that requires a specific license. a trained veterinarian was involved in the sampling and data collection, and farmer consent was obtained before the sample collection. the study was designed as an observational cohort study to monitor/screen pig health (particularly swiav infection) in a danish farrow-to-finish continuous-flow pig herd. the herd had 985 sows and a farrowing area divided into a number of units. nursery pigs were housed at a separate site with 2000 pen places, and finisher pigs were raised at a third site with 1050 pen places. according to the danish spf (specific pathogen free) herd health declaration (seges svineproduktion 2020), the herd was declared positive for mycoplasma hyopneumoniae (m. hyo), actinobacillus pleuropneumoniae (app) type 6 and 12 and porcine reproductive and respiratory syndrome (prrs) type 2 virus, and negative for app type 2, prrs type 1 virus, brachyspira hyodysenteriae, atrophic rhinitis, demodex mites and lice. the herd had contract with a large abattoir and processing company in denmark, which in 2015 initiated the "raised without antibiotics" (rwa) concept [42] . at present (march 2020), 50 danish herds including this herd are included in the rwa program, where the producers are payed a premium price to reflect the cost of the increased workload, intensified hygiene measures and other interventions to prevent disease in the effort to reduce the use of antimicrobial agents. the herd produced and recruited gilts internally. after weaning, pigs selected for gilt recruitment were housed in a separate room at the sow site, and vaccinated with a live prrs type 2 virus vaccine (ingelvac ® prrs vet.) at 7 and 10 weeks of age, and additionally with m. hyo, porcine circovirus-2 (pcv2) (porcilis ® pcv m hyo) and swiav (respiporc flu3) vaccines at 7, 10 and 24 weeks of age. all adult sows were vaccinated against swiav simultaneously four times a year (respiporc flu3). due to clinical signs like nasal discharge and swiav positive laboratory testing, piglets were vaccinated at four days of age with respiporc flu3 (0.4 ml, off label use). during the first week after weaning, pigs were vaccinated against m. hyo and pcv2 (porcilis ® pcv m hyo). the prevalence of different iav strains in danish pig herds has been reported to be around 45% [43] . based on this data, the iav prevalence in this herd was assumed to be between 10% and 45%. by using a minimum sample size of 40, this should, at a very minimum, give at least one iav-positive sample with a high probability of getting iav-positive samples from 4-18 pigs. it was assumed from the outset of the study that some piglets may be lost to follow-up or that some may die and hence it was decided to use an initial sample size of 47. all live-born piglets born within three consecutive farrowing days (n = 518) were ear-tagged at birth with consecutive unique id numbers. from every 10th piglet (id number 10, 20, etc.) nasal swabs were obtained (at five to nine day intervals) at week 2 (n = 46), week 3 (n = 38), week 4 (n = 39), week 5 (n = 45), week 6 (n = 44), week 8 (n = 42), week 12 (n = 41) and week 22 (n = 13). overall, 47 individual piglets/pigs were sampled throughout the study (table s1 ). due to the variable weaning age (week 4 or week 5) of the piglets, the week 4 sampling was done either at the farrowing unit (week 4 f, n = 24) or in the nursery unit (week 4 n, n = 15) situated at separate buffer stables at the sow site. at week 5, all weaned piglets were moved into separate nursery units and housed with other pigs from the same herd of the same age until approximately 12 weeks of age. thereafter, the nursery pigs were transferred to a finisher site (~5 kilometre away) where they were housed until slaughter at approximately 22 weeks of age. this finisher site received piglets from the nursery units of the same herd. the samples were collected from the 25 april 2018 until the 20 september 2018. small-or medium-sized sterile rayon swabs (medical wire, wiltshire, uk) were used to collect the nasal swab samples. the swab was inserted into one nostril of the individual piglet and was then turned a full 360 • . samples were stored in 2 ml phosphate buffered saline (pbs) at 4-8 • c until delivered to the laboratory within two days after sampling. at weeks 2 and 4, piglets were clinically examined before obtaining the nasal swabs. clinical signs of nasal discharge and conjunctivitis were recorded for each individual pig. all samples were processed at the centre for diagnostics (cfd), technical university of denmark (dtu). the pcr analysis was carried out at cfd-dtu, while the ngs was carried out at statens serum institute (ssi), denmark. all the collected samples were vortexed for 1 min, the rayon swab was then removed from the tube and 400 µl of the fluid transferred to a 2 ml eppendorf (ep) tube. the 2 ml ep tubes were centrifuged at 9000× g for 5 min at room temperature. finally, 200 µl supernatant was used for nucleic acid extraction. nucleic acid (both dna and rna) was extracted using the qiacube ht extraction robot (qiagen, hilden, germany) and the cador pathogen 96 qiacube ht kit (qiagen) according to the manufacturer's instructions. the extracted nucleic acids were stored at −80 • c for further use. the extracted nucleic acids were subjected to reverse transcription using a high capacity cdna rt kit (applied biosystems, foster city, ca usa). a final volume of 10 µl reaction mix was prepared by mixing 1 µl of 10x rt buffer, 0.4 µl of 100 mm dntp mix, 1 µl of 10x random hexamer, 0.5 µl of multiscribe rt enzyme, 2.1 µl of nuclease free water and 5 µl of extracted nucleic acids. finally, cdna synthesis was carried out in a t3 thermocycler (biometra, fredensborg, denmark) with the given cycling conditions: 25 • c for 10 min, 37 • c for 120 min followed by 85 • c for 5 min and finally paused at 4 • c. the cdna sample was pre-amplified using 2x taqman preamp master mix (applied biosystems). a total volume of 10 µl was prepared by mixing 2.5 µl of cdna with 5 µl of 2x taqman preamp master mix (applied biosystems) and 2.5 µl of a 200 nm primer mix (containing the different sets of primers used for the detection of different pathogens) as previously described [41] . in brief, pre-amplification was carried out in a t3 thermocycler (biometra) using the program 95 • c for 10 min followed by 14 cycles of 95 • c for 15 s and 60 • c for 4 min. finally, the amplification was paused at 4 • c and the pre-amplified product was stored at −20 • c for further use. initially, the collected samples were screened for iav by using the high-throughput rtpcr platform biomark (fluidigm, south san francisco, usa) and the 192.24 dynamic array (da) integrated fluidic circuit (ifc) chip (fluidigm). a 4 µl sample mix was prepared by mixing 2.2 µl of pre-sample mix (prepared by mixing 2 µl of 2x taqman gene expression mastermix (applied biosystem) and 0.2 µl of 20x sample loading reagent (fluidigm) for each sample) and 1.8 µl of pre-amplified sample. similarly, 2 µl primer/probe stock was mixed with 2 µl of 2x assay loading reagent (fluidigm). three µl of the assay mix and 3 µl of sample mix was loaded into the respective inlets of the 192.24da ifc chip. the 192.24 da ifc chip was placed in the ifc controller rx for loading and mixing for approximately 30 min. finally, the chip was inserted into the high-throughput rtpcr platform biomark (fluidigm) for thermal cycling with the following cycling condition: 50 • c for 2 min, 95 • c for 10 min followed by 40 cycles of 95 • c for 15 s and 60 • c for 60 s. all samples were tested in duplicates. positive and non-template (nuclease-free water) controls were included. amplification curves and cycle threshold (ct) values were obtained on the biomark system and finally analysed using fluidigm real time pcr analysis software 4.1.3 (fluidigm) as previously described [41] . samples found positive for iav using the high-throughput rtpcr system, were selected for rna extraction. the extraction was carried out for the selected nasal swabs using the qiacube extraction robot (qiagen) and the rneasy mini kit (qiagen) according to the manufacturer's instructions as described previously [16] . rna was eluted in 60 µl rnase-free water and stored at −80 • c. detection of iav in the extracted rna was performed in the rotor-gene q (qiagen) pcr platform using a previously published rtpcr assay targeting the matrix gene of iav [15, 44] . confirmed iav positive samples with ct values < 30 (using rotor-gene pcr) were used for full genome amplification of iav. a one-tube reaction amplifying each gene segment of iav was performed using a modified version [45] of a previously published assay [46] . five µl of the amplified one-tube full genome iav pcr products along with 10 µl of 1kb dna ladder (invitrogen, carlsbad, ca usa) were run on 0.8% agarose gels (invitrogen) to check if the bands representing all 8 gene segments of iav were visible on a bio-rad gel documentation system (hercules, ca, usa). only fully amplified iav pcr products were selected and were purified using a high pure pcr product purification kit (roche, mannheim, germany) following the manufacturer's protocol. the purified pcr products were sent for ngs on the illumina miseq sequencing platform at the state serum institute (copenhagen, denmark). sequence analysis was performed in clc genomic workbench 11.0.1. software (qiagen). all reads obtained from each of the samples were initially trimmed to remove short and low quality reads and primers/adaptors and then consensus sequences of each gene segment was constructed using the function "map reads to reference", using a panel of 22 sequences representing each different lineage of each gene segment known to be present in denmark. the consensus sequences of each gene segment were then aligned using the muscle algorithm [47] , and then examined for similarities using the function "create pairwise comparison". moreover, the function, blastn, was used to compare the generated consensus sequences with the online ncbi genbank database [48, 49] . further analysis was done by selecting some of the closest swiav sequences from the ncbi genbank. phylogenetic analysis was done after aligning all sequences of each gene using clustal-w [50] and using the maximum likelihood (ml) method with the best fitting substitution model in mega [51] . the ha subtype numbering was done using the influenza research database (ird) tool at http://www.fludb.org [52, 53] . the different amino acids present in the ha antigenic sites (cb, sa, sb, ca1 and ca2) were calculated by comparing our sequences with reference sequences [28, [54] [55] [56] . b-and t-cell epitopes were analysed by aligning our sequences with reference sequences [57] [58] [59] [60] using clustal-w [50] . differences present at seven antigenic sites (1, 2a, 2b, 2c, 2d, 3 and 4) of the na gene segment were calculated by comparing our sequences with reference sequences [61] [62] [63] [64] . netnglyc 1.0 (http://www.cbs.dtu.dk/services/netnglyc/) from dtu bioinformatics (department of bio and health informatics) [65] was used for the prediction of n-linked glycosylation sites in the ha gene segments only on the n-x-s/t sequons (excluding p at x) with a score threshold >0.5. fisher's exact test was used to determine any association of data using the analysis tool 2by2.xls at http://itve.dk/ [66] . the results of the high-throughput rtpcr analysis of nasal swab samples, including an assay for detection of the matrix gene of iav, allowed for estimation of the prevalence of swiav at all sampling times. swiav was detected in pigs throughout the sampling period starting from week 2 until week 22, indicating continuous circulation of iav in the herd ( table 1 ). the prevalence of swiav increased from the first sampling (week 2) (4.3%) until week 4 (33.3%). among week-4-old piglets, 41.7% (22.1-63.4% at 95% confidence interval) were iav-positive in the farrowing unit while 20% (4.3-48.1% at 95% confidence interval) were iav-positive in the nursery unit (weaned at week 4 or week 5). after weaning (week 5), the prevalence stabilized and then decreased, reaching the lowest prevalence at week 12 (2.4%). after the pigs had been transferred to the finisher site, the prevalence increased again, reaching 30.8% at 22 weeks of age. in total, 76.6% of the pigs were iav-positive at least once during the study period (table 1 ). occurrence of iav is defined as the detection of iav in nasal swabs in one or more consecutive weeks in the same pig, whereas recurrence is defined as the detection of iav from the same pig at two or more non-consecutive weeks [67] . of 47 pigs, 36 (76.6%) were found to be positive for iav at least once, whereas 11 (23.4%) pigs were found to be negative throughout the study (table s1 ). all the iav positive and negative pigs were housed together in mixed pens. among the positives, 7 of the 36 pigs (19.4%) were found to have recurrent iav (table s1 ), whereas 29 of 36 pigs (80.6%) were found to have only a single occurrence of iav. of these 29 pigs, 23 (63.9%) pigs were iav-positive only at a single sampling time, whereas nine pigs (25%) tested positive at two consecutive sampling time points (table s1 ). among the 46 piglets sampled in week two, 33 (71.7%) had nasal secretion and 20 piglets (43.5%) had conjunctivitis. however, only two (5.1%) piglets had nasal secretion, while four piglets (10.3%) had conjunctivitis in week 4. nasal secretion was recorded in both of the iav-positive piglets at week 2, while it was only present in two of the 13 positive piglets at week 4. similarly, conjunctivitis was observed in one out of two positive piglets at week 2 and was only present in one out of 13 positive piglets at week 4. no association between iav infection in the individual pigs and clinical signs of nasal secretion and conjunctivitis at week 2 and 4 was observed (supplementary tables s2, s3 , s4 and s5). fifteen samples that had a ct value < 30 in the iav rtpcr assay were selected for one-tube full genome amplification. of these, 11 samples (table s6 ) displayed clear bands representing all the iav gene segments on the agarose gel and were selected for iav whole genome sequencing (wgs) ( table s6 ). all the generated sequences for eight full gene segments from all the 11 pig samples have been deposited in ncbi genbank with accession numbers mt946974 -mt947061. eleven full length (1701 nt) ha gene segments from the study were used for phylogenetic analysis. twenty three additional ha reference sequences representing avian, human, classical swine and h1n1pdm09 subtypes were downloaded from ncbi genbank and included in the analysis. phylogenetic analysis revealed that all the 11 ha gene segments were of eurasian avian-like h1nx origin (1c.2 lineage using the global swine h1 clade classification system) [68] . all nine h1 sequences obtained from pigs sampled between week 4 and 8 were grouped together in one cluster and had pairwise sequence differences of 0 to 0.6% at the nucleotide level and 0 to 0.8% at amino acid level. this cluster had the highest sequence identity to a/swine/germany/holdorf-idt12357/2010(h1n2) (accession no.: kr699687) in a blastn search with~97% identity at the nucleotide level and~96.5% at the amino acid level [21] . the two h1 sequences obtained from pigs sampled at week 22 were 100% identical but clustered separately from the h1 sequences obtained from the younger pigs ( figure 1 ). these h1 genes had the highest sequence identity to a/swine/denmark/10-1725-1/2011(h1n2) (accession no.: kr700049) in the blastn search with identities of 95.4% at the nucleotide level and 96.3% at the amino acid level [21] . as mentioned earlier, sequences from two pigs shedding swiav at two non-consecutive sampling times were obtained (pig id 250 sampled at week 4 and week 8, pig id 380 sampled at week 5 and week 22; table s6 ). in the phylogenetic analysis, the ha sequences of pig id 250, obtained at weeks 4 and 8 were located in the same cluster and were~0.5% (9/1701) divergent at the nucleotide level and < 1% (5/566) divergent at the amino acid level. in contrast, the ha sequences of pig id 380 at weeks 5 and 22 were~12% (198/1701) divergent at the nucleotide level and 10% (56/566) divergent at the amino acid level. from the phylogenetic analysis of pig id 380 it was clearly seen that the ha sequence obtained at the first sampling were located in the cluster defined by the viruses from the younger pigs, whereas the ha sequence obtained at the last sampling (after the pigs had been transferred to the finisher site) were located outside this cluster, thereby representing another h1n2 variant (figure 1 ). the majority of the differences between the two different samplings (week 4 and week 8) for pig id 250 and week 5 and week 22 for pig id 380 were located in the antigenic sites of the ha protein between amino acid 78-240. three different amino acid differences were found in the antigenic sites (v144a and k145n at ca2 and s160h at the sa region) of pig id 250 at week 4 and week 8. whereas, 16 different amino acids differences were found in the antigenic sites of pig id 380 at week 5 and week 22 (table 2) . sequences from different individual piglets sampled at week 4 have one different amino acid (s160h) at the sa region and two different amino acids (v144a and k145n) at the ca2 region. similarly, sequences from pigs of 8 weeks of age had only one different amino acid (s160h) at the sa region. the two ha sequences from week 22 were found to have identical amino acids in the antigenic sites. the cleavage site with one arginine (psiqsr: 324-329) and fusion peptide sequence (glfgaiagfieggwtgmidgwyg: 330-352) were found to be conserved in all 11 full-length h1 ha sequences [28, 69] . the ha2 region of all the ha sequences were found to be more conserved compared to the ha1 region, as they were only approximately 0-9% divergent at the nucleotide level and 0-2% divergent at the amino acid level. however, the ha1 region of all the ha sequences were 0-13.6% divergent at the nucleotide level and 0-14.8% divergent at the amino acid level. eleven full-length (1410 nt) na gene segments were used for phylogenetic analysis (figure 2 ). twenty seven additional na reference sequences representing danish h1n2, european h1n2, h3n2 and asian and american h1n2 na were also used. the phylogenetic tree shown in figure 2 revealed that the n2 na gene segments from the present study were most identical to the na segment of the danish h1n2 and european h3n2 subtypes. the n2 sequences obtained from pigs sampled between week 4 and week 8 grouped together in one cluster (danish hxn2 type) and had pairwise sequence differences of 0 to 0.4% at the nucleotide level and 0 to 0.4% at the amino acid level. this cluster had the highest sequence identity to a/swine/germany/holdorf-idt12357/2010 (h1n2) (accession no.: kr699688) in the blastn search with an identity of 97.7% at the nucleotide level and >98% identity at amino acid level, when performing a pairwise comparison [21] . in contrast, the two n2 sequences obtained from pigs at week 22 were identical and were positioned apart from the cluster formed by the sequences obtained from the younger pigs. similar to the ha segment, this cluster had the highest sequence identity to a/swine/denmark/10-1725-1/2011 (h1n2) (accession no.: kr700051) in the blastn search, with an identity of 96.2% at the nucleotide level and >95% identity at the amino acid level [21] (figure 2 ). the n2 sequences obtained from pig id 250 at weeks 4 and 8 were found to be <0.1% (1/1410) divergent at the nucleotide level and 0.2% (1/469) divergent at the amino acid level. however, the two n2 na sequences obtained from pig id 380 at weeks 5 and 22 were found to be~14.5% (205/1410) divergent at the nucleotide level and~13.6% (64/469) divergent at the amino acid level. amino acids present in the antigenic sites (1, 2a, 2b, 2c, 2d, 3 and 4 ) of the n2 na sequences of pig id 250 at week 4 and week 8 were found to be identical. while the n2 sequence obtained from pig id 380 at week 5 was similar to the ones from pig id 250, the sequence from pig id 380 at week 22 were found to be~35.3% (12/34) divergent in the antigenic sites of n2. similarly, n2 sequences of pig id 380 at antigenic site 2d were found to be most divergent, at 75% (3/4), whereas no changes were found in the antigenic site 1 ( table 3 ). all 11 n2 na sequences have eight highly conserved amino acids (r118, d151, r152, r224, e276, r292, r371 and y406) at the inner shell of the na active site, which interact directly with sialic acids. similarly, ten highly conserved amino acids (e119, r156, w178, s179, d198, i222, e227, e277, n294 and e425) [61, 71, 72] were also present in an outer shell of the na active site. hence, all the amino acids present at na active sites were found to be conserved. table 3 . comparison of amino acid sequences of neuraminidase (na) antigenic sites of pig id 380 sampled at week 5 and week 22 from the pig herd. amino acid positions were numbered using an n2 numbering system. amino acid change all the six internal gene segments (pb2, pb1, pa, np, m1-m2 and nep-ns1) were compared with available online ncbi genbank reference sequences. all the internal gene segment sequences obtained from younger pigs (≤ 8 weeks) were found to be most identical (>98%) with the internal gene segments of h1n1pdm09 origin. in contrast, only four of the internal gene segments (pb2, pb1, pa and np) of virus sequences from older pigs (week 22) were of h1n1pdm09 origin. the remaining two internal gene segments (m1-m2 and nep-ns1) of sequences from older pigs (week 22) clustered with the h1n2dk and european h3n2 swiav subtypes, indicating that these viruses are the result of reassortment events between h1n2-like viruses and the h1n1pdm09 subtypes. in addition, respective internal gene segments obtained from pig id 250 at week 4 and week 8 were compared and were found to be 0-0.3% different at the nucleotide level and 0-0.4% at the amino acid level. a similar comparison of respective internal gene segments was also done for pig id 380 at weeks 5 and 22 and were found to be 3.7-22.1% different at the nucleotide level, while at the amino acid level they were 1.2-23.6% different ( table 4 ). the m1-m2 gene segments of pig id 380 at week 5 and 22 were found to be 6.5% divergent at the nucleotide level as shown in table 4 . similarly, the nep-ns1 gene segments of pig id 380 at weeks 5 and 22 were found to be most divergent (20.5%) ( table 4 ) and also clearly supported by two distinct clusters in the phylogenetic tree ( figure 3 ). nep-ns1 gene sequences obtained from pigs sampled between week 4 and week 8 made one cluster close to the h1n1pdm09 subtypes. in contrast, the nep-ns1 sequences obtained from pigs sampled at week 22 made a separate cluster and resembled the h1n2dk and european h3n2 subtypes (figure 3 ). table 4 . pairwise comparison of six internal gene segments of pig id 380 sampled at week 5 and week 22 both at the nucleotide and the amino acid level. week clearly supported by two distinct clusters in the phylogenetic tree ( figure 3 ). nep-ns1 gene 378 sequences obtained from pigs sampled between week 4 and week 8 made one cluster close to the 379 h1n1pdm09 subtypes. in contrast, the nep-ns1 sequences obtained from pigs sampled at week 22 380 made a separate cluster and resembled the h1n2dk and european h3n2 subtypes (figure 3 ). b-and t-cell epitopes present in all the swiav sequences between week 4 and week 8 were identical. one amino acid change (r487k) was observed in the b-cell epitope present in the ha2 part of the ha gene segment after comparing ha sequences from pig id 380 at week 5 and week 22. similarly, one (e101d) and four different amino acids (l86f, s91n, e219v and s220t) were found in the t-cell epitope sequence of np and ha gene segments, respectively. t-cell epitopes of other gene segments were found to be identical among the compared isolates. six to seven nlg sites were present in all the ha sequences obtained in this study of which five (at positions 21, 33, 276, 483 and 541) were well conserved between all analysed sequences. all the ha sequences obtained from pigs sampled between week 4 and week 8 had one separate nlg site at position 94, whereas the ha sequences obtained from pigs sampled at week 22 have two other nlg sites at position 125-126 and at position 165. the nlg site at position 94 was located near the ca2 antigenic site in the globular head domain of the ha protein sequence, whereas the nlg site at position 125-126 was located near the sa antigenic site (table 5) . similarly, the nlg site at position 165 was located within the sa antigenic site of the ha protein sequence. table 5 . n-linked glycosylation (nlg) sites of h1 hemagglutinin (ha) gene segments obtained from all the pigs from week 4 to week 8 and week 22. "+, ++, +++" indicates the nlg potential with score threshold > 0.5. "*" indicates that the nlg site is located in between amino acid 125-126 for h3 numbering system. the study was designed to describe the infection dynamics of swiav in one danish pig herd by following 47 pigs from 2 weeks of age until slaughter (approximately 22 weeks of age). using a high-throughput rtpcr system, we were able to determine the prevalence of iav, and by the use of ngs, we characterized the genetic and antigenic diversity of circulating h1n2 swiav. based on the analysis, it was found that the prevalence of iav in pigs reached a maximum around weaning (4-5 weeks) and then decreased until 12 weeks and then increased again at 22 weeks of age. at least two different h1n2 variants were circulating in the herd, with one of the h1n2 variants circulating at the sow and nursery sites, and the other circulating in the finisher site. finally, we also demonstrated that individual pigs could have recurrent iav infections either with a very similar h1n2 variant (pig id 250) or with two divergent h1n2 variants (pig id 380). this confirms previous findings that some pigs can have prolonged swiav infections and be subjected to re-infection, even with closely related swiav [15, 45] . the understanding of the epidemiology and the genetic and antigenic diversity of swiav in pigs may help to unravel the layers of swiav infection dynamics and viral evolution from birth to slaughter, thereby helping to design better health interventions for the prevention and control of swiav in the herds. the relative low prevalence of iav before week 4 might be due to the presence of maternally-derived antibodies (mdas), which in this herd were stimulated by the sows being vaccinated with respiporc flu3 four times annually [16, 74] . however, the mdas wane over time, and several studies have shown results indicating that piglets are no longer completely protected from around three to four weeks of age [15, 16, 74] . moreover, the loss of mda occurs at the same time as the piglets are weaned into the nursery, thereby mixing different litters of pigs, creating the optimal environment for iav circulation. this has also been observed in previous studies [15, 16, 45] . similarly, a higher prevalence of iav at week 4 also indicated that the piglet immune system did not respond to the vaccination at day 4, which is in accordance with a previous study showing no effect of early piglet vaccination against swiav [45] . one of the explanations for this is that the presence of mda may hinder an active immune response in the piglets, but it could also be due to the reduced vaccine dose used (0.4 ml) [75] in the piglets under study. after week 5, most of the weaned pigs likely developed immunity to the circulating iav subtype and at the same time no new pigs were introduced into the nursery, resulting in a lower prevalence of iav in accordance with other findings [76] . the increase in prevalence observed at week 22 at a separate finisher site, was most likely due to infections with a second h1n2dk variant that might have been circulating continuously in the finisher site. this site was managed as a multi-aged, continuous-flow pig herd. this h1n2dk variant from the finisher site differed significantly in antigenic regions from the h1n2dk variant circulating at the sow and nursery sites. mutations in the antigenic sites of eurasian avian-like swine h1 have previously been linked to a lack of cross protection and emphasize that the diversity within the subtypes, especially in the h1 avian-like viruses, have now reached a level where it makes no sense to consider viruses of the same subtypes as belonging to the same serotype [15] . however, the lack of a significant cross-reaction should be confirmed by hi-testing, which was not performed in this study. moreover, the na gene and the internal gene cassette were also different between the two h1n2dk variants, which could also impact the cross-protective immunity between different swiav variants of the same subtype [77, 78] . specifically, the internal gene cassette of the h1n2dk variant that infected the pigs in the sow and nursery sites had a complete internal gene cassette of h1n1pdm09 origin, whereas the h1n2dk variant circulating in pigs in the finisher site had an m1-m2 and nep-ns1 gene of eurasian avian-like h1nx origin. however, the protective role of immunity against the internal gene segments are still controversial [58, 79] . pig id 250 was infected twice with the same h1n2dk variant, which only showed minor genetic differences between samplings. however most of the differences were located in antigenic sites. hence, it can be speculated that even a small number of mutations could facilitate re-infection with the same subtype, thereby confirming the results of a previous study [45] . in summary, it can be concluded that re-infections can occur with both similar and different variants within the same subtype. the presence of prolonged (2-3 weeks consecutive) and recurrent (non-consecutive) shedding of iav within week 4 to week 8 pigs also indicated reinfection with the same subtype and this was documented by sequencing. a number of previous studies have shown reinfection with the same strain, leading to prolonged iav shedding [12, 45, 74] . the presence of mda may play role in the prolonged iav shedding, as mda may hinder an active immune response [45, 80] . pig id 380 was infected with two different h1n2dk variants, and most of the mutations were located in antigenic sites as mentioned above. similarly, the acquisition of nlg sites near/within sa and ca2 antigenic sites of the ha sequence may lead to a shielding effect on the antigenic sites and probably the emergence of new antigenic variants. a range of studies have shown that the shielding effect on ha antigenic sites may lead to an evolution of the ha sequence and be responsible for escaping the pre-existing immunity in the hosts [81] [82] [83] [84] . the presence of these major differences between variants within the same subtype emphasizes the presence of a massive genetic drift of eurasian avian-like h1 in danish herds [27] , which in turn could have consequences for vaccine efficacy as the current swiav vaccine available against the h1n1 subtype has not been updated since 2002-2003. in our study, the presence of clinical signs of nasal discharge and conjunctivitis in pigs harbouring iav at week 4 was not very evident. reduced levels of clinical signs could be due to the presence of mda. similarly, previous exposure or a low level of exposure to the virus might preclude clinical signs in pigs [85] [86] [87] . in contrast with other studies, our study did not find any association between iav infection and nasal secretion [15, 16] . however, clinical signs were only recorded at two stages (at week 2 and week 4) of the total samplings in this study. similarly, we did not find any association between iav infection and conjunctivitis, which is also supported by other studies [15, 16] . in conclusion, the complexity of swiav infection dynamics in pigs from the farrowing unit to the finisher unit has been demonstrated. a high infection pressure of swiav was identified during the end of the stay in the farrowing unit and the start of the nursery unit. in addition, it has been shown that the prolonged persistence of iav in pigs could be due to re-infection with iavs that are closely related to each other. similarly, re-infections with different strains within the same lineage can also be expected, as the genetic changes affect important antigenic epitopes. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/9/1013/s1, table s1 : occurrence and recurrence of iav in pigs from week 2 to week 22 in the pig herd using high-throughput rtpcr. 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biological functions on glycosylation site migrations in human influenza h1n1 viruses a perspective on the structural and functional constraints for immune evasion: insights from influenza virus a carbohydrate side chain on hemagglutinins of hong kong influenza viruses inhibits recognition by a monoclonal antibody effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza a/h3n2 virus hemagglutinin differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity cytokines in the pathogenesis of influenza avian and swine influenza viruses: our current understanding of the zoonotic risk this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to acknowledge the herd owner for providing access to his property for sampling, and the herd-veterinarian providing assistance during sampling. similarly, we want to acknowledge the laboratory technicians hue thi thanh tran and jonathan rahlff rogersen for their help with laboratory tasks. the authors declare no conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. key: cord-291014-cfnoxhtd authors: zheng, jian; perlman, stanley title: immune responses in influenza a virus and human coronavirus infections: an ongoing battle between the virus and host date: 2018-02-28 journal: current opinion in virology doi: 10.1016/j.coviro.2017.11.002 sha: doc_id: 291014 cord_uid: cfnoxhtd respiratory viruses, especially influenza a viruses and coronaviruses such as mers-cov, represent continuing global threats to human health. despite significant advances, much needs to be learned. recent studies in virology and immunology have improved our understanding of the role of the immune system in protection and in the pathogenesis of these infections and of co-evolution of viruses and their hosts. these findings, together with sophisticated molecular structure analyses, omics tools and computer-based models, have helped delineate the interaction between respiratory viruses and the host immune system, which will facilitate the development of novel treatment strategies and vaccines with enhanced efficacy. jian zheng and stanley perlman respiratory viruses, especially influenza a viruses and coronaviruses such as mers-cov, represent continuing global threats to human health. despite significant advances, much needs to be learned. recent studies in virology and immunology have improved our understanding of the role of the immune system in protection and in the pathogenesis of these infections and of co-evolution of viruses and their hosts. these findings, together with sophisticated molecular structure analyses, omics tools and computer-based models, have helped delineate the interaction between respiratory viruses and the host immune system, which will facilitate the development of novel treatment strategies and vaccines with enhanced efficacy. immune response: protective or pathogenic? the host immune system is composed of multiple tissues, cells and molecules and can protect hosts from infectious diseases by recognizing and eliminating pathogens efficiently. in one example, our studies of mice infected with sars-cov showed that the severity of sars correlated with the ability to develop a virus-specific immune response, while inhibitory alveolar macrophages and inefficient activation of dendritic cells (dcs) delayed this process and aggravated disease [1] . in another study, channappanavar et al. further demonstrated that dysregulated type i interferon (ifn) and inflammatory monocytemacrophage responses led to lethal pneumonia in sars-cov-infected mice [2] . in support of these data, inhibition of nuclear factor-kappab (nf-kb)-mediated inflammation in sars-cov-infected mice increased survival [3] . similar to their pathological roles in coronavirus infections, inappropriate or dysfunctional immune responses such as overactivation of nacht, lrr and pyd domains-containing protein 3 (nlrp3), high-mobility group box 1 protein (hmgb-1) and interleukin-1beta (il-1b), have been implicated in host tissue destruction [4-8] and persistent pathological changes in iav-infected hosts [9] . expression of the complex of tumor necrosis factor (tnf) superfamily 10 (tnfsf10), histone deacetylase 4 (hdac4) and hdac5 negatively correlated with the levels of tnfa, nf-kb and cyclooxygenase 2 (cox-2) and increases in their expression was correlated with improved prognosis of iav-infected hosts [10] . in addition to their cell-intrinsic properties, lung macrophage and monocyte heterogeneity in localization in iav infections also contributed to differences in outcomes [11] . of vaccinated iav individuals or mice, in which hemagglutinin (ha), neuraminidase (na) and glycosylation pattern mutations [24] , might hinder an effective antibody and t cell response. the contribution of innate immunity to immune defense is not limited in direct anti-viral effects [34] . innate immune signals such as ifn-i not only interact with other innate immune elements such as monocytes and type-ii ifn to limit iavcaused tissue inflammation [35] , but also directly modulated the adaptive immune response. both ifn-i and toll-like receptor 7 (tlr7) were also found to shape b cell-mediated immune responses against iav [36], while rig-i signaling was critical for efficient polyfunctional t cell responses [37] . moreover, the increased mortality of iav-infected mice in the absence of mitochondrial antiviral signaling (mavs) and tlr7 was found to independent of viral load or myeloid differentiation primary response 88 (myd88)-dependent signaling but dependent on secondary bacterial burden, caspase-1/11, and neutrophil-dependent tissue damage [38 ]. as for innate immune cells, a population of lung-resident innate lymphoid cells (ilcs) in mice and humans that expressed cd90, cd25, cd127 and st2 was found to contribute to airway epithelial integrity and its depletion resulted in diminished lung function and impaired airway remodeling [ broadly neutralizing antibodies generally target conserved functional regions on ha. [21] binding of antibody to an epitope masks the epitope and prevents the stimulation and proliferation of specific b cells. [30] mers-cov recombinant receptor-binding domains of multiple mers-covs induce cross-neutralizing antibodies against divergent human and camel mers-covs. [28] t cell response iav vaccine-generated lung-resident memory cd8 t cells provide heterosubtypic protection to iav infection. [31] potential challenges in translating protective memory cd4 t cell responses in experimental animal models to patients. [19] mers-cov mers-cov efficiently infects human primary t cells and induces apoptosis. [14] sars-cov memory t cell responses targeting the sars coronavirus persist for up to 11 years postinfection. [23] crosstalk between immune components iav cooperativity between cd8+ t cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic iav immunity. [20] antibody specificity plays an important role in the regulation of adcc. cross-talk among antibodies of varying specificities determines the magnitude of fc receptor-mediated effector functions. [17] ige cross-linking impairs monocyte antiviral responses and inhibits iavdriven th1 differentiation. [27] mers-cov recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses. [32] maintenance of immune memory iav levels of neutralizing antibodies against previously encountered iav strains ('original antigenic sin') increase over time. [22] low levels of circulating cd8+ t effector and central memory cells are associated with iav infection severity upon re-challenge. [16] regimen of a ctl-based vaccine/vaccine-component benefits from periodic boosting to prevent clinically evident iav infection. [26] multifunctional cd4+ t-cell responses were maintained only in patients with recurrent infections. [29] immunopathology iav iav-specific cd8+ t cells exacerbate infection following high dose challenge of aged mice. [25] different subsets of cd8+ t cells interact with subsets of innate cells through costimulatory molecules to balance protection and immunopathology. [15] identification of protective and pathogenic t cell epitopes in iav h7n9infected patients. [18] immunotherapy iav, cov high titer anti-iav or cov sera may be useful prophylactically and therapeutically in exposed and infected patients. with the increasing accumulation of knowledge of molecular interactions between host cells and viruses, additional host molecules and normal biological processes [51-65,66 ,67-69] were found to participate in the viral replication cycle (summarized in table 2 ). to clarify the roles of these molecules and processes in virus infection, host genetic determinant screening [70] [71] [72] , immunomics and public health omics [73] , host lipid omics [74 ] and characterization of the epigenetic landscape [75] were used to supplement conventional analyses. moreover, information about interaction between immune and parenchymal cells also facilitated efforts to optimize antiviral response while reducing unwanted side effects [76 ,77 ] . computer modeling of host-pathogen interactions is likely to be used more in the future, as additional parameters are identified. thus, computer modeling helped in prediction of clinical outcomes, demonstrating key roles for the innate immune response and the interval between infections [78] . these novel methodologies are likely to provide additional approaches to identifying targets for novel antiviral therapies. the iav non-structural protein ns1, perhaps the bestcharacterized viral immunoevasive protein, binds doublestranded rna (dsrna) to inhibit host innate immune responses [79] [80] [81] . recently, ns1 was also found to bind cellular dsdna and prevent the loading of transcriptional machinery onto the dna, thus attenuating expression of antiviral genes [82] . meanwhile, the c-terminal domain of ns1 blocked ifn-beta production by targeting tnf receptor-associated factor 3 (traf-3) [83] , while other domains of the protein inhibited interferon regulatory transcription factor 3 (irf3) [84] and rna-dependent protein kinase (pkr) activation [85] . another iav protein, neuraminidase (na), was shown to remove sialic immune responses in respiratory virus infections zheng and perlman 45 table 2 recent findings related to intrinsic molecules and biological processes involved in iav and cov infectionss. ref. cell cycling proteins iav competitive inhibition of iav m1-m2 interaction by cyclin d3 impairs infectious virus packaging, resulting in attenuation apoptosis-related signals iav apoptosis signaling modulates iav propagation, innate host defense, and lung injury. [60] sex hormones-related signals iav progesterone-based contraceptives reduce adaptive immune responses and protection against subsequent iav infections. [59] male mice were more susceptible to sars-cov infection compared with agematched females, while estrogen receptor signaling played a critical role in protecting females from sars-cov-mediated pathogenesis. [53] chd chromatin remodeler iav chd1 is a proviral regulator of iav multiplication. [63] nuclear import and export machinery iav iav have evolved different mechanisms to utilize importin-alpha isoforms, affecting importation on both sides of the nuclear envelope. [65] activation of the interferon induction cascade by iav requires viral rna synthesis and nuclear export. [61] human heat shock protein 40 promotes iav replication by assisting in the nuclear import of viral ribonucleoproteins. [52] preferential usage of importin-alpha7 isoforms by seasonal iav in the human upper respiratory tract makes it a target of selective pressure. [64] vesicular trafficking iav iav infection modulates vesicular trafficking and induces golgi complex disruption. [68] iav enhances its propagation through modulating annexin-a1 dependent endosomal trafficking. [51] iav ribonucleoproteins modulate host recycling by competing with rab11 effectors. [67] sars-cov a predicted beta-hairpin structural motif in the cytoplasmic tail of the sars-cov e protein is sufficient for golgi complex localization of a reporter protein and functions as a golgi complex-targeting signal. [54] mers-cov cd9-facilitated condensation of receptors and proteases allows mers-cov pseudoviruses to enter cells rapidly and efficiently. [56] exosome secretion iav exosome deficiency uncoupled chromatin targeting of the viral polymerase complex and the formation of cellular-viral rna hybrids, which are essential rna intermediates that license transcription of antisense genomic viral rnas autophagy iav autophagy induction regulates iav replication in a time-dependent manner. [58] sars-cov cov nsp6 restricts autophagosome expansion. [55] cellular senescence iav cellular senescence enhances viral replication. [62] coagulation iav beneficial effects of inflammation-coagulation interactions during iav infection [69] acid residues from nkp46, resulting in reduced recognition of ha and enhancing immune evasion of nk cells [86] . in addition, the iav m2 protein was shown to reverse bone marrow stromal antigen 2 (bst-2)-mediated restriction of virus release via proteasomal pathways [87] . to evade the host immune system, iav also inhibits host but not viral mrna nuclear export [88] , without impairing nuclear viral ribonucleoprotein (vrnp) import [89] . in addition, productive viral replication in macrophages resulted in decreased phagocytosis via downregulation of fc receptors cd16 and cd32, potentially playing a role in iav pathogenesis [90] . the crucial role of the ifn response makes it a preferred target for viral evasion. besides ns1, multiple virusencoded molecules including the nucleoprotein [64], the fusion peptide of ha2 (ha2-fp), ha1 and some variants of polymerase subunits pb1-f2, pb1, pb2, pa all counteract the interferon response [91] . interestingly, some host cellular molecules are also utilized by iav to block ifn expression. using rna interference, knockdown of a host factor, the double phd fingers 2 gene (dpf2) [92] , resulted in decreased expression of iav proteins, by releasing ifn-b production from dpf2-mediated suppression [92] . the cov endonuclease, nsp15, efficiently prevented activation of host cell dsrna sensors including melanoma differentiation-associated protein 5 (mda5), 2 0 -5 0 oligoadenylate synthetase (oas) and pkr [93, 94 ] , while coronavirus-encoded proteases countered innate immunity, including the ifn response, through diverse pathways [95] . a recent investigation further showed that sars-cov nucleocapsid inhibited type i interferon production by interfering with tripartite motif protein 25 (trim25)-mediated rig-i ubiquitination [96] . recent studies of cellular metabolic processes [97, 98] and post-transcriptional protein modification [99, 100] identified additional approaches used by viruses to evade host immune responses [101] , and to facilitate optimal replication [102] . for example, iav delayed apoptosis of infected cells by activating a signal transducer and activator of transcription 3 (stat-3)-related pathway, allowing prolonged replication [103] . nicotinamide adenine dinucleotide phosphate (nadph) oxidase 2 (nox2), critical for expression of reactive oxygen species (ros), is often activated in endocytic compartments by rna and dna viruses, exacerbating virus-mediated pathogenicity [104 ] . in addition to important roles for host proteins [105] , the role of lipids [74 ] in respiratory virus infections has also drawn increasing attention. zhao et al. reported that agerelated increases in prostaglandin d2 (pgd 2 ) expression in mouse lungs correlated with a progressive impairment in dc migration to dlns, causing diminished t cell responses upon iav or sars-cov infection [106] . in a subsequent study, vijay et al. demonstrated a critical role for phospholipase a2 group iid (pla 2 g2d) in impaired dc migration to dln and age-related susceptibility to sars-cov infection [107] . pla 2 g2d is upstream of pgd 2 in the prostaglandin synthesis pathway. both molecules may be useful targets for anti-viral therapies. as mentioned above, sars-cov nucleocapsid protein was reported to interfere with rig-i ubiquitination [96] , while decreased deubiquitination mediated by mers-cov nsp3 deubiquitinase also inhibited the host immune response [108] . recently, fehr et al. found that mutation of the macrodomain of nsp3, important for countering adp-ribosylation, resulted in virus attenuation [109] , while another report demonstrated that binding of the methyl donor s-adenosyl-l-methionine (sam) to 2 0 -omethyltransferase nsp16 enhanced mers-cov replication, promoting the recruitment of the allosteric activator nsp10 [110] . on the other hand, iav induced host histone deacetylase 1 dysregulation in lung epithelial cells, inhibiting iav infection [111] while neddylation (conjugation of a ubiquitin-like protein, neural precursor cell expressed developmentally down-regulated 8 (nedd8)) of pb2 protein reduced its stability, suppressing iav replication [112] . nevertheless, the role of epigenetic modification during respiratory virus infection is not well understood; the application of phosphoproteomics to characterization of the human macrophage response to iav infection [113] serves as a model for future studies. other putative targets for modulating the immune response are carbohydrates present on host and viral proteins. recently, integrated omics and computational glycobiology revealed the structural basis for iav glycan microheterogeneity and host interactions [114, 115, 116] . glycosylation of the ha protein not only mediated virus entry into host cells [115] [116] [117] , but also modulated iav replication and transmission [118] , and the immune response against the virus [119] [120] [121] , thus representing a potential target for vaccine and drug development [122, 123 ] . vaccines remain as the most efficient tools for preventing the occurrence and spread of viral respiratory diseases. distinct from conventional adjuvants, novel reagents are being used to shape as well as augment the strength of the induced immune responses. targeting iav ha to the chemokine receptor, xcr1, present on some dendritic cells enhanced protective antibody responses against the virus [124] . in addition, knowledge of the microbiota [125] , and manipulation of apoptosis [126] and mtor (mechanistic target of rapamycin) [127] -related signaling pathways have been used to predict or modulate responsiveness and efficiency of vaccines, respectively. a major goal of iav and cov vaccine development is to develop vaccines able to induce broadly acting antibodies; these efforts require more precise definition of useful conserved protective antibody epitopes [128] . further, monocytederived dendritic cells (modcs) [129] dominated the activation of cd8+ t cells at late times after infection of c57bl/6 mice, triggering a switch in immunodominance from pa to np-specificity. this differential expression of t cell epitopes has implications for dc-based vaccine design. additionally, neutrophil-targeting [42] and th1-targeting strategies [130] might help in establishing tissue-resident memory (trm) and heterosubtypic immunity. inducing effective resident immune memory represents an ideal strategy for protecting the host from respiratory virus infection, especially at very early phases of virus invasion. recent technical advances have facilitated distinguishing tissue-resident cell populations from those in the periphery. in a recent publication, airway memory cd4(+) t cells induced by a single conserved n proteinspecific epitope present in both sars-cov and mers-cov mediated protection against challenge with either pathogen [131 ] . iav-specific resident memory cd8 cells in the upper respiratory tract or bronchoalveolar fluid provided superior protection compared to those in the lung, although some studies questioned whether these were truly resident memory as opposed to memory cells at these sites [132] . in a recent report, slutter et al. delineated the dynamics of iav-induced lung-resident memory t cells that underlies waning heterosubtypic immunity [133] , illustrating the tight collaboration of resident and peripheral t cell memory in respiratory virus control. in the future, use of sophisticated cell sorting methods and mass spectrometric flow cytometry will provide more precise information about resident memory immune cells. the iav-specific antibody response is well known to be a key host factor in protection from subsequent challenge [134, 135] . recent work has identified nasopharyngeal protein biomarkers in immunized mice useful for predicting the severity and outcome of acute respiratory virus infection [136] . other studies have identified an important synergistic role for immune responses in inducible bronchus-associated lymphoid tissue (ibalt) and draining lymph nodes for optimal iav-specific cd4+ t cell responses [137] . in contrast, nasal-associated lymphoid tissues (nalts) have been shown to support the recall but not priming of iav-specific cytotoxic t cells [138 ] . genomics, next-generation sequencing [139] and single cell imaging and analysis [140] facilitated in-depth investigation of the evolution, recombination and spread of infectious pathogens and extend the scope of virology research. in addition to these molecular biology tools, methods such as analyses of dc responses [141] and digital cell quantification (dcq), which combine genome-wide gene expression data with immune cell functional studies, will help identify immune cell subpopulations [142] . especially critical for understanding the ecology of rna viruses, such as iav and cov, will be obtaining respiratory samples from camels (mers-cov) and patients in both cross-sectional and longitudinal studies. these samples will be useful for identifying carriers and understanding virus evolution and transmission dynamics [143, 144] . recent analyses of mers-covinfected camels [145] have also increased our knowledge of virus demography and evolution across diverse populations. although gene sequencing and crystallographic analyses have provided insight into the molecular evolution of iav, the inability to predict future virus evolution remains an obstacle in managing epidemic and pandemic spread. models using canalized evolutionary trajectory induced by selective dynamics [146] , intra-host iav dynamics [147] , sequence based epidemiology [148] , genomic diversification and adaptation during experimental serial passages [149] will help in the development of accurate prediction models. however, successful modeling to prospectively predict the emergence of new virus strains relies on solid experimental data 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influenza as a human disease antibodydependent cellular cytotoxicity and influenza virus inducible bronchus-associated lymphoid tissue (ibalt) synergizes with local lymph nodes during antiviral cd4(+) t cell responses nasal-associated lymphoid tissues (nalts) support the recall but not priming of influenza virusspecific cytotoxic t cells nalts support the recall but not priming of iav-specific cytotoxic t cells new-generation screening assays for the detection of anti-influenza compounds targeting viral and host functions visualization of iav genomes at the single-cell level human dendritic cell response signatures distinguish 1918, pandemic and seasonal h1n1 influenza viruses digital cell quantification identifies global immune cell dynamics during influenza infection natural history of highly pathogenic avian influenza h5n1 middle east respiratory syndrome longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia canalization of the evolutionary trajectory of the human influenza virus within-host influenza dynamics: a small-scale mathematical modeling approach dynamically correlated mutations drive human influenza a evolution quantitative modeling of virus evolutionary dynamics and adaptation in serial passages using empirically inferred fitness landscapes supported in part by grants from the nih (niaid, po1 ai060699, ro1 ai129269). key: cord-272117-erzpz3c0 authors: downey, jeffrey; pernet, erwan; coulombe, françois; divangahi, maziar title: dissecting host cell death programs in the pathogenesis of influenza date: 2018-04-18 journal: microbes infect doi: 10.1016/j.micinf.2018.03.005 sha: doc_id: 272117 cord_uid: erzpz3c0 influenza a virus (iav) is a pulmonary pathogen, responsible for significant yearly morbidity and mortality. due to the absence of highly effective antiviral therapies and vaccine, as well as the constant threat of an emerging pandemic strain, there is considerable need to better understand the host–pathogen interactions and the factors that dictate a protective versus detrimental immune response to iav. even though evidence of iav-induced cell death in human pulmonary epithelial and immune cells has been observed for almost a century, very little is known about the consequences of cell death on viral pathogenesis. recent study indicates that both the type of cell death program and its kinetics have major implications on host defense and survival. in this review, we discuss advances in our understanding of cell death programs during influenza virus infection, in hopes of fostering new areas of investigation for targeted clinical intervention. influenza viruses cause the most recurring respiratory disease in humans, with outbreaks likely occurring since at least the middle ages [1] . present estimates indicate that each year, seasonal influenza affects 5e10% of the world's population, resulting in 3e5 million cases of severe illness and between 250 000 to 500 000 deaths [2] . in addition to seasonal outbreaks, influenza pandemics sporadically emerge, causing markedly higher mortality. in 1918, the worst pandemic in recorded history caused more than 50 million deaths worldwide [3] . since then, three other pandemics have occurred: first in 1957, then again in 1968 and finally in 2009 [4] . currently, there are major concerns surrounding the emergence of the new highly pathogenic avian influenza (hpai) viruses of h5n1, h7n9, h10n8, or the more recent h5n8dthe only hpai strain to cross from eurasia to america [5e9]. one of the key characteristics of highly infectious iav and hpai viruses is that they are poorly glycosylated and, thus, able to bypass the upper airway defense mechanisms to reach the lower respiratory tract, leading to pneumonia [10] . histopathologic studies of influenza autopsies from 1918 outline the key changes characteristic of severe iav viral pneumonia. these include "… varying degrees of acute intra-alveolar edema and/or hemorrhage, and diffuse alveolar damage in addition to necrotizing bronchitis and bronchiolitis" [11] . similarly, in the recent 2009 h1n1 outbreak, viral antigen could be found in the alveolar space, suggesting pandemic strains of iav have the capacity to infect distal areas of the lung and directly cause alveolar damage. these occurrences collectively predispose to a clinical condition termed acute respiratory distress syndrome (ards), which may occur as a complication of primary viral pneumonia [12] . ards is precipitated by cell death among the epithelialeendothelial barrier (eeb) of the distal lung, barrier rupture, fluid leakage into the alveolar lumen and respiratory insufficiency. hence, understanding and disrupting the strategies employed by the virus to damage the lower airways is critical in preserving pulmonary architecture. the most distal structure of the lung, the alveolus, is constantly patrolled by alveolar macrophages (am4) and is delimited by the eebda bi-cellular layer composed of type i and type ii alveolar epithelial cells (aec-i and aec-ii), facing the air space, and endothelial cells that form the pulmonary capillaries. the identification of the infective target cells of severe pulmonary viruses within the alveolus is critical in evaluating the potential effects of cell damage on lung function. for example, ex vivo infection of human lungs with middle east respiratory syndrome coronavirus (mers-cov)d a recent zoonotic virus with a fatality rate of 35e50% in humansdshowed that aec-i, aec-ii and endothelial cells can all be infected and killed [13e15] . in addition, while mers-cov productively replicates in human macrophages and t lymphocytes, it is also cytotoxic in these cells [16, 17] . interestingly, the tropism of the virus appears to have a significant impact on severity of disease. for instance, in comparison to mers-cov that infects both structural cells and leukocytes and causes high mortality, severe acute respiratory syndrome (sars)-cov only infects structural cells, causing less mortality [17] . in iav infection, a number of reports identify aec-ii as the primary replicative niche in the human lung for highly pathogenic strains, while low-pathogenicity strains fail to penetrate the lower airways [18e22] . hpai also infects human endothelial cells in vitro and some evidence suggests that infection of the endothelium may occur in vivo [23, 24] . in addition, iav antigens are found in human am4 and the virus can productively infect and kill human macrophages [18,19,10,23,21,25e27] . thus, similar to the highly pathogenic mers-cov, highly virulent iav shows tropism for both human alveolar structural cells and leukocytes, highlighting the severity of pandemic strains. am4 within the airways are the first leukocytes to encounter the majority of pulmonary pathogens and their initial response to infection is critical in "setting the tone" and orchestrating an effective immune response. studies examining how am4 are altered following pulmonary viral infection suggest that the loss of eeb integrity, reduction in regulatory mediators and activation of multiple pattern-recognition receptors (prr) lead to these cells becoming pro-inflammatory, rather than regulatory [28] . upon infection with iav, am4 initiate the immune response by producing chemokines and cytokines, including type i interferons (ifn-i; primarily composed of ifn-a/b), as well as instructing adaptive immune responses [25, 29] . if adequately regulated, these responses lead to effective immunity, clearance of the virus with minimal immunopathology and return to pulmonary homeostasis. if dysregulated, however, these responses can have dramatically deleterious effects, marked by impaired viral clearance, increased immunopathology and enhanced susceptibility to infection. thus, pulmonary immune responses must be tightly regulated to effectively eliminate viruses that reach the distal airways, while minimizing immunopathology in order to maintain optimal gas exchange [29] . the death of host cells as a result of infection has been recognized for over a century and it is now understood that pathogeninduced cell death may occur through a variety of complex mechanisms. initially, cell death was discussed in dichotomy as either: (1) apoptosis, considered an activedor programmeddprocess that preserves the plasma membrane and leads to the deletion of cells with little tissue disruption and no inflammation; or (2) necrosis, a passive form of cell death leading to complete disruption of the plasma membrane and spillage of cellular contents, causing inflammation and tissue damage [30] . however, this simplification of the cell death program has altered considerably over the last couple of decades. there are now several distinct forms of programmed cell death that have been described in addition to apoptosis, including pyroptosis, paraptosis and necroptosis [31] . interestingly, some of these cell death modalities have been observed in autopsies of iav-infected humans, particularly hpai cases [11, 32] ; yet, the consequences of these cell death programs on host immunity and the pathogenesis of iav are not fully understood. therefore, a more complete characterization of the forms of cell death occurring in iav infected lungs may define why certain cell types are more, or less, susceptible to iav-induced cell death and how this impacts viral propagation. furthermore, different forms of cell death may have distinct consequences on the virulence of iav strains by differentially modulating the immune response. this suggests that regulated modulation of cell death programs during iav infection may be an appealing target for anti-iav therapies. in this review, we focus on the current state of knowledge of cell death during influenza infection and explore research avenues that aim to dissect cell death programs, as well as their ensuing consequences in the pathogenesis of this threatening virus. considering the mechanisms of cell death have been extensively reviewed elsewhere [33, 34] , herein we focus on severe influenza infection and the immunological effects of influenza-induced cell death in the lung structural cells (e.g. epithelial cells) as well as immune cells (e.g. macrophages). finally, we suggest that manipulation of specific cell death programs at different stages of infection may offer avenues for novel therapy to iav by both sequestering viral replication and promoting host resistance mechanisms through immunomodulation. iav utilizes epithelial cells as its primary replicative site and all pulmonary epithelial cells are permissive to infection and subsequent replication. however, proximity of the replication to distal portions of the lung is strongly associated with an increase in virulence capacity of iav strains. for example, seasonal influenza strains, or those of low pathogenicity, replicate primarily in the upper airways and are rapidly killed in the pharynx and proximal lung by host resistance mechanisms, causing minor acute disease. on the contrary, hpai and highly pathogenic h1n1 strains bypass immune mechanisms of the upper airways to replicate in the distal portions of the lung and this distal tropism is highly correlated with primary viral pneumonia and mortality [11] . altered tropism of iav to the lower airways is thought to be mediated by two mechanisms. first, iav infects cells by its hemagglutinin (ha) binding to terminal sialic acids on the plasma membrane of cells, attached either via an a2,3 or a2,6 linkage. both a2,3 and a2,6 linkages are present throughout the human lung; however, preferential binding of a2,3 linked residues by iav hemagglutinin favors more distal and severe infections. second, glycosylation of iav hemagglutinin is inversely correlated with pathogenicity, as highly glycosylated hemagglutinins are more efficiently trapped and cleared from the upper airways by ciliary motion [35] , while poorly glycosylated iav bypass this host defense mechanism and access the lower airways [35] . for instance, the pandemic 1918 h1n1 strain exhibited only 1 glycosylation site on its hemagglutinin protein and hpai strains are known to preferably bind a2,3 residues, due to their enhanced expression on avian epithelial cells. in the lower airways, it was shown in a human lung explants model that approximately 90% of influenza positive cells are aec-ii cells, with a minor contribution of am4 and other cell types [21] . interestingly, iav replication is enhanced in aec-ii compared to bronchial epithelial cells [36] . therefore, highly pathogenic strains of iav have mechanisms to reach their preferred replicative niche in aec-ii in the distal lung, suggesting that maintenance of pulmonary epithelial cells is beneficial to the virus in promoting its replication. however, considering that iav is also lytic in epithelial cells of humans [37] and mice [38] hints that at certain time points after infection, cell death may be involved in preventing or facilitating viral propagation, escape and reinfection. therefore, the consequences of cell death in structural cells, and in particular aec-ii, on host immunity to iav must diverge depending on the kinetics of the infection. apoptosis comes from the ancient greek word meaning "falling off" and is the most characterized form of programmed cell death. proceeding in a non-inflammatory manner, apoptosis likely evolved to meet the growing demand to remove aging or damaged cells from the body as more complex and multicellular organisms appeared and, as a result, the apoptotic machinery remains evolutionarily conserved. indeed, at homeostasis in an adult human, it is estimated that between 100 and 200 billion cells are being cleared daily via apoptosis [39] . this number can then augment dramatically in developmental stages and aging. thus, the physiological demand for cellular turnover in a non-inflammatory manner is essential to maintain homeostasis. the signature of early apoptosis involves condensing of chromatin within the nucleus, compartmentalization of cellular organelles and the shrinking of the cell membrane. ultimately, these compartments bleb off in the form of apoptotic bodies, which are phagocytosed and degraded by macrophages and other phagocytic cells in a process known as efferocytosis [39] . importantly, the cell membrane of apoptotic cells remains intact throughout and the cell shrinks rather than ruptures, as appearing in necrotic forms of cell death. thus in apoptotic cells, there is no leakage of cytosolic contents to initiate an inflammatory response [33] . there are two major pathways that lead to apoptosis: intrinsic and extrinsic, both of which primarily converge on a family of proteins called caspases that cleave cellular substrates and lead to the morphological signature of apoptosis [40] . intrinsic apoptosis is initiated by the mitochondrion in response to internal cellular stress and involves activation of caspase 9 by cytochrome c. extrinsic apoptosis, on the other hand, is dependent upon death ligands of the tnf superfamily like trail or fasl binding their cognate cell surface receptors and the subsequent activation of caspase 8 [41, 42] . in addition to its physiological role, apoptosis is involved in promoting resistance or susceptibility to intracellular pathogens. for example, virulent mycobacterium tuberculosis (mtb) contains an anti-apoptotic gene (nuog) that diminishes both innate and adaptive immunity to promote the bacterial growth [43] . in contrast to mtb, apoptosis induced by listeria monocytogenes is an immune evasion strategy, allowing the bacteria to disseminate [44] . thus, it appears that apoptosis can be both protective and detrimental to the host depending on the pathogen. interestingly, both extrinsic and intrinsic pathways of apoptosis were shown to be activated in influenza-infected cells [45] . this observation is well established, being described in human autopsies for almost a century, beginning with the 1918 pandemic, where pronounced epithelial desquamation, hyalination and sloughing were noted [37] . experimentally, apoptosis of iav-infected epithelial cells was shown to be dependent upon viral replication, as an inactivated virus failed to induce apoptosis in mice [46] and human cells [47] . moreover, the magnitude of epithelial cell apoptosis was positively associated with iav strain pathogenicity in vivo [32, 23, 48] and in vitro [38, 49] in both mice and humans. however, whether apoptosis promotes host resistance or iav dissemination remains to be determined. logically, apoptosis of epithelial cells could represent a resistance strategy to iav, as it eliminates the intracellular niche required for viral replication. support for the protective capacity of apoptosis is that critical host antiviral factors actively induce epithelial cell apoptosis. upon viral infection, ifn-i (ifn-b) is rapidly produced, following recognition of the viral genome by pattern-recognition receptors (prr). in iav infection, rna sensing is predominantly mediated by the cytosolic retinoic acid-inducible gene (rig-i), which interacts with the mitochondrial antiviral signaling protein (mavs) at the mitochondrial outer-membrane to initiate ifn-i production mainly via the transcription factor interferon regulatory factor 3 (irf3) (reviewed in ref. [50] ). ifn-i then signals through a heterodimeric receptor complex of ifnar1 and 2, leading to sustained ifn-i (largely ifn-a) production and an upregulation of a large subset of genes, together termed interferon stimulated genes (isgs). isgs then serve to halt viral replication through both direct or indirect mechanisms. interestingly, both ifn-i and isgs have been shown to induce apoptosis. following iav infection, ifn-i signaling activates caspase-dependent apoptosis in aec-ii cells [51] , and the pharmacological inhibition of these caspases blocks apoptosis and enhances viral replication. additionally, several classical apoptosis proteins, including caspase 8 and trail, are isgs upregulated by ifn-i signaling. thus, iav sensing causes the release of ifn-i that induces epithelial cell apoptosis directly through caspase activation downstream of the ifnar1/2 complex, and indirectly by promoting the transcription of pro-apoptotic isgs. other isgs have been equally implicated in promoting apoptosis in iav-infected cells as an antiviral mechanism. for example, protein kinase r (pkr) is an isg known to inhibit iav replication through a variety of mechanisms. pkr can directly sense dsrna generated during viral replication to induce fas expression and fadd-dependent apoptosis [52] , as well as inhibit host and viral protein translation through the phosphorylation of eif2a [53] in iav-infected cells [54] . moreover, pkr has been shown to increase ifn-i production in fibroblasts directly in response to another rna virus: semliki forest virus [55] . thus, it appears pkr and other isgs contribute to antiviral immunity by promoting ifn-i responses, restricting viral protein translation and inducing apoptosis. in addition to being directly antiviral, apoptosis also protects against iav infection through the resolution of the immune response. in response to hpai, bronchiolar epithelial cell apoptosis limits excess pro-inflammatory cytokine (ie. tnf) release to dampen immunopathology and avoid the onset of ards [56] . furthermore, a recent study has implicated club cells, a subset of bronchiolar epithelial cells, in augmenting immunopathology to an h1n1 iav virus, by failing to undergo apoptosis following viral clearance [57] . therefore, the protective capacity of apoptosis in structural cells is both antiviral and immunomodulatory. given the importance of apoptosis in antiviral immunity, it is not surprising that iav has encoded factors to block its induction. the principal iav virulence factor, non-structural protein 1 (ns1), is rapidly transcribed upon infection and is the first protein expressed in infected cells. ns1 blocks early apoptosis in epithelial cells and fibroblasts by inhibiting ifn-i induction [58] and activating the host pro-survival pi3k/akt pathway to facilitate viral replication [59] . importantly, these functions are critical in viral fitness, as strains lacking a functional ns1 are severely impaired in their virulence. taken together, all the above studies highlight the importance of apoptosis of structural cells in promoting immunity to influenza, by limiting the intracellular niche necessary for viral replication. however, the notion that apoptosis solely represents a hostdriven antiviral strategy is confounded by the fact that iav encodes both anti-and pro-apoptotic factors. for example, h5n1 ns1 facilitates airway epithelial apoptosis [60, 49] , suggesting a strainor expression level-specific role for this protein in iav pathogenesis. furthermore, recent study has characterized h1n1 iav nucleoprotein (np) as a pro-apoptotic viral protein in human airway epithelial cells [61] that targets the host's anti-apoptotic protein ap15 [62] . in fact, host apoptotic machinery has been shown to be essential in promoting new virion production in epithelial cells in vitro [63e65], in a caspase-3[66] and -9dependent manner [67] , and in vivo by iav-manipulation of annexin-a1 [68] . these findings outline iav as an effective regulator of the host's apoptotic machinery in structural cells, capable of both inducing and blocking apoptosis to further its pathogenesis. the paradoxical role of apoptosis in immunity to iav, which appears to both prevent and permit viral dissemination, can perhaps be explained by the kinetics of the apoptotic response in epithelial cells (fig. 1) . immediately upon infection, it is beneficial for iav to block epithelial cell apoptosis to avoid destroying its replicative niche and this is primarily mediated by viral ns1. early blockage of apoptosis by iav is counteracted by host mechanisms, such as ifn-i signaling, to induce apoptosis and resist viral replication [69] . yet, following initial replication cycles, at later time points, iav must activate apoptotic pathways to generate new infectious virions, promote budding at the cell surface and facilitate subsequent rounds of infection in neighboring cells. thus, pharmacological inhibition of apoptosis in humans during the later stages of infection may offer appealing therapeutic avenues, either by blocking pro-apoptotic pathways [65] or enhancing antiapoptotic proteins [64] . interestingly, neutralization of proapoptotic trail or fas signaling post-iav infection in aec-ii cells decreased iav load [70] . similarly, mice treated with decoy fas in vivo to block fasl signaling were protected from lethal iav infection, when compared to untreated mice [71] . our understanding of the interplay between influenza, host apoptotic machinery and resistance mechanisms has increased exponentially recently. however, much of our knowledge still derives from study in vitro using human or mouse cells and, thus, the exact effects of these pathways on disease outcome remain to be determined. like apoptosis, the observation that iav causes necrosis in epithelial cells has long been established. yet, the impact of iavinduced epithelial cell necrosis on the host immune response, and the factorsdviral or host-deriveddinvolved in this process are just beginning to become known. canonically, necrosis was considered a passive form of cell death associated with increased injury or disease severity. however, it is now well established that many forms of necrosis are, in fact, programmed. necrotic cell death is delineated from apoptosis by a disruption of the cell membrane and spilling out of cytosolic contents that act as dangerassociated molecular patterns (damps). these damps are sensed by neighboring cells, resulting in a robust inflammatory response [72, 73] , thus, generating a potential positive feedback loop of tissue damage and immunopathology. the observation that hpai [11] and pandemic h1n1 often induce necrosis in epithelial cells, while seasonal h3n2 appears only to cause it in immune compromised hosts [48] , may at least partly explain the association between severity of disease and necrosis of the epithelium. moreover, other strains of highly pathogenic viruses such as dengue virus [74] or mers-cov [75] appear to induce considerable necrosis in epithelial cells to promote viral replication and propagation, while less virulent viruses like rhinovirus, or sars-cov induce limited or no necrosis [76, 75] . in vitro, h1n1 iav induces necrosis in human lung bronchiolar epithelial cells, coupled with massive release of proinflammatory cytokines and neutrophil chemoattractant cxcl8, which is highly suggestive of a link between necrosis of the epithelium and ards [77] . collectively, these observations promote the notion that epithelial cell necrosis is detrimental to the host and is consequential of severe infection. the most characterized form of programmed necrosis is ripk3dependent programmed necrosis (termed necroptosis). necroptosis is dependent upon the formation of the necrosome, which consists of ripk1 and ripk3, interacting through their rip homotypic interaction motifs (rhim). typically, ripk1 and ripk3 are cleaved by caspase-8, causing their inactivation and leading to extrinsic apoptosis. however, when caspase-8 activity is compromised, the necrosome is activated to induce necroptosis. known mediators downstream of the necrosome include mixed lineage kinase domain like pseudokinase (mlkl). mlkl is a substrate for ripk3 and induces necroptosis by a combination of the assembly of a pore-forming complex at the plasma membrane, or as a platform for ca 2þ -mediated necrosis [78] . although the exact executioner mechanisms of necroptosis remain unknown, recent study has implicated the plasma membrane repair machinery escrt iii as a potential mediator [79] . necroptosis regulates a variety of inflammatory processes, including ischemic heart disease, bacterial infection and embryogenesis [80] . interestingly, there is accumulating evidence for a role of necroptosis in promoting immunity to viral infection. seminal work showed necroptosis to be essential in antiviral defense, as ripk3 à/à mice succumbed to vaccinia virus infection [81] . moreover, herpes simplex viruses (hsv-1/2) and murine cytomegalovirus (mcmv) encode inhibitors of necroptosis, ul39 and m45 respectively, suggesting that viruses may block necroptosis as part of their pathogenesis [82, 83] . although no iav inhibitors of necroptosis have yet been identified; most recently, ripk3-dependent necroptosis of murine fibroblasts and alveolar epithelial cells was demonstrated to be critical in promoting iav clearance from the lungs and, as a result, ripk3-deficient mice were highly susceptible to iav infection coupled with enhanced viral loads [84] . thus, this study hints at a protective role for necroptosis in immunity to iav. however, human studies are required to investigate the potential clinical targeting of the necroptotic pathway in protection against iav infection. certainly, apoptosis and, more recently, necrosis have dominated study of iav-mediated cell death. however, the role of other forms of cell death in immunity to infectious diseases, such as iav, is beginning to be recognized. pyroptosis is a form of rapid inflammatory cell death with cell membrane rupture and dna damage, which is dependent upon caspase-1 and certain nod-like receptors. pyroptosis is coupled with proinflammatory il-1b and il-18 release from pyroptotic cells, which contributes in part to its inflammatory nature [85] . caspase-1 activation and subsequent cytokine release induced by iav in murine lung fibroblasts has been shown to inhibit viral pathogenesis [86, 87] . yet, whether caspase-1 activation in these models led to pyroptosis, or rather defined a pyroptosis-independent role for caspase-1 was not carefully elucidated. thus, more study of the role of caspase-1 activation and pyroptosis in immunity to iav is needed. although epithelial cells constitute the major replicative niche for iav, the lung is a complex organ and thus other structural cells can be infected with iav. pulmonary fibroblasts are the most substantial subset of structural cells in the lung interstitium, playing a significant role in the repair process to injurious exposure and pulmonary inflammatory disorders, such as asthma and copd [88] . fibroblasts are used extensively in iav studies as a model of lung structural cells. as in epithelial cells, fibroblasts are sensitive to both iav-induced apoptosis [58] and necroptosis [84] in vitro. however, the contribution of fibroblasts in immunity to iav infection is not well understood. whether fibroblasts are a target for iav infection in vivo and/or whether their response is analogous to, or completely independent of, epithelial cells, requires further investigation. understanding the role of fibroblasts in the pathogenesis of iav may provide additional targeted therapy against iav infection. similarly, endothelial cells represent a substantial compartment of the lung and in vitro infection of human endothelial cells led to productive iav replication and increased vascular leak, mediated by apoptosis [89] , particularly upon infection with hpai (e.g. h5n1) [90] . hpai has been shown to disseminate from the lung systemically upon infection in mice [91] . this has equally been observed in humans, where viral rna was found in multiple organs, including the brain and placenta, of fatal cases of h5n1 [92, 93] . the ability of hpai viruses to disseminate to other tissues is predominately mediated by changes to the cleavage sites of the viral ha protein, which is essential for viral entry into cells. in the case of low virulence seasonal strains of iav, ha cleavage occurs at a single basic arginine by trypsin-like proteases found exclusively in cells of the respiratory tree and gastrointestinal tract. on the other hand, hpai ha proteins contain polybasic cleavage sites that permit cleavage by furin proteases that are ubiquitously expressed. thus, this allows hpai strains to disseminate and cause systemic infection [94] . additionally, pandemic and highly virulent strains of the virus, including hpai and the 1918 h1n1 strain, are known to completely exhaust the replicative niche of epithelial cells of the lung over the course of infection as a by-product of overly exuberant replication and failure of immune response to control viral propagation. thus, it is appealing to associate the infection of endothelial cells by hpai viruses with subsequent systemic dissemination, made possible by the polybasic cleavage sites. however, this is complicated by the fact that no virus was found in endothelial cells of fatal cases of h5n1 infection [93] , yet virus was found in hyaline membranes and endothelial cells of fatal cases of the 2009 h1n1 pandemic, where dissemination to other organs was not noted [23] . as there is no known evolutionary advantage for iav to disseminate to other organs, a greater level of study is required to confirm what role infection of endothelial cells in humans plays and whether endothelial cell death is a pro-host or pro-pathogen strategy. during influenza virus infection, the pulmonary inflammatory response is characterized by early infiltration of leukocytes, including neutrophils and mononuclear cells and later by adaptive immune lymphocytes [95, 96] . autopsy reports of humans infected with hpai and pathogenic strains of h1n1 exhibited severe pulmonary leukopenia, which together constituted important clinical features of severe infection and fatal cases [97, 32] . importantly, this leukopenia was mainly contributed to iav-induced death of these cells at the site of infection, rather than failed recruitment or effector function in the lung [98e100]. interestingly, pulmonary iav infection has also been shown to have profound effects on bone marrow hematopoiesis in an ifn-i-dependent manner [101] . thus, the observed leukopenia could alternatively be explained by other mechanisms, such as exhaustion of the bone marrow hematopoietic stem cells due to severe infection. hematopoietic stem cell exhaustion is a phenomenon often observed during chronic bacterial or viral infections [102, 103] , but remains poorly characterized in iav infections. nevertheless, these findings illustrate the importance of preserved leukocyte viability and function in host defense to highly pathogenic strains of iav and activation of the cell death program in these cells may represent a strategy of immune evasion by iav. although iav has been shown to contribute to cell death in the majority of leukocytesdincluding neutrophils, dendritic cells and lymphocytesdthis review will focus on cell death in pulmonary monocyte/macrophages, given their critical role in the initial response to the infection. pulmonary macrophage populations change considerably during iav infection. at homeostasis, tissue resident am4 constitute the vast majority of cells in the airways and are the first leukocytes to encounter the virus, following replication in epithelial cells [29] . am4 can then be readily infected [104, 105] and activated to secrete a spectrum of cytokines and chemokines that orchestrate both innate and adaptive immune responses to iav and other pulmonary viral infections [106, 25, 107] . over the course of infection, am4 populations are diminished, while inflammatory monocytederived macrophages (md-m4, also called exudate macrophages) expand and then contract during the resolution phase [108] . both am4 and md-m4 have been linked to host protection and disease pathogenesis depending on the magnitude of activation of the immune response, which is directly associated with disease tolerance [108] . invariably, loss of am4 causes increased susceptibility to infection, due to reduced production of antiviral ifn-i (mainly ifn-b) leading to uncontrolled viral replication and increased immunopathology [106, 25, 107, 109] . thus, am4 represent a logical target for iav to induce cell death and, indeed, several studies have reported early apoptosis post-infection in am4 [110e112]. similarly, recruited m4 are highly permissive to iav infection and susceptible to iav-induced cell death [26, 113] . collectively, these findings suggest that in contrast to the biphasic role of epithelial cell apoptosis in preventing or promoting pathogenesis, highly virulent iav rapidly infects and induces early death in pulmonary m4 to suppress antiviral responses. apoptosis occurs in m4 during iav infection and can be either triggered intrinsically by viral proteins or activated extrinsically by host factors in the pulmonary microenvironment. while the induction of apoptosis by viral proteins has been studied extensively in structural cells, fewer studies have investigated it in m4 [114, 115, 112, 116, 117] . interestingly, influenza virus contains an essential gene, polymerase basic protein 1-frame 2 (pb1-f2, a product of an alternate reading frame of the pb1 gene), which increases apoptosis-mediated pathogenicity in iav [118] . functionally, pb1-f2 has been shown to be the main inducer of apoptosis in monocytes/m4, but not epithelial cells [116, 112] . the pro-apoptotic effect of pb1-f2 is elicited by its translocation to the mitochondrion [119] . in mitochondria, pb1-f2 interacts with components of the permeability transition pore complex (ptpc) ant3 and vdac1 [120] to disrupt the mitochondrial inner membrane potential, followed by cytochrome c release and the induction of apoptosis, which subdues the m4 antiviral capacity and promotes host susceptibility [116] . iav-induced apoptosis in m4 is not solely caused by viral protein expression and may result from the activation of the extrinsic pathway by host factors [110, 115, 121] . interestingly, iav-infected m4 are the main source of the key antiviral cytokine: ifn-i [25, 122, 116] and can induce m4 cell death via the upregulation of isgs. moreover, infection of human m4 with hpai strains induces trail release to trigger the apoptotic cascade [123, 27] . equally, upregulation of fas and secretion of fasl at later time points postinfection have been shown to induce apoptosis selectively in recruited m4 [115, 121] , a process which is thought to contribute to immune contraction and the return to homeostasis. of note, although tnf has been reported to induce apoptosis in iav-infected epithelial cells, it does not appear to mediate m4 apoptosis [45] . thus, during iav infection, activation or inhibition of apoptotic pathways are cell-type specific and can either promote or prevent iav pathogenesis. therefore, this complex network certainly needs to be interrogated more intensely. the outcome of iav-induced m4 apoptosis is still controversial, as it may favor both the host and the virus. nonetheless, much of the literature suggests a host-detrimental effect of m4 apoptosis. for instance, hpai and pandemic h1n1 influenza viruses hijack innate antiviral responses by inducing apoptosis in m4 to decrease m4-antiviral cytokines (primarily ifn-i) and promote viral replication [27, 123, 124] . considering residential am4 are the first immune cells encountered by the influenza virus and are critical in mounting an immune response, it is not surprising that iav efficiently infects and kills am4 to suppress their antiviral responses. thus, shortly after severe iav infection, the original pool of resident am4 decreases, resulting in enhanced viral replication [110, 105] . similarly, limiting am4 cell death through the exogenous administration or overexpression of gm-csfdthe critical growth and maintenance factor for am4dis protective, leading to lowered pulmonary viral load, decreased immunopathology and increased survival of mice after iav infection [125, 109] . in addition to inhibiting the early antiviral responses, iav-induced m4 apoptosis might skew the balance from a protective to pathological immune response. during iav infection, it has been shown that mice lacking type i ifn signaling die from an uncontrolled inflammatory response, as ifn-i signaling was essential to induce anti-inflammatory il-10 production by "regulatory" m4 [126] da subset of m4 known to be more susceptible to iav-induced apoptosis [113] . mechanistically, iav-induced m4 apoptosis is mainly mediated by the viral protein pb1-f2 and its expression has been linked to delayed viral clearance [120] . expression of the 1918 pb1-f2 protein in a mouse-adapted h1n1 strain resulted in early increased viral titers and death of infected m4 [127] . to prevent pb1-f2-induced early m4 apoptosis, we have recently shown that the host mitochondrial nlr-member nlrx1 directly interacts with pb1-f2 within mitochondria to disarm its apoptotic function. in iavinfected nlrx1 à/à mice, or m4 in vitro, higher levels of apoptosis were observed compared to wt m4. this was associated with reduced production of ifn-i and increased viral loads in vitro and in vivo [116] . thus, by inducing m4 cell death, iav hijacks the host's key antiviral responses, resulting in both enhanced iav replication/ dissemination and impairment of the anti-inflammatory response, which drives immunopathology and potentially ards (fig. 2) . however, recent findings indicate that in some cases, apoptosis of pulmonary m4 may be beneficial for the host. indeed, early apoptosis of hpai-infected porcine am4 correlated to decreased viral replication and magnitude of the inflammatory response [112] . aside from viral proteins, our laboratory recently demonstrated that iav induces production of the host eicosanoid prostaglandin e 2 (pge 2 ) to directly inhibit early production of ifn-i, which increased viral loads through reduced m4 apoptosis. thus, whether apoptosis is protective or pathological seems, again, to be connected to the kinetics of its induction. early post-infection, m4 viability is essential to promote antiviral immunity and this is countered by iav proteins such as pb1-f2. later, however, m4 apoptosis becomes protective, as the apoptotic vesicles stimulate adaptive immunity by promoting cross-presentation [25] . to conclude, apoptotic cell death of macrophages appears to be a double-edged sworddboth beneficial and detrimental to the host, depending on iav virulence factors and host mediators. further work is needed with closer inspection of the kinetics of expression of viral proteins (e.g. pb1-f2, ns1) as well as host factors (eg. nlrx1, ifn-i, eicosanoids) and their link with kinetics of m4 cell death (early versus late induction) to fully understand whether timing or, rather, specific pathways or types of apoptosis dictate this complex phenomenon. despite apoptosis being the main m4 death modality observed in iav infections, there is also evidence of necrosis [128, 32] and, specifically, of the more recently characterized ripk3-mediated necroptosis. however, iav-induced macrophage necrosis has been poorly studied in vivo and most of our knowledge is confined to in vitro observation. at low multiplicities of infection (moi), necrosis is not substantially induced in m4 [116, 26, 129] . in addition, we and others have recently shown that iav does not induce ripk3mediated necroptosis in m4, both in vivo and in vitro, but, rather, ripk3 plays a crucial role in the production of il-1b [129] and ifn-i [130] and, as a result, mice deficient in ripk3 are extremely susceptible to iav infection. however, following severe infection with a high viral titer, iav induces necroptosis through the ripk1eripk3ecaspase 8 complex in vitro [86] , suggesting a potential role for ripk3-mediated necroptosis in depletion of m4 during infection with highly virulent strains. synergy between these two observations may come from the pleiotropic function of ifn-i, which has been also shown to induce m4 necroptosis in vitro or in vivo during bacterial infections [131e133]. thus, it can be envisioned that dysregulated ripk3-mediated ifn-i production in severe infection may, in fact, become detrimental to the host by inducing necroptosis of pulmonary m4, suggesting a threshold for protective ifn-i production and deleterious immunopathology induced by m4 necroptosis. of note, pathways known to induce ifn-i in response to iav and other viruses, such as tlr3 [134] , pkr [132] and dna-dependent activator of ifn-regulatory factors (dai) [135] have also been shown to trigger necroptosis, advocating a potential link between type i ifn production and necroptosis induction. finally, iav-infected macrophages produce il-1b through inflammasome activation and caspase-1 cleavage, which are characteristics of pyroptotic cell death [136, 129] . however, there was no difference in cell death in iav-infected nlrp3 à/à (lacking the nlrp3 inflammasome) and wt m4, in vitro [86] , suggesting that pyroptosis does not occur in m4 during iav infection. certainly, further investigation is required to fully understand the precise mechanisms involved in this process. the lung represents a diverse microenvironment filled with numerous cellecell interactions with high turnover under homeostatic and pathological conditions. in particular, epithelial cells of the ebb and am4 exist in close proximity and are known to regulate each other's functions at homeostasis and upon infection. over the last ten years, we have gained appreciable knowledge of how pulmonary macrophages induce apoptosis in infected epithelial cells. in 2008, herold et al. showed for the first time that secretion of trail by m4 induced apoptosis of aec and contributed to lung injury during iav infection [137] . further investigation also revealed that secretion of ifn-i, and specifically ifn-b, by infected am4 was responsible for autocrine trail expression and epithelium injury [138] . most importantly, an upregulation of trail expression and secretion was observed in am4 from pandemic h1n1-infected individuals [138] . in addition to potentiating lung injury, this could potentially lead to viral dissemination by promoting efferocytosis. efferocytosis is the phagocytosis of apoptotic bodies released from dying cells by neighboring m4 [139] . recently, annexin a1, a key protein involved in efferocytosis [140, 141, 148] , was shown to facilitate iav replication and dissemination in vivo [68] . interestingly, tcherniuk et al. showed that newly formed viral particles contained annexin a1 on their membranes, increasing their infectivity and viral replication through enhanced uptake [142] . in addition, activation of formyl peptide receptor 2/lipoxin a4 receptor (fpr2/alx) via annexin a1, increased pulmonary viral load and susceptibility to infection [142] . hence, one can envision a model wherein the induction of epithelial cell apoptosis by am4derived ifn-i leads to the presence of annexin a1-expressing viral particles on the surface of infected cells. these new viral particles are, in turn, recognized and taken up by bystander m4, infecting them and preventing their antiviral responses. this leads to increased viral replication that subsequently enhances the magnitude of the immune responsedthus, continuing this vicious cycle of lung injury/reinfection. this may potentially explain how hpai and pandemic h1n1 viruses strip the pulmonary epithelium and leave the host leukopenic, as seen in fatal cases. therefore, discovery of factors that can mitigate this pathological cycle would be of clinical significance in combatting highly pathogenic strains of iav. despite influenza virus infections likely dating back to the middle ages and considerable burden on global health, there is no effective therapy against iav. to prevent infection, vaccination strategies are deployed worldwide. however, the protection conferred by vaccination is highly variable, due to the ability of the virus to shift antigens, leading to vaccine mismatch and a decrease in efficacy. antiviral therapies, like neuraminidase inhibitors (i.e. oseltamivir) that block viral egress to potentially ameliorate disease are currently in use. despite considerable efficacy in murine models in lowering viral titers and improving disease outcome [143, 144] , its protective effect in humans is controversial and limited benefit has been shown in the majority of patients [145, 146] . additionally, highly resistant strains of iav to oseltamivir have recently emerged that raises growing concern [147] . thus, these factors collectively suggest that the dogma of only targeting the virus and not the host's immune response in the generation of anti-iav drugs needs to be revisited. indeed, there are several "proof-of-concept" studies highlighting the promising therapeutic potential of targeting host cell death programs during iav-infection. first, anti-trail treatments selectively attenuated epithelial cells apoptosis, lung injury and increased survival of mice after iav infection. secondly, we recently demonstrated that targeted inhibition of mpges-1, the key enzyme in pge 2 production, selectively regulated pulmonary m4 apoptosis to boost antiviral and immunoregulatory properties. importantly, mpges-1 inhibition was effective even after three days of iavinfection, coinciding with the onset of symptoms in humans. in line with this, a recent study by hung et al. showed a reduction in viral titers, hospital stay and mortality when individuals infected with h3n2 iav were given a combinatory therapy of clarithromycin (antibiotic) enaproxen (cox inhibitor) e oseltamivir (neuraminidase inhibitor) [147] , compared to oseltamivir treatment alone, highlighting the importance of combining immunomodulatory therapies and those directly targeting the pathogen. additionally, we suggest ripk3 as an attractive target for immunomodulatory therapy. recently, the antiviral capabilities of ripk3 have been shown to be at least twofold, by both inducing epithelial cell necroptosis and promoting ifn-i responses from pulmonary m4 to potentiate survival to lethal iav infection. therefore, agonists of ripk3 may promote viral clearance via both epithelial cells and macrophages and ultimately confer survival to severe disease. this shows that combining treatments that differentially target structural cell and pulmonary m4 can significantly improve the outcome of influenza virus infection. the constant exposure of humans to potentially life-threatening respiratory pathogens presents ongoing challenges to clinicians. the knowledge gained from the studies of cell death programs in epithelial cells and macrophages during iav infection provide significantly better understanding of host mechanisms involved in protective or pathological immunity to iav infection. however, further investigation is still required to identify the kinetics of cell death during influenza infection, which appears to be critical in regulating antiviral and anti-inflammatory responses. in addition, most of our knowledge comes from in vitro experiments and thus a need for greater tools to investigate cell death in vivo and discovery of new cellular markers of the various cell death programs will be required for developing a novel therapy in influenza virus infection. . all authors declare that no 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hospitalized for influenza a (h3n2) infection: an open-label randomized, controlled, phase iib/iii trial annexin1 regulates dc efferocytosis and cross-presentation during mycobacterium tuberculosis infection key: cord-321112-w7x1dkds authors: zhao, xuesen; li, jiarui; winkler, cheryl a.; an, ping; guo, ju-tao title: ifitm genes, variants, and their roles in the control and pathogenesis of viral infections date: 2019-01-08 journal: front microbiol doi: 10.3389/fmicb.2018.03228 sha: doc_id: 321112 cord_uid: w7x1dkds interferon-induced transmembrane proteins (ifitms) are a family of small proteins that localize in the plasma and endolysosomal membranes. ifitms not only inhibit viral entry into host cells by interrupting the membrane fusion between viral envelope and cellular membranes, but also reduce the production of infectious virions or infectivity of progeny virions. not surprisingly, some viruses can evade the restriction of ifitms and even hijack the antiviral proteins to facilitate their infectious entry into host cells or promote the assembly of virions, presumably by modulating membrane fusion. similar to many other host defense genes that evolve under the selective pressure of microorganism infection, ifitm genes evolved in an accelerated speed in vertebrates and many single-nucleotide polymorphisms (snps) have been identified in the human population, some of which have been associated with severity and prognosis of viral infection (e.g., influenza a virus). here, we review the function and potential impact of genetic variation for ifitm restriction of viral infections. continuing research efforts are required to decipher the molecular mechanism underlying the complicated interaction among ifitms and viruses in an effort to determine their pathobiological roles in the context of viral infections in vivo. interferon-induced transmembrane proteins (ifitms) are a family of small proteins that localize in the plasma and endolysosomal membranes. ifitms not only inhibit viral entry into host cells by interrupting the membrane fusion between viral envelope and cellular membranes, but also reduce the production of infectious virions or infectivity of progeny virions. not surprisingly, some viruses can evade the restriction of ifitms and even hijack the antiviral proteins to facilitate their infectious entry into host cells or promote the assembly of virions, presumably by modulating membrane fusion. similar to many other host defense genes that evolve under the selective pressure of microorganism infection, ifitm genes evolved in an accelerated speed in vertebrates and many single-nucleotide polymorphisms (snps) have been identified in the human population, some of which have been associated with severity and prognosis of viral infection (e.g., influenza a virus). here, we review the function and potential impact of genetic variation for ifitm restriction of viral infections. continuing research efforts are required to decipher the molecular mechanism underlying the complicated interaction among ifitms and viruses in an effort to determine their pathobiological roles in the context of viral infections in vivo. keywords: host susceptibility, interferon-induced transmembrane proteins, ifitm, single nucleotide polymorphisms, viral infection interferon-induced transmembrane proteins (ifitms) are a family of small proteins that can be found in single cell organisms and are evolutionally conserved across vertebrates (siegrist et al., 2011; zhang et al., 2012) . the human ifitm family comprises five members, including immune-related ifitm1, ifitm2, and ifitm3, as well as ifitm5 and ifitm10 with no known role in immunity. as key host defense genes, ifitms evolved under the selective pressure of microorganism infection (compton et al., 2016) . ifitm proteins are involved in many aspects of virus-host interaction and play important roles in viral pathogenesis. among the number of single-nucleotide polymorphisms (snps) in ifitm3 gene that have been identified in human populations, several are associated with disease severity and prognosis of influenza a virus (iav) and other viral infections (everitt et al., 2012; zhang et al., 2015; xu-yang et al., 2016; allen et al., 2017) . mechanistically, these snps either alter the expression of ifitm3 or result in expression of n-terminally truncated ifitm3 isoform, 21-ifitm3, with reduced antiviral activity against different viruses (everitt et al., 2012; allen et al., 2017) . in this review, we will summarize the findings on human ifitm structural features related with antiviral activity, and impact of genetic variation on ifitm antiviral function in the control and pathogenesis of viral infections in humans. to date, ifitms have been shown to inhibit the infection of enveloped rna viruses from 9 viral families , non-enveloped rna viruses, e.g., reovirus (anafu et al., 2013) and foot-and-mouth disease virus , and several dna viruses (li et al., 2018) . ifitms efficiently inhibit a number of medically important human pathogenic viruses, including iav (brass et al., 2009; bailey et al., 2012) , dengue virus (denv) (brass et al., 2009; jiang et al., 2010) , west nile virus (wnv) (brass et al., 2009; jiang et al., 2010) , zika virus (zikv) (savidis et al., 2016) , ebola virus (ebov) (huang et al., 2011; wrensch et al., 2015) , marburg virus (marv) (huang et al., 2011) , severe acute respiratory syndrome coronavirus (sars-cov) (huang et al., 2011) , rift valley fever virus (rvfv) (mudhasani et al., 2013) , hantaan virus (htnv) (mudhasani et al., 2013; xu-yang et al., 2016) , hepatitis c virus (hcv) (wilkins et al., 2013) , and human immunodeficiency virus (hiv) (lu et al., 2011; compton et al., 2014; yu et al., 2015; compton et al., 2016; foster et al., 2016; chesarino et al., 2017; tartour et al., 2017; wang et al., 2017) . in vivo studies in ifitm3 knockout mice demonstrate the critical role of ifitm3 in restricting infection and reducing disease severity of infection by iav (bailey et al., 2012; everitt et al., 2012) , wnv , chikungunya virus and venezuelan equine encephalitis virus , and respiratory syncytial virus . ifitm3 in mice not only protects lung epithelia cells from iav infection, but it was also shown to restricts iav infection of lung dendritic cells, which traffic to lymph nodes to prime cd8 + t cell anti-viral response (infusini et al., 2015) . moreover, lung resident memory cd8 + t cells in mice were programmed to retain ifitm3 expression, facilitating their survival and protection from viral infection during subsequent exposures (wakim et al., 2013) . ifitm proteins localize at the plasma membrane as well as the membranes of endocytic vesicles and lysosomes (bailey et al., 2014) . ifitms can also be incorporated into envelope membranes of many viruses (yu et al., 2015; tartour et al., 2017) . emerging evidence suggests that ifitm proteins on both viral and cellular membranes can restrict the infectious entry of diverse envelope viruses by inhibiting viral fusion at cell plasma or endolysosomal membranes, but ifitm does not impede endocytosis of virions into cells li et al., 2013; perreira et al., 2013; compton et al., 2014; desai et al., 2014) . as extensively discussed in a previous review , the potency of ifitm restriction of viral cell entry is generally correlated to the co-localization of ifitm proteins at the sites of viral fusion. for instance, ifitm1 more efficiently restricts the viruses that enter the cytoplasm via direct fusion with plasma membrane or via rab-5 positive early endosomes, whereas ifitm3 more efficiently inhibits viruses that enter via rab7-positive late endosomes or lysosomes. this rule is highlighted by the finding that mutation of ifitm3 endocytic signal results in its cell surface accumulation and gains a function to restrict the infection of human parainfluenza virus 3 (hpiv-3), which enters cells via direct fusion with the plasma membrane (rabbani et al., 2016; zhao et al., 2018) . another example is that the sensitivity of iavs to ifitm3 appears to depend on the ph value at which the iav hemagglutinin triggers membrane fusion and thus the endocytic compartments where the membrane fusion take place (gerlach et al., 2017) . however, exceptions of this rule do exist. for instance, it remains to know how moloney leukemia virus (mlv) and sendai virus that fuse at cell plasma membrane (brass et al., 2009; hach et al., 2013) as well as lassa fever virus (lasv) and lymphocytic choriomeningitis virus (lcmv) that fuse at rab7-positive late endosomes (brass et al., 2009; mudhasani et al., 2013) to escape ifitm1 and ifitm3 restriction, respectively. the mechanism of ifitm inhibition of viral fusion and cell entry is not yet resolved. one study reported that ifitm inhibited jaagsiekte sheep retrovirus envelope and iav hemagglutinin fusion of viral envelope to cellular membranes prior to coalescence of lipid-bilayers, a process known as hemifusion . however, another study using a direct viruscell fusion assay in viable cells to investigate iav entry found that overexpression of ifitm3 protein in late endosomes did not alter lipid mixing, but rather inhibited the release of viral contents into the cytoplasm, suggesting that ifitm3 inhibits the transition from hemifusion to full fusion of the respective lipid membranes (desai et al., 2014) . others studies suggested that ifitm multimerization decreases membrane flexibility by altering membrane curvature, with the consequence of interruption of the virus-cell membrane fusion (john et al., 2013; lin et al., 2013) . another group reported that ifitm expression increased cholesterol content in the endosome or lysosome via an interaction with vesicle-associated membrane protein-associated protein a (vapa), which abrogated endolysosomal fusion with the viral envelop (amini-bavil-olyaee et al., 2013). however, this later observation was not confirmed by other studies (desai et al., 2014; wrensch et al., 2014) . in addition, ifitms were reported to affect the trafficking of vacuolar atpase (v-atpase), implying that ifitms may indirectly restrict viral entry by modulating endosomal acidity (wee et al., 2012) . it is also well documented that the antiviral potency of ifitm proteins varies among different cell types (huang et al., 2011; zhao et al., 2018) , suggesting that ifitms work together with other cellular proteins to modulate viral fusion. in support of this notion, zinc metallopeptidase ste24 (zmpste24), a transmembrane metalloprotease localized in the inner nuclear membrane and cytoplasmic organelles, had been identified as a downstream partner of ifitm3 to restrict the entry of enveloped rna and dna viruses (fu et al., 2017) . in addition to the protective function of ifitm proteins to reduce viral infection of host cells, ifitm proteins, particularly ifitm2 and 3, can lead to the production of virions that package ifitms and display reduced entry into target cells (compton et al., 2014; tartour et al., 2014 tartour et al., , 2017 . one study found that ifitm proteins interact with hiv envelope protein in viral producer cells to disrupt envelope protein processing and virion incorporation, which impairs virion infectivity (yu et al., 2015) ; however, another found that ifitm3 expression in producing cells did not affect the amount of envelope protein incorporation into progeny hiv-1 virions (appourchaux et al., 2018) . both studies reported that the level of ifitm protein incorporation into progeny virions does not correlate with the extent of infectivity reduction. moreover, recent studies have also shown that ifitms can modulate viral infection and pathogenesis via mechanisms that are not directly related to the restriction of virus entry. for example, given the resistance of human papillomaviruses (hpv) to ifitms, hpv infection of keratinocytes inhibits the expression of ifitm1 and ripk3 to escape from ifnγ and tnf-α-mediated antiproliferation and necroptosis, which is essential for establishing a persistent infection (ma et al., 2016) . in addition, it was demonstrated in ifitm3 knockout mice that ifitm3 limited murine cmv (mcmv) pathogenesis without directly preventing virus replication (stacey et al., 2017) . instead, ifitm3 contributed to the antiviral cellular immunity by abrogating inflammatory cytokine-driven lymphopenia including apoptosis-independent nk cell death and t cells depletion (stacey et al., 2017) . not surprisingly, in the arms race between pathogen and host, many viruses have evolved strategies to evade the antiviral function of host restriction proteins. for instance, transmitted founder hiv-1 strains establishing de novo infection are generally capable of evading ifitm restriction (foster et al., 2016; wang et al., 2017) . mutations allow the virus to escape adaptive immune responses, and/or switches in the hiv-1 co-receptor tropism from ccr5 to cxcr4 increases viral sensitivity to inhibition by ifitm2 and ifitm3 in endosomal compartments (foster et al., 2016) . moreover, iav facilitates its infection by activating p53 or degrading eukaryotic translation initiation factor 4b (eif4b) to inhibit the expression of ifitm proteins (wang s. et al., 2014; wang et al., 2018) . in contrast, human coronavirus oc43 (hcov-oc43) and human cytomegalovirus (hcmv) hijack ifitm3 to promote its infectious entry and progeny virions assembly, respectively (zhao et al., 2014; xie et al., 2015) . the human ifitm locus, approximately 18kb long, is located on chromosome 11 and comprises five genes: ifitm1, ifitm2, ifitm3, ifitm5, and ifitm10 (figure 1) . as an ifn-stimulated gene (isg), ifitm1, ifitm2, ifitm3 genes each has an interferon stimulated response element (isre) in its promoter region besides the additional gamma-activated sequence (gas) in the promoter region of ifitm1 gene (siegrist et al., 2011) . however, the protein expression of ifitm5 and ifitm10 are not induced by ifns (zhang et al., 2012) . though ifitm1-3 proteins are ubiquitously expressed in human tissues in the absence of ifn induction, they can be robustly up-regulated by all three types of ifns (zhao et al., 2014) . the ifitm3 promoter has binding sites for dozens of transcription factors, including polr2a, myc, elf1, phf8, chd1, taf1, rest, sin3ak20, sin3a, irf1, stat1, tbp, stat3, stat2, zbtb7a, and ctcf, some of which may affect ifitm3 expression . as illustrated in figure 1 , all ifitm genes contain two coding exons interspersed by one intron. both ifitm2 and ifitm3 were predicated to encode a wild typed full-length form and a truncated isoform with 20/21 amino acid residues deletion from n-terminus. most vertebrate animals have two or more ifitm genes. in many primate species, gene duplication, and divergence of ifitm3 has been identified (zhang et al., 2012; compton et al., 2016) . ifitm locus in many modern primate species contains multiple copies of ifitm3-like genes due to gene duplication (compton et al., 2016) . for instance, marmoset, macaque, and africa green monkey (agm) each has five, six and eight copies of ifitm3 genes, respectively (compton et al., 2016) . comparative genomics studies indicate that ifitm3 is the most ancient member in the ifitm family and ifitm2 emerges as a rather recent genetic event in human, chimpanzee, and gorilla (compton et al., 2016) . the multiplicity and diversity of ifitm2/3 indicates that there is a positive selection in ifitm evolution, which is consistent with their role in restricting pathogen invasion (compton et al., 2016) . ifitm proteins have several topologies that may affect their function. as shown in figure 2a , although ifitm3 may adopt three different membrane topologies (bailey et al., 2014) , it exists predominantly as a type ii transmembrane protein with the n-terminus in cytosol and the short c-terminus exposed to cellular exterior or in the lumen of endolysosome (bailey et al., 2013) . although ifitm1 was also reported to predominately adopt a type ii transmembrane topology, other membrane topologies may exist ( figure 2b ; li et al., 2015) . as depicted in figure 3 , ifitms consist of intramembrane (imd) and transmembrane (tmd) domains separated by an intracellular loop (cil) and variable n and c terminal domains (ntd and ctd), respectively (bailey et al., 2013 (bailey et al., , 2014 chesarino et al., 2017) . while the ntd and ctd are highly variable in length and sequence among ifitm orthologs and paralogs, the canonical cd225 domain spanning imd to cil domains is evolutionally more conserved (bailey et al., 2014) . critical structure motifs and amino acid residues undergoing post-translational modifications required for ifitm oligomerization as well as their biological and antiviral functions are discussed below. compared to ifitm1, ifitm2 and ifitm3 have n-terminal 20-aa or 21-aa extension which was previously regarded to figure 1 | ifitm gene structure. human ifitm genes located in chromosome 11p15.5 are indicated, and the exons of immunity-related ifitm genes (ifitm1, ifitm2, and ifitm3) are indicated in blue color. the putative transcripts of ifitm2 and ifitm3 are also shown below. the transcription factor binding regions derived from ucsc genome browser are shown, which is verified by chip-seq from the 161 proteins tested through encode; grch37/hg19. two red vertical lines across transcription factor binding sites represent the influenza associated snps of rs12252 and rs34481144 with allele frequencies, respectively. n-terminally truncated ifitm3 isoform ( 21 ifitm3) that is presumably generated by rs12252 is indicated. be absent in rs12252-c encoding ifitm3 isoform. although deletion of this n-terminal 21-aa region significantly impaired its ability to inhibit the infection by iav, vsv, and denv, the deletion apparently did not affect its ability to enhance hcov-oc43pp infection john et al., 2013) . interestingly, 21-ifitm3 enhanced inhibition of hiv-1 fusion (compton et al., 2016) . moreover, 20-ifitm2, a n-terminal 20-aa truncated isoform of ifitm2, which is derived from an alternatively initiated rna transcript (figure 1) , demonstrated a more potent suppression on hiv-1 infection than that by fulllength wild-type ifitm2 (wu et al., 2017) . in fact, a recent study argues that the 20-ifitm2, rather than the full-length ifitm2 and ifitm3, is the effective restriction factor of hiv-1 with cxcr4-tropism (wu et al., 2017) . the 20 yxx 23 motif of ifitm3 is an endocytic signal essential for endocytosis and localization of ifitm3 to endocytic vesicles and lysosomes (jia et al., 2014) . artificial mutations of 20 yeml 23 motif (y20d, y20a, or l23a) result in an accumulation of ifitm3 in the plasma membrane and reduced inhibition of viruses that enter the cytoplasm at endocytic vesicles/lysosomes, such as iav, human coronaviruses nl63 and -229e (chesarino et al., 2014a; jia et al., 2014; williams et al., 2014; zhao et al., 2018) , but enhanced inhibition of viruses that directly enter cells at the plasma membranes, such as hpiv-3 and hiv-1 (jia et al., 2012; compton et al., 2016; rabbani et al., 2016) . intriguingly, replacement of ifitm3 tyrosine (y) 20 with either alanine (a) or aspartic acid (d) to mimic unphosphorylated or phosphorylated ifitm3 converted the antiviral protein to enhance the entry of sars-cov and mers-cov . these studies suggest that the ntd, particularly 20 yxx 23 motif, plays important roles in ifitm2/3 subcellular localization and antiviral activity. ifitm3 are phosphorylated by the protein-tyrosine kinase fyn on tyrosine 20 (y20), which results in its plasma membrane accumulation and decreased antiviral activity against influenza viruses (jia et al., 2012 (jia et al., , 2014 chesarino et al., 2014a) . this result is consistent with the mutagenesis studies of 20 yxx 23 motif, where y20 is part of an endocytosis signal that can be blocked by phosphorylation (chesarino et al., 2014a) . additionally, phosphorylation of ifitm3 by fyn and mutagenesis of y20 also lead to decreased frontiers in microbiology | www.frontiersin.org ifitm3 ubiquitination, suggesting modification of y20 as an important mechanism to control ifitm3 trafficking and degradation (chesarino et al., 2014a) . compared to ifitm2 and ifitm3, human ifitm1 has a relatively long c-terminal region of 18 amino acid residues. the 122 krxx 125 motif serves as a sorting signal for ifitm1. substitution of two basic residues 122 kr 123 with alanine (kr/aa) in ifitm1 reduced its distribution in lamp1-positive lysosomes but enriched its localization in cd63-positive multivesicular bodies . the kr/aa mutant ifitm1 executed increased activity to inhibit the infection by jaagsiekte sheep retrovirus (jsrv) and 10a1 amphotropic murine leukemia virus (mlv) . interestingly, although sars-cov and hcov-nl63 share the ace2 receptor for infection of host cells, deletion of the c-terminal 3, 6, or 9 amino acids did not apparently affect the activity of ifitm1 to inhibit sars-cov entry but enhanced the activity of ifitm1 to inhibit the entry of hcov-nl63 . however, further deletion of the c-terminal 12, 15, or 18 amino acids significantly attenuated or even abolished the ability of ifitm1 to inhibit the entry of sars-cov, but did not apparently affect the entry of nl63 . more strikingly, deletion of c-terminal 12-aa or 18-aa converted ifitm1 to a potent enhancer of mers-cov and hcov-oc43 entry (zhao et al., 2014 . a recent mutagenesis study indicated that amino acid residues 127-132 in the ctd domain of ifitm3 differentially modulates its activities in target cell protection and negative imprinting of progeny hiv-1 infectivity (appourchaux et al., 2018) . these studies clearly indicate that the ctd of ifitms contains multiple structure motifs that regulate the subcellular localization and antiviral functions. the canonical cd225 domain comprises imd to cil domains and is evolutionally conserved (bailey et al., 2014) . imd domain (residues 58-80) is a hydrophobic region and possesses an amphipathic alpha helix spanning residues 59-68 (chesarino et al., 2017) . it was demonstrated recently that either deletion of this alpha helices or mutations altering amphipathicity largely impaired or even abolished ifitm antiviral activity, suggesting figure 3 | schematic diagram of ifitm1/3 topology and structural determinants essential for their modulation of viral entry. five structural domains, including the n-terminal domain (ntd), amphipathic helix domain (ahd), intracellular loop (cil), transmembrane domain (tmd), and c-terminal domain (ctd), are illustrated. two membrane-associated domains (ahd and tmd) are embodied in light orange. in ifitm3, three hydrophilic residues (s61, n64, and t65) in amphipathic helix are labeled in violet. in ntd domain, 20 yeml 23 motif required for ifitm3 endocytosis are depicted in blue with red circle and 17 ppny 20 motif recruiting nedd4 e3 ligase are shown in green. in cil domain, 81 svks 84 motif required for ifitm3 to inhibit iav, denv, and llov infection is indicated with purple circle. the residues with post-translational modification, including phosphorylation (y20 and y99 labeled with red circle), palmitoylation (c71, c72, and c105 marked with light blue), and ubiquitination (highlighted in orange), are indicated. in addition, two residues (f75 and f78) essential for oligomerization are marked with yellow. in ifitm1, c-terminal 12-aa residues critical for modulating human coronaviruses entry is indicated in blue, and 122 kr 123 dibasic motif constituted as ifitm1 sorting signal is depicted with red circle. a critical role in ifitm antiviral function, presumably through affecting membrane physical properties (chesarino et al., 2017) . in addition, the 81 svks 84 motif residing in cil domain is essential for ifitm3 to inhibit iav and denv infection, but replacement of this motif with four residues of alanine promoted cellular entry driven by lloviu virus (llov) glycoprotein (wrensch et al., 2015) . ifitm proteins can be palmytoylated at three cystines in cd225 domain by multiple zinc finger dhhc domain-containing palmitoyltransferases (zdhhcs) . while cys105 localizes close to the amphipathic alpha helix in the imd domain, cys71 and cys72 are adjacent to the tmd domain. mutagenesis studies indicated that substitution of those three cysteine residues with alanine alters the ifitm3 distribution from punctate clusters in the cellular membrane to a more diffused pattern and the resulting palmitoylation-deficient mutant showed impaired or even abolished antiviral activity (yount et al., 2010) . interestingly, when other lipid modification sites, such as myristoylation and prenylation, were introduced in ifitm ntd or ctd domains, the antiviral function of palmitoylation-deficient ifitm3 mutant could be restored. these findings imply that anchoring ifitm protein to membranes, but not structure alteration by s-palmitoylation, is important for ifitm restriction of virus entry (yount et al., 2012; chesarino et al., 2014b) . human ifitm proteins possess four conserved lysine residues (figure 2 ) that can be ubiquitinated by e3 ubiquitin ligase such as nedd4 (yount et al., 2012; chesarino et al., 2015) . previous studies demonstrated that each lysine residue can be modified with mono-and poly-ubiquitination through lys-48 and lys-63 linkages (yount et al., 2012) . by replacing all four residues of lysine with alanine, the mutant ifitm3 demonstrated endolysosomal distribution and execute hyperactivity to restrict iav infection than wild type (yount et al., 2012) . however, our studies demonstrated that ubi-deficient ifitm3 lost antiviral activity against all the viruses tested, including iav (zhao et al., 2014 . the discrepancy of those studies is not clear. importantly, ubiquitination and endocytosis of ifitm3 is required for mtor inhibitor-induced degradation of ifitm3 (shi et al., 2018) . in addition, ifitm3 k88 can be monomethylated by lysine methyltransferase set7 and demethylated by histone demethylase lsd1 (shan et al., 2013 (shan et al., , 2017 . while vesicular stomatitis virus (vsv) and iav infection increased ifitm3-k88me1 levels by promoting the interaction between ifitm3 and set7 and disassociation from lsd1 to attenuate ifitm3 antiviral activity, ifn-α reduced ifitm3-k88me1 levels and increased its antiviral activity (shan et al., 2013 (shan et al., , 2017 . ifitm proteins function as homo-or hetero-oligomers. two phenylalanine residues (f75 and f78) are essential for ifitm homo-and hetero-oligomerization (john et al., 2013) . ifitm3 bearing f75a and f78a mutations (ifitm3/2fa) showed reduced ability to inhibit the entry of hcov-nl63, but lost ability to inhibit or enhance the entry of all other tested viruses (zhao et al., 2014 . the antiviral activity of many naturally existing human ifitm variants has been tested in cell cultures. as shown in table 1 and mentioned in previous sections, although snp rs12252 is predicted to encode a splice variant specifying a n-terminally truncated isoform ifitm3 (everitt et al., 2012) , the putative 21-ifitm3 protein has not been detected in the tissues or cells of affected subjects (wu et al., 2017; makvandi-nejad et al., 2018) . however, subcellular localization and antiviral activity of genetically engineered 21-ifitm3 have been extensively investigated in cultured cells (john et al., 2013; compton et al., 2016) . interestingly, ifitm2 was reported recently to have a similar n-terminally truncated isoform ( 20-ifitm2) expressed in human innate immune cells and cd4 + t cells (wu et al., 2017) . compared with the full-length wildtype ifitm2, 20-ifitm2 demonstrated an increased plasma membrane accumulation and enhanced antiviral activity against hiv-1, particularly, hiv-1 with cxcr4 tropism (compton et al., 2016; wu et al., 2017) . in addition, expression and antiviral activity of some human nonsynonymous ifitm3 variants have also been investigated in cell cultures (john et al., 2013) . while many of the snps results in undetectable levels of ifitm3 in the transfected cells, presumably due to the reduced stability of mutant proteins, and thus loss of antiviral activity against iav, a few snps did not apparently alter the amount of ifitm proteins but impaired the antiviral activity against iav. it will be interesting to test all the ifitm variants against a group of viruses and identify the snps that potentially impact the control and pathogenesis of specific viral infections in humans. as a potent viral restriction factor, ifitms play a pivotal role in limiting the infection by multiple viruses and any genetic variation affecting the ifitm expression or function might contribute to viral pathogenesis. thus, natural variation in ifitm genes and its association with illness severity has been extensively investigated. currently, a dozen of snp in ifitm3 have been reported, some of which may modulate ifitm expression, affect rna splicing, or result in nonsynonymous or synonymous variants ( table 1) . several snps affecting function of ifitms have been investigated for their association with morbidity, severity and prognosis of microorganism infection (everitt et al., 2012; shen et al., 2013; zhang et al., 2013 zhang et al., , 2015 allen et al., 2017) . the most studied snp associated with severe outcomes of iav infection is snp rs12252, which is a nonsynonymous variation in the first exon of ifitm3 (everitt et al., 2012) . the substitution of the major allele of t with alternative allele of c was predicted to alter ifitm3 mrna splicing and generate a n-terminally truncated variant of ifitm3 with 21 amino acid residues deletion ( 21-ifitm3) (everitt et al., 2012) . the snp rs12252 has a higher prevalence in the population of east asia than europe (0.528 vs. 0.041) (everitt et al., 2012) . moreover, the homozygous cc genotype and heterozygous tc genotype have significantly higher frequency in east asia than that in europe (0.300 vs. 0.000 and 0.456 vs. 0.082, respectively) (everitt et al., 2012) . rs34481144 located at the ifitm3 promoter is associated with severe influenza in three human cohorts . snp rs34481144, wherein the majority g allele is replaced with a minor a allele, controls ifitm3 promoter activity and determines the expression level. compared to the g allele, a allele has activities of decreased irf3 binding and increased ctcf binding, thus resulting in lower ifitm3 expression. snp rs34481144 has diverse allele and genotypic frequencies in different human populations ( table 1) . the allele frequency of rs34481144-g is higher in the population of east asia and africa than that in europe (0.957 and 0.994 vs. 0.538, respectively) . also, the homozygous gg genotype has several groups previously reported that ifitm3 snp of rs12252 is associated with the susceptibility and severity of patients with seasonal influenza and pandemic h1n1 and h7n9 iav infection (everitt et al., 2012; zhang et al., 2013; pan et al., 2017) . as summarized in table 2 , everitt et al. (2012) first discovered that a higher allelic frequency of rs12252-c exists in caucasian hospitalized influenza patients than healthy control. moreover, they observed a remarkably higher frequency of the homologous cc genotype in hospitalized influenza patients compared to the normal european population (5.7% vs. 0.3%) (everitt et al., 2012) . importantly, another group reported that the minor c allele of rs12252 in the caucasian population is much more prevalent in han chinese and japanese and is associated with disease severity in patients with ph1n1/09 iav infection (zhang et al., 2013) . in line with this observation, results obtained from study of h7n iav infection revealed that h7n9 infected patients with rs12252-cc genotype exhibited accelerated disease progression and increased mortality rate than patients with the tc and tt genotype . these studies collectively suggest that rs12252-c is a risk allele associated with severe iav infection. however, several recent studies from european or africa-american ethnic groups did not support the association of rs12252 with severe influenza infection (mills et al., 2014; lopez-rodriguez et al., 2016; randolph et al., 2017) . the rare frequency of rs12252-c in european population may account for this discrepancy. through meta-analysis of 11 studies, rs12252 t > c was associated with risk to severe influenza infection with odds ratio of 1.69 (95% ci 1.23, 2.33) in both european and east asian populations, but for the mild infection, the results remained uncertain (prabhu et al., 2018) . although the association of rs12252 with the susceptibility to influenza infection and disease severity was observed by many studies, the mechanism for this association is largely unknown. as mentioned above, although rs12252-c is speculated to encode a n-terminal truncated ifitm3 with attenuated antiviral activity to impede iav entry, the truncated variant has not been detected so far (everitt et al., 2012; makvandi-nejad et al., 2018) . moreover, the homozygous cc genotype was demonstrated to only express full-length ifitm3 at a similar level to tt genotype (wu et al., 2017) . thus rs12252-c genetic variant does not appear to affect the biochemical nature and expression of ifitm3. obviously, further investigation to determine whether rs12252-c influences ifitm3 gene splicing or protein levels in a cell type-specific manner and whether this snp co-segregates with a different causative allele is warranted. a recent study found that another ifitm3 snp rs34481144 has a strong association with disease severity in three influenza cohorts . rs34481144 is located in the promoter region of ifitm3 (figure 1) . the substitution of the majority allele of g with minority allele of c reduces ifitm3 expression level and consequently results in the reduced number of antiviral cd8 + t cells in lung tissue upon iav infection . in cohort flu09, a cohort of naturally acquired influenza infection, a higher frequency of homozygosity of risk a allele was observed in patients with severe illness than the mild cases (33.3% vs. 1.3%) ( table 3 ; allen et al., 2017) . also, an increased frequency p-value refers to the differences of allele frequencies between mild influenza patients and severe influenza patients. frontiers in microbiology | www.frontiersin.org of the a allele was found in patients who suffered with severe influenza in other two cohorts (table 3 ; allen et al., 2017) . thus, rs34481144-a is a risk allele associated with severe iav infection and its association with other viral infection disease deserves to be investigated in future. in addition to influenza, snp of rs12252 was also observed to have association with development of aids and hantaan virus associated hemorrhagic fever with renal syndrome (hfrs) (zhang et al., 2015; xu-yang et al., 2016) . zhang et al. (2015) reported that rs12252 is associated with rapid progression of aids, but not the susceptibility to hiv infection. patients of aids rapid progressors had a higher frequency of rs12252 cc and ct genotype than non-progressors (zhang et al., 2015) . compared with tt genotype, more patients with cc/ct genotypes were characterized with higher viremia level and more significantly reduced cd4 t + cells (zhang et al., 2015) . in addition, the association of rs12252-c and homozygous cc genotype with disease severity in hfrs patients infected by hantaan virus was reported recently (xu-yang et al., 2016) . a higher allelic frequency of rs12252-c was observed in severe hfrs patients hospitalized than healthy han chinese controls (68.29% vs. 52.16%). also, severe hfrs patients had a higher frequency of rs12252 cc than patients with mild hfrs and healthy han chinese. along these lines, this group also reported that the viral titer detected in the plasma of hfrs patients with rs12252-cc genotype was more significantly alleviated than those with the tc and tt genotype (xu-yang et al., 2016) . thus, rs12252 c is a risk allele associated with severity of aids and hfrs, indicating that snp rs12252 may have a profound impact on the pathogenesis of multiple viral diseases. ifitms are a group of small fusogenic proteins that restrict the infection of broad spectrum of viruses by inhibiting the fusion of viral and cellular membranes. however, whether ifitms inhibit hemifusion, the process whereby the outer, but not the inner, leaflet of the viral and cellular membranes merge, or the transition from hemifusion to pore formation remains to be rigorously determined. it is also important to note that ifitms do not always inhibit virus entry, but may promote membrane fusion under selected conditions, such as in the case of hcov-oc43 infection (zhao et al., 2014) . moreover, our recent studies indicated that specific mutations can flip the biological activity of ifitms, from inhibiting to promoting the infection of selected human coronaviruses . those later findings strongly suggest that the fusogenic activity of ifitms might be bidirectional and could be regulated by viral and host factors. the elucidation of molecular mechanisms underlying ifitm-mediated innate immunity via modulation of viral entry into host cells as well as the negative imprinting of progeny virions (virions incorporating ifitms display decreased infectivity) will open new avenues for future research. we have only just begun to appreciate the role of ifitm proteins in innate antiviral defenses. genetic association studies of rare and common variants may explain population-specific and individual variance in susceptibility to common viral pathogens and identify specific ifitm -virus interactions. expansion of genetic studies incorporating gene knock-down or knockoff screens, single cell transcriptomics, epigenetic modification, and targeted sequencing for different viral infections will provide needed insights into cellular mechanisms and pathways involved in ifitm-mediated host response to viral exposure and infection. all the authors participated in the draft and revision of this review article. the project was supported by grants from the u.s. national institutes of health (ai113267), the national natural science foundation of china (81571976 and 81772173), the commonwealth of pennsylvania through the hepatitis b foundation as well as federal funds from the national cancer institute, national institutes of health, under contract hhsn26120080001e. this project was supported in part by the intramural research program of the nih, national cancer institute, center for cancer research. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor do mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. snp-mediated disruption of ctcf binding at the ifitm3 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and pathogenesis of arthritogenic and encephalitic alphaviruses association between ifitm3 rs12252 polymorphism and influenza susceptibility and severity: a meta-analysis identification of interferon-stimulated gene proteins that inhibit human parainfluenza virus type 3 evaluation of ifitm3 rs12252 association with severe pediatric influenza infection the ifitms inhibit zika virus replication histone demethylase lsd1 restricts influenza a virus infection by erasing ifitm3-k88 monomethylation negative regulation of interferon-induced transmembrane protein 3 by set7-mediated lysine monomethylation a functional promoter polymorphism of ifitm3 is associated with susceptibility to pediatric tuberculosis in han chinese population mtor inhibitors lower an intrinsic barrier to virus infection mediated by ifitm3 the small interferon-induced transmembrane genes and proteins the antiviral restriction factor ifn-induced transmembrane protein 3 prevents cytokine-driven cmv pathogenesis ifitm 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membrane organizers by influencing clathrin and v-atpase localization and function interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms ifitm1 is a tight junction protein that inhibits hepatitis c virus entry ifitm3 polymorphism rs12252-c restricts influenza a viruses interferon-induced transmembrane protein-mediated inhibition of host cell entry of ebolaviruses ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms delta20 ifitm2 differentially restricts x4 and r5 hiv-1 human cytomegalovirus exploits interferon-induced transmembrane proteins to facilitate morphogenesis of the virion assembly compartment swine interferoninduced transmembrane protein, sifitm3, inhibits foot-and-mouth disease virus infection in vitro and in vivo interferon-induced transmembrane protein 3 inhibits hantaan virus infection, and its single nucleotide polymorphism rs12252 influences the severity of hemorrhagic fever with renal syndrome s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (ifitm3)-mediated resistance to influenza virus palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 ifitm proteins restrict hiv-1 infection by antagonizing the envelope glycoprotein interferon-induced transmembrane protein-3 rs12252-c is associated with rapid progression of acute hiv-1 infection in chinese msm cohort interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals evolutionary dynamics of the interferon-induced transmembrane gene family in vertebrates interferon induction of ifitm proteins promotes infection by human coronavirus oc43 identification of residues controlling restriction versus enhancing activities of ifitm proteins on entry of human coronaviruses we want to thank the critical comments and suggestions by dr. jinhong chang and julia ma on this manuscript. key: cord-288705-f3zqhpx1 authors: slaine, patrick; kleer, mariel; duguay, brett; pringle, eric s.; kadijk, eileigh; ying, shan; balgi, aruna d.; roberge, michel; mccormick, craig; khaperskyy, denys a. title: thiopurines activate an antiviral unfolded protein response that blocks viral glycoprotein accumulation in cell culture infection model date: 2020-10-01 journal: biorxiv doi: 10.1101/2020.09.30.319863 sha: doc_id: 288705 cord_uid: f3zqhpx1 enveloped viruses, including influenza a viruses (iavs) and coronaviruses (covs), utilize the host cell secretory pathway to synthesize viral glycoproteins and direct them to sites of assembly. using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-tg) and 6-thioguanosine (6-tgo), that selectively disrupted the processing and accumulation of iav glycoproteins hemagglutinin (ha) and neuraminidase (na). selective disruption of iav glycoprotein processing and accumulation by 6-tg and 6-tgo correlated with unfolded protein response (upr) activation and ha accumulation could be partially restored by the chemical chaperone 4-phenylbutyrate (4pba). chemical inhibition of the integrated stress response (isr) restored accumulation of na monomers in the presence of 6-tg or 6-tgo, but did not restore na glycosylation or oligomerization. thiopurines inhibited replication of the human coronavirus oc43 (hcov-oc43), which also correlated with upr/isr activation and diminished accumulation of orf1ab and nucleocapsid (n) mrnas and n protein, which suggests broader disruption of coronavirus gene expression in er-derived cytoplasmic compartments. the chemically similar thiopurine 6-mercaptopurine (6-mp) had little effect on the upr and did not affect iav or hcov-oc43 replication. consistent with reports on other cov spike (s) proteins, ectopic expression of sars-cov-2 s protein caused upr activation. 6-tg treatment inhibited accumulation of full length s0 or furin-cleaved s2 fusion proteins, but spared the s1 ectodomain. dbeq, which inhibits the p97 aaa-atpase required for retrotranslocation of ubiquitinated misfolded proteins during er-associated degradation (erad) restored accumulation of s0 and s2 proteins in the presence of 6-tg, suggesting that 6-tg induced upr accelerates erad-mediated turnover of membrane-anchored s0 and s2 glycoproteins. taken together, these data indicate that 6-tg and 6-tgo are effective host-targeted antivirals that trigger the upr and disrupt accumulation of viral glycoproteins. importantly, our data demonstrate for the first time the efficacy of these thiopurines in limiting iav and hcov-oc43 replication in cell culture models. importance secreted and transmembrane proteins are synthesized in the endoplasmic reticulum (er), where they are folded and modified prior to transport. during infection, many viruses burden the er with the task of creating and processing viral glycoproteins that will ultimately be incorporated into viral envelopes. some viruses refashion the er into replication compartments where viral gene expression and genome replication take place. this viral burden on the er can trigger the cellular unfolded protein response (upr), which attempts to increase the protein folding and processing capacity of the er to match the protein load. much remains to be learned about how viruses co-opt the upr to ensure efficient synthesis of viral glycoproteins. here, we show that two fda-approved thiopurine drugs, 6-tg and 6-tgo, induce the upr in a manner that impedes viral glycoprotein accumulation for enveloped influenza viruses and coronaviruses. these drugs may impede the replication of viruses that require precise tuning of the upr to support viral glycoprotein synthesis for the successful completion of a replication cycle. secreted and transmembrane proteins are synthesized in the endoplasmic reticulum (er), where 51 they are folded and modified prior to transport. during infection, many viruses burden the er with 52 the task of creating and processing viral glycoproteins that will ultimately be incorporated into 53 viral envelopes. some viruses refashion the er into replication compartments where viral gene 54 expression and genome replication take place. this viral burden on the er can trigger the cellular 55 unfolded protein response (upr), which attempts to increase the protein folding and processing 56 capacity of the er to match the protein load. much remains to be learned about how viruses co-57 opt the upr to ensure efficient synthesis of viral glycoproteins. here, we show that two fda-58 approved thiopurine drugs, 6-tg and 6-tgo, induce the upr in a manner that impedes viral 59 glycoprotein accumulation for enveloped influenza viruses and coronaviruses. these drugs may 60 impede the replication of viruses that require precise tuning of the upr to support viral 61 glycoprotein synthesis for the successful completion of a replication cycle. 62 enveloped viruses encode integral membrane proteins that are synthesized and post-65 translationally modified in the endoplasmic reticulum (er) prior to transport to sites of virion 66 assembly. when er protein folding capacity is exceeded, the accumulation of unfolded proteins 67 in the er causes activation of the unfolded protein response (upr) whereby activating 68 transcription factor-6 (atf6), inositol requiring enzyme-1 (ire1) and pkr-like endoplasmic 69 reticulum kinase (perk) sense er stress and trigger the synthesis of basic leucine zipper (bzip) 70 transcription factors that initiate a transcriptional response (1) . upr gene expression causes the 71 accumulation of proteins that attempt to restore er proteostasis by expanding er folding capacity 72 and stimulating catabolic activities like er-associated degradation (erad) (2). erad ensures 73 that integral membrane proteins that fail to be properly folded are ubiquitinated and 74 retrotranslocated out of the er for degradation in the 26s proteasome. there is accumulating 75 evidence that bursts of viral glycoprotein synthesis can burden er protein folding machinery, and 76 that enveloped viruses subvert the upr to promote efficient viral replication (3, 4) . 77 influenza a viruses (iavs) encode three integral membrane proteins: hemagglutinin (ha) 78 neuraminidase (na) and matrix protein 2 (m2). ha adopts a type i transmembrane topology in 79 the er, followed by addition of n-linked glycans, disulfide bond formation, and trimerization prior 80 to transport to the golgi and further processing by proteases and glycosyltransferases (5-11); na 81 adopts a type ii transmembrane topology in the er, is similarly processed by glycosyltransferases 82 and protein disulfide isomerases, and assembles into tetramers prior to traversing the secretory 83 pathway to the cell surface (12, 13). the small m2 protein also forms disulfide-linked tetramers in 84 the er, which is a prerequisite for viroporin activity (14-16). iav replication causes selective 85 activation of the upr; ire1 is activated, but perk and atf6 are not (17), although the precise 86 mechanisms of regulation remain unknown. furthermore, chemical chaperones and selective 87 chemical inhibition of ire1 activity inhibit iav replication, suggesting that ire1 has pro-viral 88 effects. ha is sufficient to activate the upr (18) and is subject to erad-mediated degradation 89 (19) . by contrast, little is known about how na and m2 proteins affect the upr. however, these inhibitors triggered sg formation and cytotoxic effects in uninfected cells as well, 137 limiting their potential utility as antivirals. because sg formation correlates with antiviral activity, 138 we conducted an image-based high-content screen to identify molecules that selectively induce 139 sg formation in iav infected cells. we identified two fda-approved thiopurine analogs, 6-140 thioguanine (6-tg) and 6-thioguanosine (6-tgo), that blocked iav and hcov-oc43 replication 141 in a dose-dependent manner. unlike pateamine a and silvestrol, these thiopurines selectively 142 disrupted the processing and accumulation of viral glycoproteins, which correlated with upr 143 activation. synthesis of viral glycoproteins could be partially restored in 6-tg treated cells by the 144 chemical inhibition of the upr or isr. our data suggest that upr-inducing molecules could be 145 effective host-targeted antivirals against viruses that depend on er processes to support efficient 146 replication. induction of upr by 6-tg and 6-tgo represents a novel host-directed antiviral 147 mechanism triggered by these drugs and reveals a previously unrecognized unique mechanism of 148 action that distinguishes them from other closely related thiopurines and nucleoside analogues. through this screen, we identified two thiopurines, 6-thioguanine (6-tg) and 6-166 thioguanosine (6-tgo) (fig. 1a) , that triggered dose-dependent sg formation in iav-infected 167 cells (fig. 1b) . specifically, sgs formed in approximately 10 % of 6-tg-treated or 6-tgo-treated 168 infected cells; no sgs were detected in mock infected cells treated with either drug at the highest 169 concentration (fig. 1b) . these findings were confirmed in parental a549 cells infected with iav 170 strain a/california/07/2009 (h1n1; iav-ca/07); 6-tg treated cells displayed the formation of 171 foci that contained sg constituent proteins g3bp1 and poly a binding protein (pabp) (fig. 1c) . 172 these foci also contained canonical sg proteins tiar and eif3a (fig. 1d) , supporting their 173 identity as bona fide sgs. 174 next, we wanted to determine whether thiopurine-mediated sg formation indicated a 176 disruption of viral replication. a549 cells were infected with iav strain a/puertorico/8/1934 177 (h1n1; iav-pr8) and treated with 6-tg, 6-tgo or controls at 1 hpi. cell supernatants were 178 harvested at 24 hpi and infectious virions enumerated by plaque assay. despite sg induction in 179 only a fraction of virus-infected cells, we observed a sharp dose-dependent decrease in virion 180 production following treatment with either thiopurine analog. treatment with 2 m 6-tg reduced 181 virion production by ~10-fold, whereas 2 m 6-tgo reduced virion production by ~100-fold (fig. 182 2a) . furthermore, treatment of iav infected cells with 10 µm concentrations of either 6-tg or 6-183 tgo led to even greater inhibition of iav production ( fig. 2a ). this suggests that sg formation 184 correlates with the disruption of the viral replication cycle. however, the sharp decrease in 185 infectious virion production in 6-tg/6-tgo-treated cells suggests that sg formation is not 186 required for their antiviral effect. the nucleoside analog 5-fluorouracil (5-fu) had no effect on 187 iav replication at 2 m and 10 m doses ( fig. 2a) . using an alamarblue assay, we observed a 188 ~30% reduction in a549 cell viability in the presence of 10 m doses of 6-tg/6-tgo (fig. 2b) . 189 compared to sg-inducing translation inhibitor silvestrol, which causes apoptosis in a549 cells 190 upon prolonged exposure, we did not observe significant disruption of cell monolayer by 6-tg 191 treatment ( fig. 2c ) or induction of apoptosis as measured by parp cleavage (fig. 2d ). this is 192 consistent with a recent report of 6-tg-mediated cytostatic rather than cytotoxic effects on a549 193 cells (46). in vero cells, 6-tg treatment partially protected cellular monolayers from iav-induced 194 cell death over 72-h incubation (fig. 2e ). taken together, our data suggest that 6-tg and 6-tgo 195 elicit a broad dose-dependent antiviral effect against iav that was not shared by the nucleoside 196 analog 5-fu. faster-migrating, presumably un-glycosylated species, but these were difficult to visualize as they 211 migrated to the same position as np on immunoblots probed with polyclonal anti-iav antibodies 212 that concurrently detect np, m1 and ha. 5-fu, which had no effect on viral replication over the 213 24 h time course in these cells ( fig. 2a) , likewise had no effect on the accumulation of these iav 214 proteins (fig. 3a) . consistent with the notion of selective inhibition of iav glycoprotein synthesis 215 and maturation, we observed that 6-tg had no effect on the accumulation of iav-pr8 ha or na 216 transcripts or function of the rdrp in genome replication, as 6-tg had little effect on the 217 accumulation of ha and na genome segments (fig. 3b ). taken together, these data support a 218 novel mechanism of action for thiopurine analogs in selectively inhibiting processing and 219 accumulation of iav glycoproteins and significantly impairing iav replication. 220 6-tg and 6-tgo activate the upr and chemical mitigation of er stress restores synthesis of 221 by inhibiting n-linked glycosylation, tm impedes proper processing of secreted and 223 transmembrane proteins in the lumen of the er, which elicits er stress and activates the upr 224 (47). indeed, we observed that tm treatment of a549 cells caused accumulation of xbp1s and 225 the er chaperone binding immunoglobulin protein (bip) (fig. 4a ). bip upregulation is an 226 excellent measure for upr activation because it requires both atf6(n)-dependent transcription 227 of bip and perk-mediated activation of the isr and uorf-skipping-dependent translation (1) . 228 we observed that both 6-tg and 6-tgo caused bip and xbp1s accumulation in a549 cells, 229 whereas the chemically similar thiopurine 6-mercaptopurine (6-mp) did not. nucleoside analogs 230 5-fu and ribavirin also did not affect bip or xbp1s levels (fig. 4a ). these data demonstrate that 231 6-tg and 6-tgo, but not all thiopurines, activate the upr in a549 cells. 232 to determine whether thiopurines could activate the upr during iav infection, a549 cells 233 infected with iav-pr8 were treated with 6-tg. we observed that 6-tg caused strong 234 accumulation of bip which coincided with diminished accumulation of ha (fig. 4b ). by contrast, 235 co-administration of 6-tg and the chemical chaperone 4-phenylbutyrate (4-pba) (48) diminished 236 accumulation of bip and partially restored ha levels in infected cells, without affecting levels of 237 np and m1 proteins. this suggests that thiopurine-mediated activation of the upr/isr is at least 238 partially responsible for the diminished accumulation of ha glycoproteins in infected cells. 239 to corroborate our observation of 6-tg-mediated upr activation, a549 cells were mock 240 infected or iav-pr8 infected, and treated with 6-tg, 6-mp, or tm for 24 h before harvesting 241 rna for rt-qpcr analysis of upr gene expression. we analyzed transcripts produced from 242 target genes linked to each arm of the upr; atf4 target gene chop, xbp1s target genes edem1 243 and erdj4, and atf6(n) target genes bip and herpud1. as expected, tm treatment caused 244 strong induction of all 3 arms of the upr and increased transcription of all five target genes in 245 mock-infected cells and infected cells alike (fig. 4c ). this strong and consistent transcriptional 246 output between mock-infected and infected cells suggests that the upr remains largely intact 247 during iav-pr8 infection. treatment with 6-mp had little effect on upr gene expression (fig. 248 4c ). by contrast, 6-tg treatment caused statistically significant increases in transcription from all 249 five upr target genes (fig. 4c) . these observations confirm that 6-tg activates all three arms of 250 the upr, whereas the chemically similar thiopurine 6-mp does not. 251 inhibition of the integrated stress response does not restore na processing and 252 oligomerization in 6-tg treated cells 253 iav glycoproteins are translocated into the er, where they are modified with n-linked 254 glycans and organize into oligomeric complexes. upon synthesis in the er, the type ii 255 transmembrane protein na is glycosylated and forms dimers linked by intermolecular disulfide 256 bonds in the stalk region (13) that then assemble into tetramers (49). we investigated the effect of 257 6-tg on na processing and oligomerization using sds-page/immunoblotting procedures in the 258 presence or absence of the disulfide bond reducing agent dithiothreitol (dtt). since na tetramers 259 are known to dissociate into dimers during electrophoresis (12, 50) we annotated the ~120 kda 260 band as dimers/tetramers (fig. 5a ). we observed intact glycosylated na dimers/tetramers and 261 monomers in mock-treated iav-pr8-infected cells, which were resolved into ~60 kda 262 glycosylated na monomers in the presence of dtt (fig. 5a ). unglycosylated na monomers 263 were undetectable in mock-treated cells at steady state, confirming that n-glycosylation is a rapid 264 initial step in na processing in the er. tm treatment eliminated na dimers/tetramers, leaving a 265 minor fraction of unglycosylated na monomers. the 6-tg treatment diminished accumulation of 266 all forms of na, yielding a distinct residual band that migrated closer to the size of the 267 unglycosylated na monomers from tm-treated cells; this suggests that 6-tg treatment interferes 268 with proper n-glycosylation of nascent na. treatment with integrated stress response inhibitor 269 (isrib), which prevents isr-mediated translation arrest by maintaining eif2b activity (51, 52), 270 rescued accumulation of na monomers in both tm-and 6-tg-treated cells. however, isrib was 271 not able to restore na glycosylation and oligomerization. these data provide further evidence that 272 6-tg inhibits iav glycoprotein accumulation via upr/isr activation and extends our 273 understanding by demonstrating that isr suppression does not fully reverse these effects. this is 274 further supported by our observations that administration of isrib alone had no impact on iav 275 replication while co-administration of isrib with tm-or 6-tg failed to restore virion production 276 in single-cycle infection assays (fig. 5b) . 277 in vitro studies have shown that thiopurines 6-tg and 6-mp can reversibly inhibit sars-279 cov-1 and mers-cov papain-like cysteine proteases pl(pro) (53-55); however, whether these 280 thiopurines could inhibit viral replication was not assessed. our observations of upr activation 281 and selective inhibition of iav ha and na processing and accumulation by low micromolar doses 282 of 6-tg and 6tgo, but not 6-mp, suggest a distinct antiviral mechanism of action for these 283 thiopurines. if true, the antiviral activity of 6-tg and 6-tgo may be broadly applicable to other 284 viruses with envelope glycoproteins like coronaviruses. to test this directly, we performed hcov-285 with the strong effect on infectious virion production, we also observed significant, dose-294 dependent reductions in viral protein accumulation due to 6-tg and 6-tgo treatment in hcov-295 oc43-infected hct-8 cells (fig. 6c , the main band recognized by the anti-oc43 antibody is 296 consistent with the size of the nucleocapsid n protein). treatment with higher dose of 6-mp caused 297 detectable decline in n protein levels in hcov-oc43-infected cells, but not to the levels observed 298 with 6-tg or 6-tgo treatment (fig. 6c ). in hcov-oc43-infected hct-8 cells, bip and chop 299 expression was upregulated following thiopurine treatment (fig. 6c ). this is consistent with the 300 significant induction of bip proten and chop mrna levels that was observed in thiopurine-301 treated a549 cells (fig. 4c ). to test the effects of 6-tg on cov replication, we analysed hcov-302 oc43 mrna synthesis by harvesting rna from infected cells treated with 6-tg or vehicle 303 control. we observed that 6-tg treatment caused significant decreases in steady-state levels of (+) 304 genomic rna (orf1ab) as well as (+) subgenomic rna (sgrna) that encodes n (fig. 6d) . 305 thus, despite the previously reported effects of 6-tg and 6-mp on hcov cysteine protease 306 activity in vitro, we observed that 6-mp had only modest effects on hcov-oc43 replication 307 whereas 6-tg and 6-tgo had clear antiviral effects similar to our previous observations of 308 inhibition of iav replication. thiopurine antiviral activity in our hcov-oc43 infection assays 309 correlated with upr activation and hampered viral genome synthesis and viral protein production. 310 because 6-tg activates the upr/isr and inhibits the processing and accumulation of iav 312 glycoproteins, we reasoned that coronavirus glycoproteins would be similarly affected by 6-tg 313 treatment. due to the ongoing sars-cov-2 pandemic, numerous reagents and constructs have 314 been rapidly developed to study this virus, including expression plasmids. we therefore sought to 315 determine if sars-cov-2 s glycoprotein is sensitive to 6-tg in ectopic expression experiments. 316 s is first translated as full-length s0 proprotein, before cleavage to s1 and s2 domains by cellular 317 proprotein convertases like furin (56). we observed that the s protein co-localised with the er 318 marker calnexin when expressed alone or co-expressed with m protein (fig. 7a ). m also caused 319 some of the s protein to accumulate in distinct regions of the cytoplasm proximal to, but not 320 overlapping with, calnexin-stained er, which likely represents the ergic (fig. 7a) . ectopic 321 expression of s led to accumulation of ~230 kda full-length n-glycosylated s0 monomers; 322 detection of ~110 kda s1 ectodomains demonstrated efficient s n-glycosylation and trimerization 323 in the er and transport to the golgi for furin cleavage (fig. 7b ). we observed that ectopic s 324 expression was sufficient to activate the upr/isr, as indicated by accumulation of bip (fig. 7b) , 325 consistent with previous reports of sars-cov-1 s (26, 31). 6-tg causes a loss of membrane-326 bound s0 and s2, but spared the cleaved s1 subunit (fig 7b) . co-expression of s with m altered 327 s processing leading to different accumulation of s1 protein species, possibly due to altered s 328 trafficking by m and retention at the ergic compartment (57). pngase f treatment of the lysates 329 to remove n-glycosylations confirmed that m and 6-tg altered glycosylation of s, but did not 330 affect cleavage (fig. 7c) . treatment with either the chemical chaperone 4pba or dbeq, a 331 selective chemical inhibitor of the p97 aaa-atpase, led to partial restoration of s0 and s2 (fig 332 7d ). together, these observations suggest that like iav glycoproteins, sars-cov-2 s 333 glycoprotein is vulnerable to 6-tg mediated activation of the upr/isr and suggest a mechanism 334 involving accelerated turnover of membrane-anchored s0 and s2 proteins by erad. 335 336 discussion 337 compared to current direct-acting antiviral drugs, effective host-targeting antivirals may 338 provide a higher barrier to the emergence of antiviral drug-resistant viruses. however, it remains 339 challenging to identify cellular pathways that can be targeted to disrupt viral replication without 340 causing adverse effects on bystander uninfected cells. here, we report that two chemically similar 341 fda-approved thiopurine analogues, 6-tg and 6-tgo, have broad antiviral effects that result 342 from activation of upr and disruption of viral glycoprotein synthesis and maturation. importantly, 343 our data demonstrate for the first time that 6-tg and 6-tgo are effective antivirals against 344 influenza virus and coronavirus and may be effective against other glycoprotein-containing 345 viruses. 6-tg is currently used in clinical settings to treat acute lymphoblastic leukemia and other 346 hematologic malignancies, with the main mechanism of action involving conversion into 347 thioguanine nucleotides and subsequent incorporation into cellular dna, which preferentially kills 348 cycling cancer cells (58, 59). furthermore, the active 6-tg metabolite, 6-thioguanosine 5′-349 triphosphate, was shown to inhibit small gtpase rac1 (60), which is believed to be largely with minimal effects on other viral proteins suggests that upr induction by 6-tg and 6-tgo is 357 the main antiviral mechanism. indeed, chemical chaperones and the isr inhibitor isrib partially 358 restored iav ha, na, and sars-cov-2 s protein accumulation in cells treated with 6-tg. 359 inhibition of the erad pathway with dbeq also restored accumulation of er membrane-360 anchored subunits of sars-cov-2 s protein (uncleaved precursor s0 and cleaved s2) in 6-tg-361 treated cells. this indicates that the 6-tg-induced upr causes both the phospho-eif2a dependent 362 decrease in viral glycoprotein mrna translation and the erad-mediated degradation of newly 363 synthesized er-anchored proteins. in the case of iav which replicates in the nucleus of infected 364 cells, depletion of viral envelope glycoproteins blocks infectious virion production but minimally 365 affects replication of viral nucleic acids. by contrast, synthesis of coronavirus genomic rna is 366 inhibited by thiopurine-induced upr. this reduction could be due to inhibition of pl(pro) activity 367 as previously suggested (53-55); however, we suggest that thiopurines likely inhibit viral 368 replication due to its occurrence on the multivesicular network generated from virus-rearranged 369 er membranes which may be highly sensitive to upr-induced alterations. despite multiple mechanisms deployed by iav to block sg formation, 6-tg and 6-tgo 382 treatment induced sgs in infected cells, which allowed us to identify these molecules in our image-383 based screen. we previously reported that in a549 cells, iav inhibited sg formation triggered by 384 treatment with thapsigargin, a potent inducer of er stress, with only 9% of infected cells forming 385 sgs compared to 35% of mock-infected cells (44). thus, induction of sgs in approximately 10% 386 of infected cells by 6-tg and 6-tgo treatment appears consistent with our previous observations. 387 however, unlike thapsigargin, 6-tg and 6-tgo did not trigger sg formation in uninfected cells. 388 the levels of upr induction by these drugs were similar between infected and uninfected cells, 389 highlighting that in iav-infected cells sg formation may not be triggered exclusively by er stress 390 and perk activation and may only partially contribute to antiviral effects of thiopurines. indeed, 391 sgs formed in a fraction of infected cells while accumulation of viral glycoproteins ha and na 392 was nearly completely blocked by 6-tg and 6-tgo. 393 consistent with previous reports, ectopic expression of sars-cov-2 s protein was 394 enhanced during m co-expression and s alone was sufficient to trigger er stress. in this system, 395 6-tg treatment further potentiated upr responses, as measured by increased bip accumulation. 396 our data also highlight the sensitivity of membrane anchored viral proteins to 6-tg treatment. it 397 is currently unknown if host glycoproteins will be similarly affected by 6-tg treatment, but this 398 is an important question to answer due to the prevalent use of thiopurines clinically. while we 399 suspect that 6-tg and 6-tgo will be effective against a wide-range of enveloped viruses, our 400 future studies will investigate if sars-cov-2 replication can be negatively impacted following what is the mechanism of upr induction by 6-tg and 6-tgo? our results suggest that 406 the effects are unlikely to be mediated through dna or rna incorporation of 6-tg because 1) 407 replicative stress does not specifically induce upr; 2) among viral proteins, glycoprotein 408 accumulation and processing was preferentially disrupted; 3) messenger rna levels of ha and 409 na were not affected. furthermore, the closely related thiopurine 6-mp that can be converted 410 into 6-thioguanosine triphosphate and incorporated into nucleic acids did not induce upr and 411 had no effect on iav glycoproteins or oc43 replication. another nucleoside analogue, 5-fu, 412 that is also incorporated into nucleic acids and can even trigger sg formation upon prolonged 413 48-hour incubation (62), was similarly inactive in our assays. the second previously described 414 antiviral mechanism of action of 6-tg and 6-mp that involves direct inhibition of viral cysteine 415 proteases is similarly unlikely to have major contribution to the observed phenotypes because 416 upr induction was triggered in both infected and uninfected cells and because, as mentioned 417 above, 6-mp was not active in our assays. thus, by process of elimination, we speculate that the 418 mechanism of upr induction by 6-tg and 6-tgo could involve gtpase inhibition. numerous 419 gtpases regulate er homeostasis, including rab gtpases that govern vesicular trafficking 420 events and dynamin-like gtpases that regulate homotypic er membrane fusion events required 421 for the maintenance of branched tubular networks (63) . future studies will focus on identifying 422 specific molecular targets of these upr-inducing thiopurines using orthogonal biochemical and 423 genetic screens. 424 human lung adenocarcinoma a549 cells, human embryonic kidney (hek) 293t and 293a cells, table 1 . table 1 . primer sequences for rt-qpcr analysis 691 primer sequences (5'-3') 5s rrna hpi, cells were treated with 0, 1, 10 and 30 um doses of thiopurine analogs 6-thioguanine (6-tg) or 6-thioguanosine (6-tgo). at 8 hpi, cells were fixed and stained with hoeschst 33342. automated image capture was performed using a cellomics arrayscan vti hcs reader. 15 images were captured for each well and average punctate egfp-g3bp1 intensity was calculated. (c) a549 cells were infected with iav-ca/07 at a moi of 1. at 4 hpi, cells were treated with 6-tg or mock-treated. at 8 hpi, cells were fixed and immunostained with antibodies directed to stress granule marker proteins g3bp1 (red), pabp (green) and a polyclonal iav antibody (blue) that detects antigens from np, m1, and ha, followed by staining with alexa-conjugated secondary antibodies. (d) a549 cells were infected with iav-ca/07 at a moi of 1. at 4 hpi, cells were treated with 6-tg (10µm). at 8 hpi, cells were fixed and immunostained with antibodies directed to stress granule marker proteins g3bp1 (red), tiar (green) and eif3a (green), followed by staining with alexa-conjugated secondary antibodies. images captured on a zeiss axioimager z2 fluorescent microscope. representative images shown. scale bars represents 20 µm. a549 cells were treated with 6-thioguanine (6-tg), 6-thioguanosine (6-tgo), 6-mercaptopurine (6-mp), 5-fluorouracil (5-fu) or ribavirin at the indicated concentrations for 6 h (xbp1s) or 24 h (bip) prior to harvesting lysates for immunoblotting. 5 µg/ml tunicamycin (tm) served as positive control for upr activation, whereas dmso was mock treatment. membranes were probed with anti-bip and anti-xbp1s antibodies to measure upr activation. -actin served as a loading control. (b) a549 cells were mock-infected or infected with iav-pr8 at moi of 1. after 1 h, cells were washed and incubated with 20 µm 6-tg or vehicle control, with or without 10 mm 4pba, a chemical chaperone. at 20 hpi, cell lysates were harvested and probed with antibodies for the indicated target proteins. western blots are representative of 3 independent experiments. (c) a549 cells were infected with iav-pr8 at moi of 1, washed and overlaid with media containing 6-mp, 6-tg, or tm. cell lysates were collected at 24 hpi and rna was isolated and processed for rt-qpcr. changes in chop, bip, edem1, erdj4, and herpud1 mrna levels were calculated by the δδct method and normalized using 18s rrna as a reference gene and standardized to mock. error bars represent the standard deviation between biological replicates (n=3); circles represent biological replicates; lines represents the average value. statistical significance was calculated via a two-way anova followed by a dunnett multiple comparisons test. the unfolded protein response: from stress pathway to homeostatic 694 regulation mechanistic insights into er-associated protein degradation herpesviruses and the unfolded protein response cellular proteostasis during influenza 700 a virus infection-friend or foe? cells 8 structure of the haemagglutinin membrane 702 glycoprotein of influenza virus at 3 a 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stress and upregulation of intracellular chemokine 746 mrna concentrations the perk arm of the unfolded 748 protein response negatively regulates transmissible gastroenteritis virus replication by 749 suppressing protein translation and promoting type i interferon production a human coronavirus oc43 variant 751 harboring persistence-associated mutations in the s glycoprotein differentially induces the 752 unfolded protein response in human neurons as compared to wild-type virus comparative host gene transcription by 756 microarray analysis early after infection of the huh7 cell line by severe acute respiratory 757 syndrome coronavirus and human coronavirus 229e modulation of the 759 unfolded protein response by the severe acute respiratory syndrome coronavirus spike 760 protein transcriptional profiling of vero e6 cells over-expressing sars-cov s2 subunit: insights 763 on viral regulation of apoptosis and proliferation coronavirus infection modulates the 765 unfolded protein response and mediates sustained translational repression post-translational modifications of coronavirus proteins: roles and 768 function assembly of coronavirus spike protein into trimers and its role 770 in epitope expression comparative analysis of the 772 activation of unfolded protein response by spike proteins of severe acute respiratory 773 syndrome coronavirus and human coronavirus hku1 a sars-cov protein er stress and jnk-dependent apoptosis coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-779 independent downregulation of the type 1 interferon receptor the 8ab protein of sars-781 cov is a luminal er membrane-associated protein and induces the activation of atf6 sars-coronavirus replication is supported 785 by a reticulovesicular network of modified endoplasmic reticulum membrane topology of murine 787 coronavirus replicase nonstructural protein 3 localization and membrane topology of coronavirus nonstructural protein 4: 790 involvement of the early secretory pathway in replication molinari m. 2010. coronaviruses hijack the lc3-i-positive edemosomes derived vesicles exporting short-lived erad regulators, for replication translation inhibition and stress granules in the 796 antiviral immune response the mechanism of eukaryotic translation 798 initiation and principles of its regulation rna-binding proteins tiar link the phosphorylation of eif-2 alpha to the assembly of mammalian stress 801 granules complexes mediate stress granule condensation and associate with 40s subunits influenza a virus inhibits cytoplasmic 807 stress granule formation influenza a virus host shutoff disables antiviral stress-induced translation arrest eukaryotic translation initiation factor 4a inhibitors block influenza a virus replication antioxidant antagonises chemotherapeutic drug effect in lung cancer cell line a549 mammalian stress response: induction of the glucose-regulated protein 818 family clinical and experimental applications of sodium 820 phenylbutyrate three-dimensional structure of the neuraminidase of 822 influenza virus a/tokyo/3/67 at 2.2 a resolution antigenicity of the n8 824 influenza a virus neuraminidase: existence of an epitope at the subunit interface of the 825 neuraminidase pharmacological brake-release of mrna 829 translation enhances cognitive memory the small molecule isrib 831 reverses the effects of eif2α phosphorylation on translation and stress granule assembly thiopurine analogues inhibit papain-like protease of severe acute respiratory 835 syndrome coronavirus thiopurine analogue inhibitors of severe acute 837 respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and 838 deisgylating enzyme thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like 841 protease of middle east respiratory syndrome coronavirus cleavage inhibition 843 of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not 844 virus-cell fusion the cytoplasmic tail of the severe acute respiratory 846 syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal 847 that binds copi and promotes interaction with membrane protein oxidation-mediated dna cross-linking contributes to the toxicity 849 of 6-thioguanine in human cells incorporation of 6-thioguanine into nucleic acids mediate immunosuppressive effects by interfering with rac1 protein function drug insight: 856 pharmacology and toxicity of thiopurine therapy in patients with ibd 5-fluorouracil affects 859 assembly of stress granules based on rna incorporation fusion of the endoplasmic reticulum by membrane-bound 862 new low-viscosity overlay 864 medium for viral plaque assays mammalian stress granules and processing bodies key: cord-333655-lylt7qld authors: van breedam, wander; pöhlmann, stefan; favoreel, herman w.; de groot, raoul j.; nauwynck, hans j. title: bitter‐sweet symphony: glycan–lectin interactions in virus biology date: 2013-12-06 journal: fems microbiol rev doi: 10.1111/1574-6976.12052 sha: doc_id: 333655 cord_uid: lylt7qld glycans are carbohydrate modifications typically found on proteins or lipids, and can act as ligands for glycan‐binding proteins called lectins. glycans and lectins play crucial roles in the function of cells and organs, and in the immune system of animals and humans. viral pathogens use glycans and lectins that are encoded by their own or the host genome for their replication and spread. recent advances in glycobiological research indicate that glycans and lectins mediate key interactions at the virus‐host interface, controlling viral spread and/or activation of the immune system. this review reflects on glycan–lectin interactions in the context of viral infection and antiviral immunity. a short introduction illustrates the nature of glycans and lectins, and conveys the basic principles of their interactions. subsequently, examples are discussed highlighting specific glycan–lectin interactions and how they affect the progress of viral infections, either benefiting the host or the virus. moreover, glycan and lectin variability and their potential biological consequences are discussed. finally, the review outlines how recent advances in the glycan–lectin field might be transformed into promising new approaches to antiviral therapy. many emerging and re-emerging viral diseases in animals and humans pose significant global health problems for which novel antiviral measures are in urgent demand. in general, rational design of new prophylactic and curative antiviral strategies requires a detailed knowledge of the viral infection mechanism and the host's innate and adaptive immune defense. historically, cell biological and microbiological research was mainly focussed on the nucleic acid and protein level. however, over the last few decades it has become clear that also glycans account to a great extent for the structural and functional diversity displayed by animal and human cells and their pathogens. at this moment, glycobiology is one of the most rapidly expanding disciplines in biology. a rapidly evolving array of powerful, novel techniques for the analysis of glycan structure and function (glycomics) has prompted many scientists to apply these tools to the field of virology, and this glycovirological approach has yielded a wealth of information on the various glycobiological aspects of viral infection and antiviral immunity. particularly fascinating in this context are the many distinct glycan-lectin interactions that may occur during viral infection of a host. virion-associated glycans often serve as ligands for specific host lectins. conversely, the glycan portions of host glycoconjugates function as receptors for various viruses that employ viral lectins for host cell entry. this review reflects on glycan-lectin interactions in the context of viral infection and antiviral immunity. following a general introduction on glycan and lectin biology, specific glycan-lectin interactions are highlighted and discussed within the larger framework of viral infection and immunity. distinction is made between interactions that benefit the host and interactions that benefit the virus. in addition, different factors that contribute to glycan and lectin variationand that consequently affect glycan-lectin interactionsare explored and various approaches to modulate specific glycan-lectin interactions in antiviral therapies are briefly discussed. glycans like nucleic acids, proteins and lipids, glycans are essential components of the animal cell and organism. the term 'glycan' refers to the carbohydrate portion of glycoproteins and glycolipids typically found at cell surfaces, in extracellular matrices and in cell secretions. unlike nucleic acid and protein synthesis, the biosynthesis of glycans is not a template-driven process. instead, glycosylation depends on the concerted action of different glycosyltransferase, glycosidase, and other enzymes. several important glycan types are exclusively assembled by the enzyme sets present in the endoplasmic reticulum (er) and the golgi network: glycan/glycoconjugate synthesis is typically initiated in the er or early golgi and gradual processing and diversificationor 'maturation'occurs as these molecules move further through the different enzyme-equipped compartments of the secretory pathway. the variability inherent to glycan synthesis and maturation forms the basis of the considerable diversity and complexity of the glycan repertoires found on animal glycoconjugates (varki et al., 2009; taylor & drickamer, 2011) . according to the basic glycan 'core' structure, the type of molecule the glycans are linked with and the type of the linkage, glycans and glycoconjugates can be categorized in different classes. two major classes of proteinlinked glycosylation are the n-and o-linked glycans. n-linked glycans are covalently linked to the nitrogen atoms of specific amino acid (aa) residues (typically asparagine) via an n-glycosidic bond. n-acetylglucosamine-to-asparagine (glcnacb-asn) type glycans represent the most common form of n-linked protein glycosylation (weerapana & imperiali, 2006; varki et al., 2009; larkin & imperiali, 2011; schwarz & aebi, 2011; taylor & drickamer, 2011) . o-linked glycans are covalently attached to the oxygen atoms of specific amino acid residues (typically serine or threonine) via an o-glycosidic bond. a common o-glycan typeand potentially the one most studiedis the n-acetylgalactosamine-to-serine/threonine (galnaca-ser/thr) type or mucin-type o-glycan. other (nonmucin) o-glycan types include a-linked o-mannose, a-linked o-fucose, b-linked o-xylose, b-linked o-glcnac (n-acetylglucosamine), a-/b-linked o-galactose, and a-/b-linked o-glucose glycans (van den steen et al., 1998; peter-katalinic, 2005; varki et al., 2009; jensen et al., 2010; gill et al., 2011; taylor & drickamer, 2011) . interestingly, various proteins are also modified with glycosaminoglycans (gags), linear polysaccharide chains composed of repeated disaccharide subunits consisting of a uronic acid/galactose residue and an amino sugar. glycosaminoglycan-carrying glycoproteins are generally referred to as proteoglycans (prydz & dalen, 2000; varki et al., 2009; taylor & drickamer, 2011) . figure 1 illustrates the general structure and classification of some common types of protein glycosylation. similarly as for protein-linked glycosylation, different types of lipid-linked glycosylation can be discerned: the glycosphingolipids (varki et al., 2009; taylor & drickamer, 2011; yu et al., 2011) and the glycophospholipid anchors or glycosylphosphatidylinositol (gpi) anchors (paulick & bertozzi, 2008; varki et al., fig. 1 . classification and basic structure of common types of protein-linked glycosylation. (a) glcnacb-asn type n-linked glycans are covalently attached to the amide nitrogen atoms of asn side chains and are almost exclusively found on asn residues within the sequence asn-x-ser/thr, in which x can be any amino acid except pro. the nature of the glycan structures that decorate the common glycan corethe glycan part shown in a dashed boxdictates classification of n-linked glycans as high-mannose type, hybrid type or complex type glycans, examples of which are shown in the panel. (b) galnaca-ser/thr type o-linked glycans have a galnac residue a-linked to the oxygen atom of the hydroxyl group of ser or thr residues. unlike for glcnacb-asn type n-linked protein glycosylation, there are no clear amino acid motifs that mark these o-linked glycosylation sites. a single galnac residue linked to the ser/thr is termed the 'tn antigen'. depending on the basic structure of the glycan core, more complex (extended) o-linked glycans are categorized into different 'core types'. cores 1-4 are the most common core structures, but also other core types exist. the tn antigen and examples of extended core 1, 2, 3, and 4 o-glycans are shown in the panel. the distinct glycan cores are shown in dashed boxes. (c) glycosaminoglycans (gags) are linear polysaccharide chains composed of repeated disaccharide subunits of a uronic acid/galactose residue and an amino sugar. glycosaminoglycans are classified as hyaluronan (ha), heparan sulfate/heparin (hs), chondroitin sulfate (cs), dermatan sulfate (ds), or keratan sulfate (ks), depending on the structure of their basic disaccharide subunits (shown in square brackets) and further modification (e.g. sulfation at different positions) of the glycan chain. with exception of hyaluronan, all major glycosaminoglycan types are sulfated and occur covalently linked to proteins. hs, cs, and ds are found on ser-linked xylose residues. although no unambiguous consensus sequence for xylosylation exists, the ser attachment site is consistently flanked by a gly residue at its carboxyterminal side. as depicted in the figure, heparan sulfate and heparin have the same basic structure. although they share a common biosynthesis, heparin generally undergoes more extensive sulfation and epimerization of uronic acid to iduronic acid. moreover, heparin is synthesized only in connective tissue mast cells as part of serglycin proteoglycans, whereas heparan sulfate is synthesized in virtually all mammalian cells. ks is found on asn-linked n-glycan core structures (ks i) or ser/thr-linked o-glycan core 2 structures (ks ii). capping or further modification of the glycosaminoglycan chainssulfation exceptedis not depicted (adapted from varki et al., 2009 ). 2009; taylor & drickamer, 2011) represent important glycolipid classes. their basic structure is introduced in fig. 2 . despite their different core structures and linkages to carrier molecules, distinct glycan types can still share conserved structural characteristics as they often follow partially overlapping biosynthetic pathways. although some glycan features may be exclusively found in one specific glycan class, many (sub)terminal glycan modifications can be found in different glycan classes. common (sub)terminal modifications include poly-n-acetyllactosamine chains, abh and lewis histo-blood group antigens (hbga), and sialic acids in different linkages (varki et al., 2009; taylor & drickamer, 2011) . consequently, the glycan moieties of glycoproteins and glycolipids often have more in common than one would expect based on their core structure. apart from the different glycan types introduced here, several other forms of glycosylation exist. however, an in-depth discussion of these is beyond the scope of this review. for more detailed information on glycan structure and biosynthesis, readers may refer to recent reference works on this topic (varki et al., 2009; taylor & drickamer, 2011) . not only the animal and human hosts, but also their pathogens can benefit from the fine-tuned cellular biosynthetic pathways that govern glycosylation. this is most obvious in the case of obligatory intracellular pathogens such as viruses. glycans form an important part of the surface of many viruses. as the glycosylation of viral components is facilitated by the cellular glycosylation machinery, viral glycosylation is similar to that of the host. however, important differences have been noted: viral glycoproteins are often more heavily glycosylated than their cellular counterparts and the composition of individual glycan chains can diverge greatly between virus and host. the biological basis of this variability is further classification and basic structure of major types of lipid-linked glycosylation. (a) glycosphingolipids consist of a hydrophilic glycan moiety linked to a hydrophobic sphingolipid. in higher animals, a ceramide lipid molecule is initially modified with a b-linked glucose or galactose residue, after which further extension and modification of the glycan moiety can occur. extension to larger glycan chains is common on ceramide-linked glucose residues, whereas further glycan extension on ceramide-linked galactose residues is more rare. depending on their glycan core structure, glycosphingolipids are classified in 'series'. the figure depicts a number of glycosphingolipid core structures. the key features that characterize each series are shown in dashed boxes. core structures can be further modified with sialic acids or sulfate groups, which allows subclassification of glycosphingolipids as neutral (lacking charged carbohydrates or ionic groups), sialylated or sulfated. (b) glycosylphosphatidylinositol (gpi) anchors are found in association with certain membrane proteins and serve as linkers between the protein and the lipid membrane. glycosylphosphatidylinositol anchors have a common core structure comprising ethanolamine-po 4 -6mana1-2mana1-6mana1-4glcna1-6myo-inositol-1-po 4 -lipid. differential derivatization of this common core structure through lipid remodeling and modification of the glycan moiety can cause significant glycosylphosphatidylinositol anchor heterogeneity. the protein is linked to the glycosylphosphatidylinositol anchor via an amide linkage between the c-terminal carboxyl group of the protein and the amino group of phosphatidylethanolamine (adapted from varki et al., 2009). situated later in this review (see 'glycan and lectin variation at the virus level'). in line with the similarity between viral and host glycosylation, many of the basic functions covered by glycans in normal animal and human physiology and in viral infection biologywhich is intrinsically linked to the biology of the hostare essentially the same. glycans play important structural roles and are for instance implicated in protein folding and solubility, protease resistance, and masking of highly immunogenic protein stretches ('glycan shielding') (varki et al., 2009; taylor & drickamer, 2011) . alternatively, glycans can also have nonstructural roles and take part in specific recognition events, in which they usually interact with complementary glycan-binding proteins called lectins (varki et al., 2009; taylor & drickamer, 2011) . lectins may simply be defined as carbohydrate-binding proteins, although some definitions are more restrictive and exclude mono-/oligo-saccharide transport proteins, enzymes, glycan-specific antibodies, and even glycosaminoglycan-binding proteins (elgavish & shaanan, 1997; weis, 1997; loris, 2002; varki et al., 2009; gabius et al., 2011) . according to the more strict definitions, glycosaminoglycan-binding proteins and lectins are distinguished based on different factors, including their ligand range, the structural basis of their glycan recognition, and their conservation (varki et al., 2009) . in general, glycosaminoglycan-binding proteins interact with negatively charged glycosaminoglycans via clusters of positively charged aa residues andwith exception of hyaluronan-binding proteins, which seem to share an evolutionarily conserved fold do not appear to be evolutionarily related to each other (varki et al., 2009) . in contrast, most strict sense lectins belong to protein families with defined 'carbohydrate recognition domains' (crd). the crds within a lectin family share structural and functional properties and selectively recognize specific portions of n-glycans, o-glycans, or glycolipids (sometimes also glycosaminoglycans) (varki et al., 2009) . although some crds can efficiently bind monosaccharides, other crds show no apparent affinity for monosaccharides and favor oligosaccharide ligands. the latter crd type often has a preference for ligands with specific linkages between the monosaccharide subunits, as these linkages determine the 3d structure of the glycan ligand and therefore the portions of the glycan that are available for interaction with the crd. interactions between a single crd and a single mono-/ oligo-saccharide (affinity) are often weak, and strong interactions are usually the result of multivalent binding, i.e. the interaction of multiple crds with multiple ligands (avidity). whereas some lectins contain multiple crds that can participate in ligand binding, others contain only a single crd and rely on clustering of individual lectin molecules for high avidity binding. clustering of crds does not only allow stronger interactions with ligands, but also contributes to the specificity/selectivity of interactions at the multivalent level. the relative spacing of the crds allows highly avid, multivalent binding to specific saccharide ligands in a certain density and particular presentation. ultimately, the avidity of lectins for specific glycoconjugates depends on the structure, multivalency, and density of glycans on these molecules (elgavish & shaanan, 1997; weis, 1997; loris, 2002; varki et al., 2009; gabius et al., 2011) . animal lectins are typically expressed in a cell-and/or tissue-specific manner. they are involved in many different biological processes, including glycoprotein trafficking, cell adhesion and signaling, and their expression is usually tightly regulated (varki et al., 2009) . particularly striking is the great number of membrane-associated and soluble lectins that are linked with host immunity. immune system lectins are involved in intercellular communication, positive and/or negative regulation of activation, regulation of inflammation, disposal of damaged and apoptotic cells, etc. several of these lectins have also been identified as 'pattern recognition receptors' (prrs), which act as molecular sensors for pathogens and endogenous stress signals and often trigger specific immune reactions/mechanisms in response to their detection (gordon, 2002; janeway & medzhitov, 2002; cambi & figdor, 2003; mcgreal et al., 2004; cambi et al., 2005; mcgreal et al., 2005; crocker et al., 2007; crocker & redelinghuys, 2008; van kooyk & rabinovich, 2008; garcia-vallejo & van kooyk, 2009; geijtenbeek & gringhuis, 2009; sato et al., 2009; bottazzi et al., 2010; dam & brewer, 2010; kumagai & akira, 2010; svajger et al., 2010; davicino et al., 2011; osorio & reis e sousa, 2011; sancho & reis e sousa, 2012) . the capacity of many immune system lectins to couple glycan recognition events with specific signaling and/or effector functions gives them a key regulatory position in the immune system. figure 3 gives a schematic overview of different types of animal lectins that are considered in this review. lectins play a pivotal role in different aspects of the physiology, including the immune defense against (viral) pathogens. however, it has become apparent that several viruses exploit host lectins to promote their spread. in addition, many viruses encode lectins for the recognition of and infectious entry into target cells. the following section explores how glycan-lectin interactions shape the virus-host interplay, mainly focussing on glycan-lectin interactions directly involving infectious virions. glycan-lectin interactions that benefit the host lectins cover essential roles in the animal host's immune defense. importantly, several membrane-associated host (immune system) lectins act as pathogen recognition molecules: they can bind pathogens and activate signaling mechanisms or capture pathogens for subsequent degradation and presentation to cells of the adaptive immune system (e.g. mhcii-restricted presentation of antigens to t cells), resulting in the induction of a pathogen-specific adaptive immune response. alternatively, pathogens attached to such cell surface lectins may also be directly presented to neighboring immune cells in trans, a process that seems especially significant at sites with a high density of immune cells (e.g. the lymph nodes). hence, binding of a viral pathogen to membrane-associated (immune system) lectins can lead to its clearance and degradation. a prominent example is the interaction between the human immunodeficiency virus type 1 (hiv-1) and human langerin, a c-type lectin mainly expressed on langerhans cells (de witte et al., 2007; . de witte et al. (2007) reported that hiv-1 interacts with langerin via high-mannose glycans on the gp120 envelope protein and is subsequently internalized into birbeck granules, leading to virus degradation (de witte et al., 2007) . also dc-sign, a mannose-binding c-type lectin mainly expressed on dendritic cells (dcs), can bind and internalize hiv-1 virions for degradation and promotes mhcii-restricted as well as exogenous mhci-restricted presentation of hiv-1 antigens (moris et al., 2004 (moris et al., , 2006 . similarly, various other membrane-associated host lectins can aid as prrs in the defense against viral pathogens. however, growing evidence illustrates that many of these lectins, including dc-sign, are also abused by viruses to gain access to their target cells and facilitate viral spread, as is discussed further below. in contrast to the dual role played by different membrane-associated host lectins, soluble host lectins have mainly been associated with protection against viral infection. several soluble host lectins have been reported to aid in neutralization and clearance of various viral pathogens. table 1 gives an overview of membrane-associated and soluble host lectins that are linked with the host's defense against different viruses. current experimental data strongly implicate these host lectins in the defense against the listed viral pathogens and do not attribute explicit proviral effects to the host lectinunlike for the lectin-virus pairs listed further in table 2 . the basic principles of soluble lectin-mediated antiviral protection are most easily conveyed using some specific, wellcharacterized examples. for instance, various studies point out an important role of surfactant protein a (sp-a), surfactant protein d (sp-d), and mannose-binding lectin (mbl) in the defense against influenza a virus (iav) infection. sp-a, sp-d, and mbl are all soluble c-type lectins and share a similar basic structure: lectin monomersconsisting of an n-terminal cysteine-rich domain, a collagen-like domain, a coiled coil neck domain, and a c-terminal crdassemble into trimers which, under physiologic conditions, further multimerize via their n-termini to form typical cruciform-(sp-d) or bouquet-(sp-a and fig. 3 . schematic overview of different types of membrane-associated (a) and soluble (b) animal lectins that are considered in this review. the lectin domains are highlighted and listed in the key. c-type lectin/c-type lectin domain: lectins are classified as c-type lectins based on their ca 2+ -dependency and shared primary structure. in the c-type crd, a ca 2+ ion is directly involved in carbohydrate binding by making coordination bonds to both the crd surface and key hydroxyl groups of the carbohydrate. the c-type lectin family contains both membraneassociated (a.1) and soluble (b.1) lectins. the collectins are soluble c-type lectins characterized by the presence of collagen-like domains. r-type lectin domain: this term refers to a crd that is structurally similar to the crd in ricin, a toxin found in the plant ricinus communis. i-type lectin/ i-type lectin domain: i-type lectins are glycan-binding proteins that belong to the ig superfamily, but are not antibodies or t-cell receptors. the 'sialic acid-binding ig-like lectin (siglec)' family of membrane-associated lectins is currently the only well-characterized group of i-type lectins (a.2). ficolin: ficolins (b.2) are soluble lectins characterized by the presence of collagen-like domains and fibrinogen-like globular domains with a lectin activity. galectin/s-type lectin (domain): galectins (b.3) are soluble lectins that typically bind b-galactose-containing glycoconjugates and show primary structural homology in their crds. galectins were initially referred to as s-type lectins to reflect their sulfhydryl dependency, the presence of cysteine residues and their solubility; however, at present, not all identified galectins fit this initial description anymore. pentraxin/pentraxin domain: pentraxins (b.4) are characterized by the presence of pentraxin domains, which contain an eight amino acid long conserved 'pentraxin signature' (hxcxs/twxs, where x is any amino acid) and display an l-type (legume-type) lectin fold. sap is a soluble lectin that requires ca 2+ ions for carbohydrate ligand binding (adapted from fujita, 2002; varki et al., 2009; bottazzi et al., 2010) . (van de wetering et al., 2004; veldhuizen et al., 2011) . despite their similar structure, these lectins show distinct glycan ligand specificities (veldhuizen et al., 2011) and also their interaction with iav and their effects on infection and spread appear to differ. studies using distinct iav isolates indicate that sp-d and mbl bind mannose-rich glycans on the viral surface glycoproteins hemagglutinin and neuraminidase (a viral lectin and a viral glycosidase, involved in iav entry and release; see discussion on viral lectins) through their crds (malhotra et al., 1994; reading et al., 1997; kase et al., 1999; hartshorn et al., 2000; hillaire et al., 2011) . although sp-a may interact with some iav isolates in a similar manner (malhotra et al., 1994) , binding of this molecule to most of the iav variants tested to date appears not to involve the lectin activity of sp-a; in contrast, virus binding depends on the interaction of the viral hemagglutinin with a sialylated n-glycan on the sp-a crd (hartshorn et al., 1994; benne et al., 1995 benne et al., , 1997 hartshorn et al., 1997; van eijk et al., 2003; mikerov et al., 2008) . in other words: sp-d and mbl binding depend on viral glycosylation, whereas sp-a binding mainly depends on the specificity of the hemagglutinin, and this is mirrored in the spectrum of iav variants these lectins can effectively bind (hartshorn et al., 1994; malhotra et al., 1994; benne et al., 1995 benne et al., , 1997 hartshorn et al., 1997; reading et al., 1997; kase et al., 1999; hartshorn et al., 2000; van eijk et al., 2003; mikerov et al., 2008; hillaire et al., 2011) . interestingly, the porcine variant of sp-d appears to combine the above-mentioned iav-binding functionalities: this molecule may not only bind iav virions through interaction of its crd with high mannose glycans on the virion surface (similar to other sp-d molecules), but also through interaction of a sialylated glycan on the lateral surface of its crd with the iav hemagglutinin (similar to sp-a) and can therefore bind to a broader array of iav variants (van eijk et al., 2002 (van eijk et al., , 2003 (van eijk et al., , 2004 hillaire et al., 2011) . in vitro assays show that sp-a and sp-d can directly neutralize iav infectivity (benne et al., 1995; reading et al., 1997; hartshorn et al., 2000; van eijk et al., 2003; hawgood et al., 2004; hillaire et al., 2011) . both sp-a and sp-d inhibit the viral hemagglutinating activity that is required for iav attachment to target cells (hartshorn et al., 1994; malhotra et al., 1994; benne et al., 1995; hartshorn et al., 1996 hartshorn et al., , 1997 hartshorn et al., , 2000 van eijk et al., 2002 van eijk et al., , 2003 van eijk et al., , 2004 mikerov et al., 2008; hillaire et al., 2011) and sp-d was reported to inhibit the viral neuraminidase (reading et al., 1997; hillaire et al., 2011) . interestingly, both lectins also induce viral aggregation (hartshorn et al., 1994 (hartshorn et al., , 1996 (hartshorn et al., , 1997 van eijk et al., 2003) and function as potent opsonins. sp-a for instance was identified as an opsonin for iav phagocytosis by alveolar macrophages (benne et al., 1997) . moreover, sp-a and sp-d were shown to enhance iav binding to neutrophils (hartshorn et al., 1994 (hartshorn et al., , 1996 (hartshorn et al., , 1997 van eijk et al., 2003) and sp-d-iav complexes were found to internalize upon attachment to neutrophils (hartshorn et al., 1997; van eijk et al., 2003) . pre-incubation of iav with sp-a or sp-d also enhances the virus-induced h 2 o 2 responses in neutrophils (hartshorn et al., 1994 (hartshorn et al., , 1996 (hartshorn et al., , 1997 van eijk et al., 2003) and pre-incubation of the virus with sp-d can protect neutrophils from iav-induced deactivation (hartshorn et al., 1994 (hartshorn et al., , 1996 (hartshorn et al., , 1997 . mbl can counteract iav by roughly the same mechanisms as sp-d (hartshorn et al., 1993; anders et al., 1994; malhotra et al., 1994; hartshorn et al., 1996 hartshorn et al., , 1997 reading et al., 1997; kase et al., 1999) , although its ability to activate the complement cascade expands its capabilities (anders et al., 1994; reading et al., 1995; chang et al., 2010) . ligand binding by mbl (or alternatively ficolins) can lead to activation of mbl-associated serine proteases (masps) and initiate the complement cascade via the so-called lectin pathway (blue et al., 2004; bottazzi et al., 2010) . complement deposition on a virus may interfere directly with crucial steps in the viral infection process (e.g. receptor binding), but can also trigger complement receptor-mediated uptake of the pathogen into immune cells. in addition, for enveloped viruses, complement activation may result in membrane attack complex (mac) formation on the viral envelope and subsequent virolysis (blue et al., 2004; bottazzi et al., 2010) . in line with the available in vitro data, recent work with sp-a-, sp-d-, and mbl-knockout mice also confirmed the antiviral potential of these soluble lectins in vivo (levine et al., 2001 (levine et al., , 2002 zhang et al., 2002; li et al., 2002; hawgood et al., 2004; levine et al., 2004; kingma et al., 2006; chang et al., 2010) . noteworthy caveats regarding these in vivo studies are, however, that direct antiviral effects of these lectins can be hard to uncouple from othere.g. immune-regulatoryeffects and that mice do not represent natural hosts for iav. another interesting example of antiviral activity mediated by soluble host lectins was recently reported for nipah virus (niv): the physiologic, homodimeric form of the soluble lectin galectin-1 can inhibit niv envelope protein-mediated membrane fusion (levroney et al., 2005; garner et al., 2010) . niv encodes two viral membrane although capture of a virus by these lectins may have certain antiviral effects or promote the specific immunity against this pathogen, current experimental data suggest that the listed viruses may also employ these lectins to promote viral infection, spread or persistence. references in table 2 are listed in supporting information, data s2. dimt: discussed in main text. glycoproteinsthe attachment protein niv-g and the fusion protein niv-fthat mediate viral entry and direct the endothelial cell syncytia formation typically associated with niv infection (levroney et al., 2005; garner et al., 2010; lee & ataman, 2011) . binding of niv-g to cell surface receptors induces a conformational change in niv-f, thereby activating its fusogenic activity (levroney et al., 2005; garner et al., 2010; lee & ataman, 2011) . galectin-1 associates with glycans on the niv envelope proteins and interferes with the membrane fusion process in multiple ways (levroney et al., 2005; garner et al., 2010) . not only does galectin-1 binding directly inhibit the crucial conformational change in niv-f associated with membrane fusion, it also reduces the lateral mobility of niv-f and niv-g in the lipid membrane and consequently counteracts the physical separation of niv-f and niv-g that is essential for this conformation change (garner et al., 2010) . moreover, galectin-1 binding impedes endocytosis and maturation of the niv-f precursor niv-f 0 in infected cells (garner et al., 2010) , further illustrating the capacity of this lectin to block different stages of the viral infection/ replication process. in sum, soluble host lectins can counteract viral infection in various ways: they may directly neutralize virus by destabilizing or aggregating virions, interfere with crucial steps in the viral infection process (e.g. entry), and/or opsonize virus to facilitate uptake and degradation. upon virus binding, some soluble lectins can trigger complement deposition on the virus, which may inhibit viral infection, enhance viral uptake via complement receptors and subsequent degradation in immune cells, and/or cause virolysis. considering the above, it is clear that lectins contribute significantly to the host's antiviral defense. host lectins are involved in neutralization and clearance of free virus, immune regulation, andalthough not extensively discussed in this overviewthe detection and clearance of virus-infected cells. figure 4 illustrates how membraneassociated and soluble host lectins can aid in antiviral defense. like their animal hosts, also viruses can benefit from interactions between glycans and glycan-binding proteins. for instance, many viruses, including hiv-1, herpes simplex virus-1, and dengue virus, have been shown to interact with glycosaminoglycan molecules present on target cells (patel et al., 1993; chen et al., 1997; krusat & streckert, 1997; summerford & samulski, 1998; dechecchi et al., 2000 dechecchi et al., , 2001 vanderheijden et al., 2001; delputte et al., 2002; trybala et al., 2002) . viral association with glycosaminoglycans is usually attributed to charge-based attractions between clusters of positively charged aa residues on the virion surface and the negatively charged glycosaminoglycan chains. the interaction with glycosaminoglycans often constitutes the first contact between a virus and its target cell and typically increases infection efficiency. however, it has been documented for several viruses that the ability to interact with glycosaminoglycans can result from adaptation to growth in cell culture (sa-carvalho et al., 1997; klimstra et al., 1998; hulst et al., 2000; mandl et al., 2001) . clearly, use of primary virus isolates is of crucial importance when assessing the occurrence and relevance of such interactions in vivo. aside from potential glycosaminoglycan-binding capacity, several viruses are endowed with a true lectin activity: they carry virally encoded lectins on their surface and use these as keys to gain entry into their target cells (fig. 5a ). similar to animal lectins, such virally encoded lectins often possess characteristic glycan binding regions ('glycan binding pockets') and recognize specific portions of protein-and lipid-linked glycans. the hemagglutinin protein of iav is generally regarded as the prototype of a viral lectin. hemagglutinin is a membrane glycoprotein that forms noncovalently linked fig. 4 . schematic overview of how membrane-associated (a) and soluble (b) host lectins are implicated in antiviral defense. (a.1) binding of virion-associated glycans with membrane-associated host lectins can lead to virus uptake, degradation, and presentation of viral antigens to cells of the adaptive immune system. binding may trigger specific signaling that promotes an effective antiviral immunity. (a.2) binding of virionassociated glycans with membrane-associated host lectins may promote direct presentation of the virus to immune cells in trans. binding may trigger specific signaling that promotes an effective antiviral immunity. (b.1) binding of soluble host lectins to virion-associated glycans may interfere directly with viral infection by destabilizing virions, blocking interaction of the virus with its receptors or interfering with other crucial steps in the infection process (e.g. membrane fusion). soluble host lectins may also aggregate virions, which often negatively impacts viral infectivity (not depicted). (b.2) soluble host lectins can act as opsonins: lectin binding to virion-associated glycans may facilitate viral uptake in immune cells via lectin receptors, leading to viral degradation and potential presentation of viral antigens to cells of the adaptive immune system. lectin binding may also trigger complement deposition on the virus (through the lectin pathway) and facilitate viral uptake via complement receptors. (b.3) detection of virion-associated glycans by soluble host lectins may trigger complement deposition on the virus (through the lectin pathway), which may directly inhibit viral infection and/or elicit lysis of the (enveloped) virus. homotrimers in the (viral) membrane and is responsible for both virus attachment and penetration (skehel & wiley, 2000; harrison, 2008; gamblin & skehel, 2010) . the mature hemagglutinin protein consists of two disulfide-linked subunits, termed ha1 and ha2 (skehel & wiley, 2000; harrison, 2008; gamblin & skehel, 2010) . the ha1 subunit forms the globular 'head' region of hemagglutinin that covers the lectin function of this molecule: sialic acid-binding pockets in the membrane distal part of ha1 allow interaction with sialic acidcontaining receptors on target cells (skehel & wiley, 2000; harrison, 2008; gamblin & skehel, 2010) . variation in the ha1 subunit determines the affinity and specificity (e.g. a2-3vs. a2-6-linked sialic acids) of this molecule (skehel & wiley, 2000; gamblin & skehel, 2010) . as seen for animal lectins, also hemagglutinin binds with its glycan counterparts with a relatively low affinity and efficient virus attachment and entry depends on the interaction of multiple hemagglutinin molecules with multiple sialic acid-containing receptors (skehel & wiley, 2000; gamblin & skehel, 2010) . the stalk-like ha2 subunit of hemagglutinin mediates the ph-dependent fusion process upon internalization of the iav virion in the endosomal compartment of the target cell (skehel & wiley, 2000; harrison, 2008; gamblin & skehel, 2010) . similar to iav, various other enveloped [e.g. influenza b and c viruses (nakada et al., 1984; herrler et al., 1985a; rogers et al., 1986; vlasak et al., 1987; herrler et al., 1988; herrler & klenk, 1991; rosenthal et al., 1998; lamb & krug, 2001; suzuki & nei, 2002; wang et al., 2007b; wang et al., 2008a) , mumps virus (bowden et al., 2010; harrison et al., 2010; chang & dutch, 2012) ] as well as nonenveloped [e.g. murine norovirus (taube et al., 2009 (taube et al., , 2012 , feline calicivirus (stuart & brown, 2007) , and rhesus rotavirus (dormitzer et al., 2002) , among others (taube et al., 2010) ] viruses employ sialic acid-binding viral lectins to infect target cells. importantly however, viral lectins with a different glycan specificity have also been identified. for instance, many virusesincluding human noroviruses (estes et al., 2006; le pendu et al., 2006; cao et al., 2007; bu et al., 2008; choi et al., 2008; donaldson et al., 2008; shirato, 2011) , rabbit hemorrhagic disease viruses (ruvoen-clouet et al., 2000; rademacher et al., 2008; guillon et al., 2009; nystrom et al., 2011) , and the rhesus monkey tulane virus (farkas et al., 2010) were found to interact with specific hbga types. although a broad spectrum of viruses has evolved to use viral lectins to secure efficient target cell infection, the use of viral lectins for cellular attachment comes with a price. the glycan receptors for viral lectins are not necessarily target cell-specific and, whereas low affinity/avidity interactions may be reversible, high affinity/avidity binding of viral lectins to nontarget cell-associated glycoconjugates ('decoy receptors') can prevent the virus from efficiently targeting susceptible host cells. in line with this, it was shown for iav that interaction of the viral lectin hemagglutinin with soluble, sialylated host glycoproteins e.g. sp-a (cfr. supra) or a2-macroglobulincan interfere with the viral hemagglutinating activity that is crucial for receptor binding (rogers et al., 1983; pritchett & paulson, 1989; ryan-poirier & kawaoka, 1991; matrosovich et al., 1992; ryan-poirier & kawaoka, 1993; hartshorn et al., 1994; malhotra et al., 1994; benne et al., 1995; gimsa et al., 1996; benne et al., 1997; hartshorn et al., 1997; matrosovich et al., 1998; van eijk et al., 2003; mikerov et al., 2008; chen et al., 2010; cwach et al., 2012) . although the presence of 'high avidity' glycan decoys invariably puts a strain on viral infection efficiency, this burden may be lighter on viruses that are equipped with a receptor destroying enzyme (rde) that matches the specificity of the viral lectin. intriguingly, the best-known examples in this context are again influenza viruses. as situated above, both influenza a and b viruses display hemagglutinin proteins on their surface, which bind to sialic acids displayed on the host cell surface and mediate ph-dependent fusion of the viral membrane with the host cell membrane (skehel & wiley, 2000; lamb & krug, 2001; wang et al., 2007b; wang et al., 2008a; harrison, 2008; gamblin & skehel, 2010 ). an rde activity was mapped to another viral membrane glycoprotein, designated neuraminidase (gottschalk, 1957; colman, 1994; fig. 5. (a) illustrates how viral lectins promote target cell infection. (b) shows how many viruses that employ viral lectins also benefit from a matching receptor-destroying enzyme (rde) activity, which provides a counterweight against (high avidity) lectin activity. (a) interaction of viral lectins with glycosylated receptors on a target cell promotes viral entry and infection (attachment/internalization/fusion, depending on specific virus biology). (b) although they clearly benefit the virus, the use of (high avidity) viral lectins comes with a price. for instance, viral lectin activity can cause virions to aggregate (b.1) and can impair efficient release of newly formed virions from (glycosylated) infected cells (b.2). moreover, binding of viral lectins to nontarget cell-associated glycoconjugates (decoy receptors) can prevent the virus from efficiently targeting susceptible host cells (b.3). intriguingly, several lectin-carrying viruses are also equipped with an rde that matches the specificity of the viral lectin and provides a counterweight against lectin-mediated glycan binding. in fact, for viruses equipped with both viral lectins and rdes, a functional balance between these molecules appears to be an important determinant of the viral (replicative) fitness. lamb & krug, 2001; gamblin & skehel, 2010) . this enzyme removes sialic acid moieties from glycoproteins and glycolipids by catalyzing the hydrolysis of the a-ketosidic linkage to the subterminal sugar residue and consequently destroys potential receptors for the viral hemagglutinin (gottschalk, 1957; colman, 1994; lamb & krug, 2001; gamblin & skehel, 2010) . viral use of an enzyme that can actually destroy receptors for the virus may seem peculiar at first. importantly, however, the neuraminidase activity can prevent virions from aggregating via hemagglutinin-sialic acid interactions, promotes efficient release of newly formed virions from (sialic acidcarrying) infected cells, and provides a counterweight to the interaction of hemagglutinin molecules with nontarget cell-associated glycans: neuraminidase-mediated removal of sialic acids from decoy receptors prevents virions from establishing high-avidity interactions with these glycoconjugates and may even provide an escape route for virions after hemagglutinin-mediated binding to nontarget cellassociated glycans (colman, 1994; suzuki et al., 1994; gimsa et al., 1996; barrere et al., 1997; gamblin & skehel, 2010) . conceivably, a functional balance between the hemagglutinin and neuraminidase activities is an important determinant of the (replicative) fitness of iav variants in vivo. using iav as a paradigm, fig. 5b illustrates how viral rde activity can balance the high avidity of viral lectins where favorable and consequently improve viral infection efficiency. in contrast to influenza a and b viruses, other viruses combine both lectin and rde functions in one protein complex. for example, the viral membranes of mumps virus, newcastle disease virus, sendai virus, and human parainfluenza virus 3 and 5 are studded with hemagglutinin-neuraminidase proteins (bowden et al., 2010; harrison et al., 2010; chang & dutch, 2012) . another example is the influenza c virus, which carries the hemagglutination, rde and fusion protein functions in one single envelope protein named the hemagglutininesterase-fusion protein (nakada et al., 1984; herrler et al., 1985a, b; rogers et al., 1986; vlasak et al., 1987; herrler et al., 1988; schauer et al., 1988; herrler & klenk, 1991; rosenthal et al., 1998; pekosz & lamb, 1999; lamb & krug, 2001; suzuki & nei, 2002) . whereas the rde of influenza a and b viruses is a neuraminidase, which cleaves off entire sialic acid residues, the rde of influenza c functions as a sialate-o-acetylesterase and cleaves off specific o-acetyl groups (herrler et al., 1985b; vlasak et al., 1987; herrler et al., 1988; schauer et al., 1988; pekosz & lamb, 1999) . a similar situation is seen for certain coronaviruses and toroviruses that carry an accessory protein called hemagglutinin-esterase on their surface (de groot, 2006) . as the name implies, functional hemagglutinin-esterase proteins combine a hemagglutinating activity with a sialate-o-acetylesterase activity (de groot, 2006) . interestingly, for some coronaviruses, the hemagglutinin-esterase protein is not the only envelope protein endowed with a lectin activity. for example, the spike proteins of bovine coronavirus and human coronavirus oc43 have been shown to be potent sialic acid-binding lectins kunkel & herrler, 1993) . also the spike protein of the transmissible gastroenteritis coronavirus of pigs has been shown to possess such a lectin activity (schultze et al., 1996; krempl et al., 1997 krempl et al., , 2000 schwegmann-wessels et al., 2011) . however, transmissible gastroenteritis coronavirus has no hemagglutinin-esterase protein that serves as rde to counteract the hemagglutinating activity of the spike protein. viruses do not only benefit from virally encoded lectins, but can also use host lectins to their advantage. paradoxically, many of the host lectins that are exploited by viruses form part of the immune system. although capture of viral pathogens by these lectins may have certain antiviral effects or promote the specific immunity against this pathogen (cfr. supra), many viruses can also employ such interactions to promote efficient infection and spread or to facilitate persistence (table 2) . virus binding to membrane-associated lectins can lead to concentration of virions at the cell surface and can facilitate infection of target cells. in many cases, host lectins appear to function as true portals for viral entry: the virus binds to the lectin, which drives subsequent internalization of the virus into specific cellular compartments from which the virus can initiate the next stage of infection. however, it has also been shown that lectins present on non-target cells may facilitate infection of target cells, a process called trans-infection. many membrane-associated (immune system) lectins also participate in specific signaling pathways and engagement of such lectins by a virus may modulate both viral infection and the immune response in favor of the pathogen. an immune system lectin that can be used as a paradigm in this context is dc-sign. this molecule is mainly expressed on dendritic cells (dcs), but expression on distinct other cell-typesincluding macrophages, b lymphocytes, platelets, and (immortalized) podocyteshas also been described (geijtenbeek et al., 2000a, b; soilleux et al., 2002; granelli-piperno et al., 2005; gurney et al., 2005; chaipan et al., 2006; rappocciolo et al., 2006; mikulak et al., 2010; svajger et al., 2010) . prototypic dc-sign molecules consist of a c-terminal c-type crd, a neck region made up of 7 and a half 23 aa residue repeats, a transmembrane domain, and a cytoplasmic domain containing motifs involved in receptor internalization and signaling (svajger et al., 2010; tsegaye & pohlmann, 2010) . lectin monomers typically multimerize via their neck regions to form tetramers, which in turn organize in nanoclusters on the cell membrane (svajger et al., 2010; tsegaye & pohlmann, 2010; manzo et al., 2012) . dc-sign functions as a receptor for various ligands. the t cell-expressed molecule icam-3 is probably its most prominent endogenous ligand: dc-sign binds icam-3 on the t cell surface and thereby contributes to the transient, nonantigen-specific interaction of dc with t cells that is required for efficient screening of mhcii-peptide complexes and eventual t cell priming (geijtenbeek et al., 2000a; svajger et al., 2010) . moreover, dc-sign has been implicated in various other processes, including dc differentiation, migration, and antigen capture (svajger et al., 2010) . over the last decade, it has become apparent that dc-sign interacts with a wide variety of viral pathogens. a textbook example of a virus that recruits dc-sign is hiv-1 (lekkerkerker et al., 2006; wu & kewalramani, 2006; piguet & steinman, 2007; tsegaye & pohlmann, 2010; da silva et al., 2011; van der vlist et al., 2011) . dc-sign can bind the enveloped hiv-1 particle and mainly recognizes mannose-rich glycans on the viral envelope glycoprotein gp120 (curtis et al., 1992; geijtenbeek et al., 2000a, b; feinberg et al., 2001; hong et al., 2002; lin et al., 2003; su et al., 2004; hong et al., 2007) . the efficiency of hiv-1 capture by dc-sign has been linked to receptor density. experiments in 293 t-rex cells using an inducible dc-sign expression system have shown that high surface expression levels of dc-sign correlate with optimal binding of hiv-1 particles, and that lowering the dc-sign expression levels can significantly reduce the efficiency of hiv-1 binding . these data suggest that the high dc-sign surface expression levels on certain (immature) dc subsets are compatible with optimal capture of hiv-1 virions, whereas lower dc-sign expression levels on b lymphocytes and especially platelets may be mirrored in a less efficient hiv-1 capture (baribaud et al., 2002; boukour et al., 2006; chaipan et al., 2006; rappocciolo et al., 2006) . nevertheless, studies have shown that also b lymphocytes and platelets can effectively bind hiv-1 virions via dc-sign (boukour et al., 2006; chaipan et al., 2006; rappocciolo et al., 2006) . evidently, the interaction between hiv-1 and dc-sign is also critically dependent on the viral glycome. recent research has shown that virion-associated gp120 of peripheral blood mononuclear cell (pbmc)grown virus predominantly carries oligomannose n-glycans (doores et al., 2010; bonomelli et al., 2011) , which constitute optimal ligands for dc-sign (van liempt et al., 2006) . nevertheless, virus originating from different host cell types can display a different glycosylation profile, and this may modulate the efficiency of dc-sign recruitment (lin et al., 2003) . binding of hiv-1 to dc-sign can entail both negative and positive effects for the virus. in dc-signexpressing antigen-presenting cells, most of the dc-sign-captured virions appear to be internalized into the endolysosomal pathway and rapidly degraded (moris et al., 2004; turville et al., 2004) . in line with this, dc-sign was found to promote mhcii-restricted presentation of hiv-1 antigens (moris et al., 2006) . intriguingly however, in cells that co-express cd4 and ccr5/ cxcr4 (hiv-1 receptor and co-receptors, respectively), dc-sign expression also facilitates hiv-1 fusion: dc-sign efficiently captures and concentrates viral particles at the cell surface, and binding of this lectin to the hiv-1 envelope protein appears to increase exposure of the cd4 binding site nobile et al., 2005; burleigh et al., 2006; hijazi et al., 2011) . although dc-sign-mediated enhancement of hiv-1 fusion may promote mhci-restricted presentation of hiv-1 antigen (proteasome and tap-dependent pathway) and activation of cytotoxic t lymphocytes (moris et al., 2004) , enhanced hiv fusion inevitably leads to more efficient infection. indeed, several studies confirm that dc-sign facilitates productive (cis-) infection in dc-signexpressing cells that also co-express cd4 and ccr5/ cxcr4 nobile et al., 2005; burleigh et al., 2006; hijazi et al., 2011) . dc-sign has also been implicated in hiv-1 transinfection. an initial study showed that dc-sign can efficiently capture hiv-1 particles and transfer them to adjacent target t cells, without the need for productive infection of the dc-sign-expressing cell (geijtenbeek et al., 2000b) . subsequent studies on the subject reported dc-sign-mediated internalization of infectious hiv-1 virions into low ph nonlysosomal compartments, and advocated that the virus traffics in intracellular compartments towards the zone of t cell contact, where it is released into the infectious synapse (i.e. the contact zone between the virus-loaded cell and the target t cell) (kwon et al., 2002; mcdonald et al., 2003) . dc-captured hiv-1 virions as well as t-cellexpressed cd4 and ccr5/cxcr4 were found to concentrate at the dc-t-cell interface, rendering it an ideal micro-environment for efficient infection of target t cells (mcdonald et al., 2003) . moreover, it was postulated that dc-sign-mediated capture of hiv-1 virions temporarily protects them from degradation and preserves viral infectivity (geijtenbeek et al., 2000b; kwon et al., 2002) . the above data supporting dc-sign-mediated capture, uptake, intracellular transport and ultimately transfer of intact hiv-1 particles from dcs to target t cells were united in the 'trojan horse model' of mucosal hiv-1 transmission. this model posits that submucosal dcs capture and internalize hiv-1 virions via dc-sign and, by homing to the lymph nodes, provide a means of transport for the virus to a compartment rich in target cells. the virus-loaded dcs then interact with cd4 + t cells and the virions are transferred to the target t cell via the infectious synapse, ultimately resulting in efficient target cell infection (geijtenbeek et al., 2000b; baribaud et al., 2001; sewell & price, 2001) . however, recent research is not always in line with this initial model and has challenged several of its key features. cavrois et al. (2007) reported that hiv-1 trans-infection does not require intracellular virus trafficking, but primarily depends on cell surface-associated virions that reach the infectious synapses via transport on the cell surface (cavrois et al., 2007) . in line with this, yu et al. (2008) reported that hiv-1 traffics towards the infectious synapse through a specialized, surface-accessible intracellular compartment (yu et al., 2008) . in addition, reports stating that virus capture by dc-sign mainly leads to virus internalization into the endolysosomal pathway and subsequent degradation (moris et al., 2004; turville et al., 2004; moris et al., 2006) and that dc-sign-mediated trans-infection can only occur within the first hours after virus attachment (turville et al., 2004) seem to downplay the importance of dc-sign-mediated trans-infection for efficient hiv-1 infection and spread. these and other data counter the theory that hiv-1 capture by dcs preserves viral infectivity, and suggest that the presenceand transferof infectious virus at later time points may be ascribed to productive dc infection (turville et al., 2004; nobile et al., 2005; burleigh et al., 2006; wang et al., 2007a) . it is also noteworthy that, whereas initial studies identified dc-sign as the main factor involved in hiv-1 capture and transmission by dcs (geijtenbeek et al., 2000b) , recent studies also implicate other lectins in this process (turville et al., 2002; izquierdo-useros et al., 2012) or even conclude that dc-sign is not involved in dc-mediated hiv-1 trans-infection (boggiano et al., 2007) . differences in virus strains, cell types, and experimental setup might in part explain these conflicting data. intriguingly, other recent work indicates that dc-sign-expressing b lymphocytes and platelets may effectively capture and transfer infectious hiv-1 via dc-sign (boukour et al., 2006; chaipan et al., 2006; rappocciolo et al., 2006) , potentially implicating these cells/cell fragments in hiv-1 dissemination in infected patients, although recent work suggests that platelets might negatively regulate viral spread by secretion of cxcl4 (auerbach et al., 2012; tsegaye et al., 2013) . clearly, further research is necessary to allow a better understanding of dc-sign-mediated hiv-1 trans-infection and its relevance for viral infection and spread in vivo. importantly, the role of dc-sign in hiv-1 infection appears not to be restricted to purely physical capture of virions for subsequent degradation or cis-or transinfection. recruitment of dc-sign by hiv-1 also triggers signal transduction that modulates immune responses and infection of dcs and adjacent target cells more indirectly. for example, hodges et al. (2007) reported that binding of hiv-1 to dc-sign compromises dc maturation and primes these cells for trans-infection: upregulation of cd86 and mhcii is suppressed, whereas synapse formation between dcs and cd4 + t cells is promoted (hodges et al., 2007) . moreover, hiv-1 binding to dc-sign was shown to activate cdc42 and promote formation of membrane extensions that facilitate hiv-1 transfer to cd4 + lymphocytes (nikolic et al., 2011) . other work by gringhuis et al. (2007 gringhuis et al. ( , 2010 showed that binding of hiv-1 to dc-sign triggers raf-1 dependent signaling, which modulates toll-like receptor (tlr)-elicited signals to induce synthesis of fulllength hiv transcripts as well as production of the immunosuppressive cytokine il-10 ( gringhuis et al., (2007 gringhuis et al., ( , 2010 . in general, the data discussed above suggest that dc-sign recruitment by hiv-1 might affect viral infection and transmission, as well as the host defense against this pathogen in several ways. over the last decade, dc-sign has become a prototype for lectin-mediated cis-and trans-infection and has been implicated in the infection process of various viruses, including hiv, dengue virus, ebola virus, and iav (see table 2 ). importantly, however, dc-sign is not the only host lectin that is (ab)used by viruses to promote target cell infection or avoid immune recognition and clearance. various other membrane-associated host lectins seem to be exploited by virusesin ways similar to dc-signto aid cis-infection, trans-infection and/or viral persistence. analysis of recent literature suggests that membrane-associated host lectins may constitute weak links in the host's defense against viral pathogens (table 2) . although generally implicated in antiviral defense, soluble host lectins may also support viral infection (table 2) . for example, galectin-1 has been proposed to promote hiv-1 infection (ouellet et al., 2005; mercier et al., 2008; st-pierre et al., 2011; sato et al., 2012) . in vitro experiments pointed out that galectin-1 can enhance hiv-1 infection of different cell typesincluding human lymphoid cell lines, pbmc, cd4 + t lymphocytes, and monocyte-derived macrophages (ouellet et al., 2005; mercier et al., 2008) and increase hiv-1 infection in an ex vivo lymphoid tissue model (ouellet et al., 2005) . further experiments showed that galectin-1 accelerates virion binding to the target cell surface, probably by crosslinking viral and cellular glycans (ouellet et al., 2005; mercier et al., 2008) . a more recent study confirmed these findings and showed that galectin-1 binds to clusters of n-linked glycans on the viral gp120 envelope protein in a b-galactoside-dependent manner (st-pierre et al., 2011) . data from the same study identify the hiv-1 receptor cd4 as a ligand for galectin-1 and suggest that galectin-1 can cross-link gp120 and cd4 (st-pierre et al., 2011) . in sum, it appears that the dimeric lectin galectin-1 can enhance hiv-1 infection efficiency by cross-linking viral and host cell glycans and thereby promoting firmer adhesion of the virus to the target cell surface and facilitating virus-receptor interactions (ouellet et al., 2005; mercier et al., 2008; st-pierre et al., 2011; sato et al., 2012) . some studies have also attributed proviral effects to the collectins mbl, sp-a, and sp-d. for some viruses, it was reported thatunder certain conditionsviral recognition by collectins may enhance cis-or trans-infection (hickling et al., 2000; sano et al., 2003; gaiha et al., 2008; brudner et al., 2013; madsen et al., 2013) . it is conceivable that these collectins can bind the virus and subsequently associate with collectin receptors on the surface of target/ transmitting cells, thereby concentrating virions at the cell surface and facilitating infection or viral transfer. nevertheless, involvement of other mechanisms (e.g. collectinmediated cross-linking of virus-and host cell-displayed glycans, cfr. the galectin-1-hiv-1 example described above) can currently not be excluded. further research is necessary to elucidate the biology behind the potential proviral effects of collectins and to estimate the occurrence and relevance of these events in an in vivo context. figure 6 gives a schematic overview of how membraneassociated and soluble host lectins can be implicated in interactions that benefit the virus and facilitate viral infection and spread. as glycan-lectin interactions often represent key events in viral infection and/or antiviral immunity, variation in glycan or lectin expression and structureeither at the host or at the virus levelmay significantly shift the balance between host and pathogen. a basic insight into the nature and origin of this variability is therefore germane to a proper understanding of glycan-lectin interactions in the context of viral infection biology and immunology. glycan formation is a very complex and versatile biosynthetic process. in contrast to the primary amino acid sequences of proteins, glycan structures are not directly encoded in the host genome. instead, they are synthesized in a step-wise manner via the concerted action of various host-encoded glycosyltransferase, glycosidase, and other enzymes. the availability of these glycoenzymes, the availability of precursor molecules and the accessibility of specific glycosylation sites govern (the efficiency of) glycan addition and modification and hence determine glycan variability. the genetic make-up of the host evidently has a major impact on glycosylation, but also other host-related factors can have pronounced effects. recent research has shown that different cell types within a host can assemble radically different glycomes (roth, 1996; haslam et al., 2008) and that factors such as the activation (comelli et al., 2006; bax et al., 2007; haslam et al., 2008) or infection (lanteri et al., 2003) status of a cell can significantly influence glycosylation processes. clearly, various biological factors contribute to the high glycan heterogeneity that is seen for many animal glycoconjugates. although host glycan variation may influence virtually all viral infections in several ways, its potential impact is probably most evident for viruses that are equipped with viral lectins. a notable example in this context are the noroviruses, a major cause of nonbacterial gastroenteritis in humans. it is well known that the viral capsids of most human noroviruses display an affinity for hbgas, structurally related but highly polymorphic carbohydrate structures found on proteins and lipids of epithelial cells in the gastrointestinal and respiratory tract, on the surfaces of red blood cells and as free antigens in body fluids such as saliva, blood, and intestinal contents (bu et al., 2008; choi et al., 2008; shirato, 2011) . different noroviruses display distinct hbga specificities and can be categorized according to the (range of) hbga structures they preferentially bind shirato, 2011) . human hbga synthesis is controlled by various enzymes, including the glycosyltransferase enzymes encoded in the abo, fut2, and fut3 gene loci. the presence of variant (functional or nonfunctional) alleles at these and other relevant gene loci is a key determinant of hbga phenotype, as it controls which abh and lewis antigens an individual can synthesize (le pendu et al., 2006; shirato, 2011) . although it is still unclear whether they function as primary receptors for noroviruses, current data indicate that hbgas are important determinants of the noroviral tissue specificity. moreover, several studies have established a link between hbga geno-/ phenotype and individual susceptibility to (clinical) infection with specific norovirus variants: hbga phenotypes matching the specificity of the viral lectin correlate with a higher risk of (clinical) infection, whereas nonmatching hbga phenotypes correlate with relative resistance (le pendu et al., 2006; shirato, 2011) . several studies have revealed significant heterogeneity relating to animal lectins. lectin expression is governed by various genetic and nongenetic (e.g. hormone balance, immune status) factors. importantly, gene polymorphisms that affect protein expression and/or functionality have been described for several animal lectins, including mbl and dc-sign. for mbl, mutations in the promoter region of the mbl2 gene were found to affect protein expression levels, probably by influencing binding of transcription factors (eisen & minchinton, 2003; dommett et al., 2006; heitzeneder et al., 2012) . moreover, specific polymorphisms in mbl2 exon 1, encoding the collagen-like domain of mbl, appear to hinder correct and stable oligomerization of mbl protein chains and impede efficient ligand binding and activation of the lectin complement pathway (eisen & minchinton, 2003; dommett et al., 2006; heitzeneder et al., 2012) . several studies suggest a correlation between mbl deficiency and susceptibility to hiv infection, but conflicting data have been reported and further research is clearly necessary to corroborate this link (eisen & minchinton, 2003; dommett et al., 2006; heitzeneder et al., 2012) . similar findings have been recorded for dc-sign. polymorphisms in the promoter region of the dc-signencoding cd209 gene can affect protein expression levels and have been linked with altered susceptibility to and/or altered disease progression after infection with several viral pathogens, including hiv-1 and dengue virus (martin et al., 2004; sakuntabhai et al., 2005; koizumi et al., 2007; selvaraj et al., 2009; wang et al., 2011; boily-larouche et al., 2012) . moreover, distinct gene polymorphisms in the dc-sign-encoding region as well as alternative splicing events give rise to different isoforms of the protein, ranging from variants containing single nucleotide polymorphisms (snps) to variants with truncated lectin domains, variable numbers of 23-aa-residue repeats in the neck domain, alternative cytoplasmic domains or a lacking transmembrane region (mummidi et al., 2001; liu et al., 2004; serrano-gomez et al., 2008; boily-larouche et al., 2012) . information on the expression and biological activity of most of these dc-sign variants is rather limited. recently, however, boily-larouche et al. (2012) reported that naturally occurring genetic variants of dc-sign, carrying specific snps in the neck domain-encoding exon 4, have an enhanced capacity to capture and transfer hiv-1 virions to cd4 + t lymphocytes. moreover, liu et al. (2004) described differ-ent neck domain length variants of dc-signcarrying variable numbers of 23-aa-residue repeats in the neck regionand correlated neck domain length heterozygosity with a reduced risk of hiv-1 infection. recent experimental data provide evidence that naturally occurring dc-sign neck domain variants can differ in multimerization competence in the cell membrane and display altered glycan binding capacity (serrano-gomez et al., 2008) . moreover, the presence of such neck domain variants appears to modulate multimerization of the prototypic dc-sign molecule (serrano-gomez et al., 2008) . the fact that neck domain variation may influence the presence, stability, and functionality of dc-sign multimers on the cell surface can provide a molecular explanation for the link between dc-sign polymorphisms and susceptibility to hiv-1 and other pathogens, although further research is needed to substantiate this (serrano-gomez et al., 2008) . although viruses rely on the host cell machinery for glycoconjugate synthesis, viral glycosylation profiles can significantly differ from the standard glycosylation profile of their host cell. for instance, it is well known that viral glycoproteins are often more heavily glycosylated than host glycoproteins, and that also the nature of their glycan modifications can significantly differ. a prototypic example in this context is the gp120 glycoprotein of hiv-1. the hiv-1 envelope is studded with trimers of noncovalently associated gp120/gp41 heterodimers . gp120 is one of the most heavily n-glycosylated proteins in nature: it contains more than 20 n-linked glycosylation sites, and n-glycans account for about half of its molecular weight (zhu et al., 2000; wei et al., 2003; pantophlet & burton, 2006; scanlan et al., 2007) . intriguingly, whereas mammalian glycoproteins typically carry mainly complex type n-glycans, this is not the case for the viral gp120 glycoprotein. recent reasearch has shown that virion-associated gp120 of pbmc-grown virusas opposed to recombinantly expressed monomeric gp120predominantly carries oligomannose n-glycans (doores et al., 2010; bonomelli et al., 2011) . the synthesis of this unusual glycosylation profile appears to be partially directed by the structure of the gp120/gp41 spike itself: the presence of a dense n-glycan cluster in gp120, combined with the steric consequences of gp120/gp41 trimerization, seems to hinder further processing of (normally transient) biosynthetic glycan intermediates by er and golgi a-mannosidases, ultimately yielding hiv-1 virions with oligomannose-enriched gp120 glycoproteins (zhu et al., 2000; doores et al., 2010; eggink et al., 2010; bonomelli et al., 2011) . clearly, although viral glycosylation is critically dependent on the glycosylation machinery of the host cell, the genetic and structural background of a virus can have a decisive influence in this process. importantly, viral infection itself may also have strong repercussions on the glycosylation biology of a host cell. considering the restricted glycosylation enzyme and precursor availabilities, it is conceivable that overexpression of viral glycoproteins in an infected target cell can result in an increased glycan heterogeneity of both viral and cellular glycoconjugates. moreover, viruses may also actively modify the host and viral glycome by modulating the expression of host cell glycoenzymes (hiraiwa et al., 1997; cebulla et al., 2000; hiraiwa et al., 2003) or via expression of virally encoded glycoenzymes in infected cells (jackson et al., 1999; willer et al., 1999; nash et al., 2000; sujino et al., 2000; vanderplasschen et al., 2000; markine-goriaynoff et al., 2003 , 2004a . an additional source of glycan variation can be discerned for viruses that can infect multiple cell types, or even different host species. for instance, it is well known that hiv can productively infect multiple cell types, and that hiv glycosylation is cell type-dependent (liedtke et al., 1994; willey et al., 1996; liedtke et al., 1997; lin et al., 2003) . cell type-dependent glycosylation differences for hiv have been shown to impact viral interaction with and trans-infection via dc-sign (lin et al., 2003) , as well as viral sensitivity to antibody neutralization (willey et al., 1996) . another, particularly fascinating example in this context is dengue virus (dv). dv is a mosquito-borne flavivirus that can replicate in mosquitos as well as in humans (navarro-sanchez et al., 2003; dejnirattisai et al., 2011) . in humans, immature skin dcs are considered the primary target cell for the virus after a mosquito bite, and dc-sign is believed to be the main dv receptor on these cells (navarro-sanchez et al., 2003; dejnirattisai et al., 2011) . in a recent study, it was shown that insect cell-derived dv can efficiently infect dcs, whereas dc-derived dv is not able to reinfect dcs (dejnirattisai et al., 2011) . similarly, insect cell-derived dv could efficiently bind and infect a dc-sign-expessing cell line, whereas this was not the case for dcderived dv (dejnirattisai et al., 2011) . finally, it was found that insect cell-derived dv predominantly contains high-/pauci-mannose-type n-glycans, whereas dcderived virus contains only complex type n-glycans (dejnirattisai et al., 2011) . projected against the background of dv infection, these data outline a tentative model of the first stages of dv infection in humans: during dv replication in mosquito cells, newly formed dv virions obtain mannose-rich glycans. upon viral transfer to a human host, these virions efficiently infect immature skin dcs via dc-sign. importantly, dv replication in skin dcs yields virions with complex type n-glycans, thus creating a 'glycan mismatch' with dc-sign. due to this mismatch, newly synthesized dc-derived virus will not readily reinfect dcs via dc-sign, but preferentially infect other potential host cells via other receptors (dejnirattisai et al., 2011) . although much more research is needed to verify this tentative model, it elegantly illustrates how cell type-dependent glycan variability may impact a viral infection process. another notable factor to be considered in the context of virus-related variability is the rapid evolution of many viral pathogens. this seems especially significant for rna viruses, as these viruses generally evolve more rapidly than dna viruses due to factors inherent to their biology and infection strategy (belshaw et al., 2008; holmes, 2009; lauring & andino, 2010) . the higher mutation frequency of many rna viruses directly implies a higher chance for addition or deletion of putative glycosylation sites. as has been shown for iav, acquisition or deletion of glycosylation sites may affect crucial steps in the viral infection/replication process (e.g. receptor binding, fusion, release of newly formed virions) (ohuchi et al., 1997; wagner et al., 2000; tsuchiya et al., 2002; kim & park, 2012) , alter the capacity of the virus to avoid induction of/recognition by virus-specific antibodies (glycan shielding) wei et al., 2010; wanzeck et al., 2011; kim & park, 2012; job et al., 2013; sun et al., 2013) , and modulate viral interaction with various immune system lectins (reading et al., 2007; vigerust et al., 2007; reading et al., 2009; tate et al., 2011a, b) . clearly, mutational changes in the viral glycome may affect the virus-host interactome in various ways. ultimately, the net benefit of a specific glycome change will determine if a glycosylation variant may become dominant in the virus population. however, not only the viral glycosylation status, but also the affinity and specificity of viral lectins for specific glycoconjugates may change as a result of mutations. for instance, ample data show that amino acid changes at specific sites of the iav hemagglutinin protein can significantly alter its affinity and/or specificity for particular sialic acid-containing receptorsa factor that is crucial for the virus to infect new host species (skehel & wiley, 2000; wagner et al., 2002; suzuki, 2005; gamblin & skehel, 2010) . finally, functional alterations in the viral rde due to mutations may also have a strong impact on the interaction of viral lectins with host cell glycans. in the case of iav, balanced lectin and rde functions appear to be crucial for efficient viral replication. for iav variants that are well adapted to a certain host species, the substrate specificity and activity of the neuraminidase generally match the ligand specificity and affinity of the hemagglutinin . disruption of this balancefor instance due to reassortment or transmission to a new host speciesoften results in a decreased replicative fitness . interestingly, however, the virus may overcome this hurdle and evolve towards replicative competence by selecting for compensatory mutations in hemagglutinin and/or neuraminidase that restore the functional balance between these molecules . considering these data on iav, it is conceivable that the balance between viral lectin and rde is also an important determinant of the replicative fitness of several other viruses. on a related note, glycan-lectin interactions in virus biology are typically studied using a limited number of (prototypic) virus variants. although the information obtained in these studies can often be extrapolated to include other virus variants, there are important exceptions. for iav, for instance, it is well documented that different virus variants can carry hemagglutinin lectins with distinct glycan ligand specificities and therefore associate with distinct spectra of (decoy) receptors. moreover, iav variants can display different glycan arrays on their surface, which has been shown to modulate viral infection, glycan shielding, and recognition by various immune system lectins (cfr. supra). the fact that the specific genetic make-up of a virus determines specificity/ affinity of viral lectins, co-directs viral glycosylation, etc., and that this can be mirrored in a distinct virus-host interactome remains an important issue in glycovirology. in sum, an intricate web of glycan-lectin interactions can modulate viral infection, and host and virus inherent variability in glycans and lectins adds a further layer of complexity to this matter. considering the pivotal roles of glycan-lectin interactions in many viral infections, interfering with these interactions seems an attractive strategy in the combat against these pathogens. conversely, strategies that promote recognition of viruses by specific immune system lectinsinvolved in viral inhibition and clearancemay also prove useful in antiviral therapies. several possibilities have been and are currently being explored. perhaps the most obvious strategy to modulate glycanlectin binding is the use of molecules that can physically interfere with these interactions. glycan decoys (e.g. carbohydrate-containing drugs, sugar analogs/glycomimetics) or carbohydrate-binding agents (cbas) may be used in antiviral therapies to directly block key glycan-lectin interactions at the side of the lectin and at the side of the glycan, respectively. binding of glycan decoys with a specific lectin can inhibit binding of other ligands by this lectin. for instance, it has been shown that multivalent mannosecontaining molecules or mannose-based glycomimetics can compromise binding of hiv-1 gp120 with dc-sign (wang et al., 2008b; luallen et al., 2009; martinez-avila et al., 2009b; becer et al., 2010) and inhibit dc-sign-mediated trans-infection of cd4 + t lymphocytes (martinez-avila et al., 2009a; sattin et al., 2010; berzi et al., 2012) . likewise, sialic acid-containing glycan decoys and sialic acid analogs are being evaluated for their capacity to block the sialic acid binding site of iav hemagglutinin to inhibit interaction of the virus with sialic acid-containing receptor molecules on the surface of target cells (landers et al., 2002; matrosovich & klenk, 2003; matsubara et al., 2010; papp et al., 2010 papp et al., , 2011 . alternatively, binding of cbas to glycans displayed on the virion surface can inhibit viral cis-or trans-infection via host cell lectins. this is elegantly exemplified in several recent studies, showing that mannose-as well as n-acetylglucosamine-specific cbas can effectively prevent dc-sign-mediated hiv-1 capture and subsequent transmission to t lymphocytes balzarini et al., 2007a; bertaux et al., 2007; huskens et al., 2010; hoorelbeke et al., 2011; alexandre et al., 2012) . although cba binding to host cell-associated glycans may inhibit viruses that employ viral lectins, the potential of this strategy for antiviral therapy remains virtually unexplored. it is noteworthy that the antiviral activity of cbas or glycan decoys that bind to virion surfaces is not necessarily limited to direct inhibition of crucial glycan-lectin interactions, as they can mask greater portions of the virus and interfere with other crucial (including non-glycanlectin) interactions or steps in the infection process. moreover, recent research on hiv-1 highlights the antiviral potential of cbas from yet another angle. although the heavily glycosylated hiv-1 gp120 protein generally provides multiple ligands for mannose-and glcnac-specific cbas, prolonged cba pressure selects for hiv-1 variants with multiple n-glycosylation site deletions in the gp120 protein that are less sensitive to cba-mediated neutralization (balzarini et al., 2004 (balzarini et al., , 2005a witvrouw et al., 2005; balzarini et al., 2006; balzarini, 2007b, c; balzarini et al., 2007b; huskens et al., 2007) . interestingly, however, deletion of n-glycosylation sites can also increase the immunogenicity of the virus and weaken the glycan shield that protects the virus from recognition by virus-specific antibodies and other (nonlectin) immune receptors (botarelli et al., 1991; back et al., 1994; reitter et al., 1998; bolmstedt et al., 2001; kang et al., 2005) , and may decrease the efficiency of hiv-1 trans-infection via immune system lectins like dc-sign (hong et al., 2007; liao et al., 2011) . in addition, it appears that accumulation of mutations under cba pressure is often paralleled by a significant reduction of the viral fitness, which is obviously advantageous in the context of an antiviral treatment (balzarini et al., 2005a; balzarini, 2007b, c) . for further information on cbas and their potential in antiviral therapy, readers may refer to recent expert reviews on this topic (balzarini, 2007c; francois & balzarini, 2012) . although the use of glycan decoys and cbas may seem the most intuitive strategy to interfere directly with glycan-lectin interaction, other options exist as well. for instance, similar to glycan decoys and cbas, lectin-or glycoconjugate-specific immunoglobulins may be used to block specific interactions. evidently, the increasing availability of such 'direct' modulators of glycan-lectin interaction is mirrored in an increasing number of potential antiviral applications. apart from direct modulation, also strategies that influence glycan-lectin interactions more indirectly can be employed. in fact, many of the molecules used to examine glycan-lectin interactions in vitro suggest themselves as potential therapeutics. one approach to indirectly govern glycan-lectin interaction is via the use of drugs that alter the host and/or viral glycome. glycosidases and other enzymes may be used to alter the glycan portions of fully formed and matured glycoconjugates. alternatively, various drugs may be employed to directly modify glycan synthesis: glycoconjugates produced in the presence of such molecules will obtain aberrant glycosylation, which may promote or annihilate their interaction with specific lectins. promising results in glycovirological research have highlighted the antiviral potential of such compounds. for instance, ample data indicate that sialidases may be used to counteract infections where sialic acids play important roles as cellular receptors for viral lectins [e.g. iav binds to sialic acid receptors on the airway epithelium (skehel & wiley, 2000; malakhov et al., 2006; belser et al., 2007; harrison, 2008; chan et al., 2009; triana-baltzer et al., 2009a , 2011 or as viral ligands for host lectins that serve as portals for viral entry [e.g. sialic acids on the porcine reproductive and respiratory syndrome virus bind the macrophage-specific entry mediator sialoadhesin (delputte & nauwynck, 2004; delputte et al., 2007; van breedam et al., 2010a, b) ]. sialidase treatment may also enhance recognition of viral glycoconjugates by mannose-specific immune system lectins that can limit viral infection: in vitro experiments have shown that enzymatic removal of sialic acids from the hiv-1 virion surface can significantly enhance virus binding and neutralization by mbl (hart et al., 2002 (hart et al., , 2003 . in line with this, production of hiv-1 in the presence of the golgi a1-2-mannosidase i inhibitor 1-deoxymannojirimycinwhich blocks the biosynthesis of complex-type, sialylated oligosaccharidesincreased susceptibility of the virus to mbl-mediated neutralization (hart et al., 2003) . interestingly, 1-deoxymannojirimycin treatment of hiv-1-infected cells was also shown to potentiate the antiviral effects of mannose-specific plant cbas towards the newly produced hiv-1 virions (balzarini, 2007a) . these and other examples illustrate the potential of these molecules as antiviral drugs. considering the various structural and nonstructural roles of glycans, it is clear that glycome modulation can also have effects beyond the alteration of glycan-lectin binding events. for instance, interfering with host cell glycosylation processes using specific inhibitors may inhibit assembly of infectious virions (leavitt et al., 1977; katz et al., 1980; pizer et al., 1980; herrler & compans, 1983; montefiori et al., 1988; pal et al., 1989; mehta et al., 1998; dwek et al., 2002; wu et al., 2002; durantel et al., 2007; lazar et al., 2007; scanlan et al., 2007; durantel, 2009; merry & astrautsova, 2010) . moreover, glycome modulation may significantly alter the capacity of the virus to evade recognition by virus-specific antibodies and b-and t-cell receptors via glycan shielding (botarelli et al., 1991; back et al., 1994; willey et al., 1996; reitter et al., 1998; bolmstedt et al., 2001; kang et al., 2005; aguilar et al., 2006; wang et al., 2009; francica et al., 2010; kobayashi & suzuki, 2012) . clearly, glycome-modifying drugs can counteract viruses in various ways and constitute versatile tools in the control of viral infection. also other strategies that can indirectly influence glycan-lectin interactions are certainly worth exploring. for instance, drugs that alter host or viral lectin expression (e.g. cytokines or rnai) may prove useful in antiviral strategies (ochiel et al., 2010; relloso et al., 2002; ge et al., 2003; arrighi et al., 2004; ge et al., 2004; nair et al., 2005; yagi et al., 2010; raza et al., 2011) . furthermore, patients infected with a virus that employs both viral lectins and rdes may also benefit from treatment with rde-specific inhibitors, as these can alter the balance between viral lectin and rde activity which is often crucial for efficient viral replication and spread. notable examples in this context are the several neuraminidase inhibitors that have been used successfully for the treatment of iav infections (kim et al., 1999; lew et al., 2000; roberts, 2001; garman & laver, 2004; alymova et al., 2005; von itzstein, 2007; von itzstein & thomson, 2009; gamblin & skehel, 2010; ikematsu & kawai, 2011) . in sum, drugs that modulate glycan-lectin interactionseither directly or indirectlycan be powerful instruments in the combat against viral pathogens. however, the antiviral strategies suggested above can also have drawbacks. intensive use of antiviral therapeutics may elicit rapid selection of drug-resistant virus variants. another possible drawback relates to the potential off-target effects of these therapies: therapeutics aimed at influencing specific glycan-lectin interactions that play key roles in viral infection processes may also affect general host glycosylation, lectin expression or glycan-lectin interactions that are crucial for normal functioning of the host and its immune defense. it is also conceivable that such drugs, despite their antiviral effects, may benefit the virus in some ways. for instance, although a glycome-modifying drug may promote viral recognition by specific immune system lectins that aid in viral clearance, it may also promote viral interaction with host lectins that can aid in cis-or transinfection. ultimately, the potential of specific agents as antiviral drugs depends on their net (antiviral) effects in vivo. other potential disadvantages concern the pharmacokinetic properties of specific drugs. for instance, if glycan decoys or cbas used in antiviral therapy show a broad reactivity and can respectively bind with multiple (nontarget) lectins or glycans in the host, it is possible that much of the antiviral effect is lost. also, when using peptidic cbas that are not native to the host, an antibody response might be mounted against these components, leading to neutralization and/or faster clearance of the active compound. in spite of these and other potential pitfalls, it is clear that glycan decoys and cbas, as well as various indirect approaches to modulate glycan-lectin interaction, show potential for the treatment of diverse viral infections, either or not in combination with other antiviral strategies. a good understanding of relevant glycan-lectin interactions facilitates specific targeting of these binding events and can help to minimize possible off-target effects and to reduce the risk of drug resistance. glycans and lectins cover crucial roles in virus biology and their interplay often shapes the virus-host interaction. in general, the nature of the glycan, the lectin, and the specific conditions under which their interaction occurs determines the outcome of a specific binding event and directs the virus to a certain fate. based on current knowledge, it is clear that viral lectins generally facilitate viral infection and spread. on the other hand, although it may seem intuitive that host lectin prrs and other immune system lectins exclusively act in defense of the host, there is ample evidence that contradicts this. although many host lectins are involved in the induction of an efficacious immune response against viral pathogens, many viruses can also abuse these lectins to promote infection and spread. intriguingly, analysis of the many studies regarding the role of host lectins in viral infections suggests that soluble host lectins tend to be associated with antiviral activity, whereas membrane-associated host lectins seem to play a more dubious role and are often implicated in pro-as well as antiviral mechanisms (tables 1 & 2) . it is however noteworthy that the information provided in this manuscript reflects current views on glycan-lectin interactions in virus biology, and that future research may alter our understanding and interpretation of specific interactions. the fact that (aspects of) viral glycobiology may change during virus-host co-evolution even advocates periodic re-evaluation of specific glycan-lectin interactions. the biology of glycans and lectins is complex and has long been poorly accessible to virologists and other scientists outside this field. this situation is changing with the emergence of international glycomics consortia (e.g. consortium for functional glycomics), which can provide state-of-the-art techniques and expertise to analyze and interpret virologically/immunologically relevant glycanlectin interactions. still, there are specific pitfalls associated with glycovirological research. in vitro experiments need to be designed and interpreted considering key issues like glycan and lectin variability, the cell-type dependency of host and virus glycosylation and the influence of the lectin-expressing cell type on the final outcome of a glycan-lectin interaction. in addition, the results of in vitro experiments must ultimately be compared withand re-interpreted in the context ofdata obtained in animal models. in fact, our current understanding of specific glycan-lectin interactions in viral infection is mostly based on in vitro experiments, underlining the need for experimental validation of these results in the context of the infected host. in the context of viral infections, many different (lectin-dependent or -independent) interactions and processes take place simultaneously, resulting in a complex network of virus-host factor interaction, signaling, and effector mechanisms, the net effect of which may benefit the host or the virus. many of the interactions taking place are not yet well defined and probably more are still unknown. key to combating viral disease is to make these black boxes more transparent. synergisms between different branches of life sciences are essential to sustain and advance our knowledge in this important field of research. n-glycans on nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry the lectins griffithsin, cyanovirin-n and scytovirin inhibit hiv-1 binding to the dc-sign receptor and transfer to cd4(+) cells neuraminidase inhibitors as antiviral agents complement-dependent neutralization of influenza virus by a serum mannose-binding lectin lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells identification of the platelet-derived chemokine cxcl4/pf-4 as a broad-spectrum hiv-1 inhibitor an n-glycan within the human immunodeficiency virus type 1 gp120 v3 loop affects virus neutralization the alpha(1,2)-mannosidase i inhibitor 1-deoxymannojirimycin potentiates the antiviral activity of carbohydrate-binding agents against wild-type and mutant hiv-1 strains containing glycan deletions in gp120 carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses? targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy profile of resistance of human immunodeficiency virus to mannose-specific plant lectins carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120: a new therapeutic concept to hit the achilles heel of hiv marked depletion of glycosylation sites in hiv-1 gp120 under selection pressure by the mannose-specific plant lectins of hippeastrum hybrid and galanthus nivalis mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type 1 strains with n-glycan deletions in their gp120 envelopes carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign)-directed hiv-1 transmission to t lymphocytes pradimicin a, a carbohydrate-binding nonpeptidic lead compound for treatment of infections with viruses with highly glycosylated envelopes, such as human immunodeficiency virus pradimicin s, a highly soluble nonpeptidic small-size carbohydrate-binding antibiotic, is an anti-hiv drug lead for both microbicidal and systemic use the role of dc-sign and dc-signr in hiv and siv attachment, infection, and transmission quantitative expression and virus transmission analysis of dc-sign on monocyte-derived dendritic cells a novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of mdck cells in culture dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins high-affinity glycopolymer binding to human dc-sign and disruption of dc-sign interactions with hiv envelope glycoprotein das181, a novel sialidase fusion protein, protects mice from lethal avian influenza h5n1 virus infection pacing a small cage: mutation and rna viruses interactions of surfactant protein a with influenza a viruses: binding and neutralization surfactant protein a, but not surfactant protein d, is an opsonin for influenza a virus phagocytosis by rat alveolar macrophages entry of hepatitis c virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions a glycomimetic compound inhibits dc-sign-mediated hiv infection in cellular and cervical explant models the relevance of complement to virus biology dendritic cell-mediated trans-enhancement of human immunodeficiency virus type 1 infectivity is independent of dc-sign naturally-occurring genetic variants in human dc-sign increase hiv-1 capture, cell-transfer and risk of mother-to-child transmission enhanced immunogenicity of a human immunodeficiency virus type 1 env dna vaccine by manipulating n-glycosylation signals. effects of elimination of the v3 n306 glycan the glycan shield of hiv is predominantly oligomannose independently of production system or viral clade n-glycosylation of hiv-gp120 may constrain recognition by t lymphocytes an integrated view of humoral innate immunity: pentraxins as a paradigm lentivirus degradation and dc-sign expression by human platelets and megakaryocytes shared paramyxoviral glycoprotein architecture is adapted for diverse attachment strategies lectin-dependent enhancement of ebola virus infection via soluble and transmembrane c-type lectin receptors structural basis for the receptor binding specificity of norwalk virus infection of dendritic cells (dcs), not dc-sign-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to t cells dual function of c-type lectin-like receptors in the immune system how c-type lectins detect pathogens structural basis for the recognition of blood group trisaccharides by norovirus in vitro derived dendritic cells trans-infect cd4 t cells primarily with surface-bound hiv-1 virions cytomegalovirus induces sialyl lewis (x) and lewis(x) on human endothelial cells dc-sign and clec-2 mediate human immunodeficiency virus type 1 capture by platelets das181 inhibits h5n1 influenza virus infection of human lung tissues paramyxovirus fusion and entry: multiple paths to a common end lack of the pattern recognition molecule mannose-binding lectin increases susceptibility to influenza a virus infection dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate the essentiality of alpha-2-macroglobulin in human salivary innate immunity against new h1n1 swine origin influenza a virus atomic resolution structural characterization of recognition of histo-blood group antigens by norwalk virus influenza virus neuraminidase: structure, antibodies, and inhibitors activation of murine cd4+ and cd8+ t lymphocytes leads to dramatic remodeling of n-linked glycans siglecs as positive and negative regulators of the immune system siglecs and their roles in the immune system envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens specificity and affinity of sialic acid binding by the rhesus rotavirus vp8* core celgosivir, an alpha-glucosidase i inhibitor for the potential treatment of hcv infection glucosidase inhibitors as antiviral agents for hepatitis b and c targeting glycosylation as a therapeutic approach lack of complex n-glycans on hiv-1 envelope glycoproteins preserves protein conformation and entry function impact of mannose-binding lectin on susceptibility to infectious diseases lectin-carbohydrate interactions: different folds, common recognition principles noroviruses everywhere: has something changed? genetic diversity and histo-blood group antigen interactions of rhesus enteric caliciviruses structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein potential of carbohydrate-binding agents as therapeutics against enveloped viruses evolution of the lectin-complement pathway and its role in innate immunity from lectin structure to functional glycomics: principles of the sugar code surfactant protein a binds to hiv and inhibits direct infection of cd4+ cells, but enhances dendritic cell-mediated viral transfer influenza hemagglutinin and neuraminidase membrane glycoproteins endogenous ligands for c-type lectin receptors: the true regulators of immune homeostasis controlling influenza by inhibiting the virus's neuraminidase endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription use of sirnas to prevent and treat influenza virus infection signalling through c-type lectin receptors: shaping immune responses identification of dc-sign, a novel dendritic cell-specific icam-3 receptor that supports primary immune responses dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells location, location, location: new insights into o-galnac protein glycosylation two evolutionary strategies of influenza viruses to escape host non-specific inhibitors: alteration of hemagglutinin or neuraminidase specificity pattern recognition receptors: doubling up for the innate immune response neuraminidase: the specific enzyme of influenza virus and vibrio cholerae dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin/cd209 is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction c-type lectin dc-sign modulates toll-like receptor signaling via kinase-dependent acetylation of transcription factor nf-kappab hiv-1 exploits innate signaling by tlr8 and dc-sign for productive infection of dendritic cells association between expression of the h histo-blood group antigen, alpha1,2fucosyltransferases polymorphism of wild rabbits, and sensitivity to rabbit hemorrhagic disease virus binding and transfer of human immunodeficiency virus by dc-sign+ cells in human rectal mucosa viral membrane fusion paramyxovirus assembly and budding: building particles that transmit infections high mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (mbl) to hiv type 1 glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type 1 binding and neutralization by mannose-binding lectin human mannose-binding protein functions as an opsonin for influenza a viruses evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses neutrophil deactivation by influenza a viruses: mechanisms of protection after viral opsonization with collectins and hemagglutination-inhibiting antibodies mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins mechanism of binding of surfactant protein d to influenza a viruses: importance of binding to haemagglutinin to antiviral activity characterizing the glycome of the mammalian immune system pulmonary collectins modulate strain-specific influenza a virus infection and host responses mannan-binding lectin deficiency -good news, bad news, doesn't matter? posttranslational modification and intracellular transport of mumps virus glycoproteins structure and function of the hef glycoprotein of influenza c virus neuraminic acid is involved in the binding of influenza c virus to erythrocytes the receptor-destroying enzyme of influenza c virus is neuraminate-o-acetylesterase the glycoprotein of influenza c virus is the haemagglutinin, esterase and fusion factor 9-o-acetylated sialic acid, a receptor determinant for influenza c virus and coronaviruses lung surfactant protein a provides a route of entry for respiratory syncytial virus into host cells dc-sign increases the affinity of hiv-1 envelope glycoprotein interaction with cd4 assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro human t-cell leukemia virus-1 encoded tax protein transactivates alpha 1->3 fucosyltransferase fuc-t vii, which synthesizes sialyl lewis x, a selectin ligand expressed on adult t-cell leukemia cells transactivation of the fucosyltransferase vii gene by human t-cell leukemia virus type 1 tax through a variant camp-responsive element activation of the lectin dc-sign induces an immature dendritic cell phenotype triggering rho-gtpase activity required for hiv-1 replication the evolution and emergence of rna viruses human immunodeficiency virus envelope (gp120) binding to dc-sign and primary dendritic cells is carbohydrate dependent but does not involve 2g12 or cyanovirin binding sites: implications for structural analyses of gp120-dc-sign binding identification of the optimal dc-sign binding site on human immunodeficiency virus type 1 gp120 differences in the mannose oligomer specificities of the closely related lectins from galanthus nivalis and zea mays strongly determine their eventual anti-hiv activity norovirus and histo-blood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein e(rns) resistance of hiv-1 to the broadly hiv-1-neutralizing, anti-carbohydrate antibody 2g12 microvirin, a novel alpha (1,2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv-1 activity comparable with that of cyanovirin-n but a much higher safety profile laninamivir octanoate: a new long-acting neuraminidase inhibitor for the treatment of influenza siglec-1 is a novel dendritic cell receptor that mediates hiv-1 trans-infection through recognition of viral membrane gangliosides myxoma virus encodes an alpha2,3-sialyltransferase that enhances virulence innate immune recognition mucin-type o-glycosylation-putting the pieces together addition of glycosylation to influenza a virus hemagglutinin modulates antibody-mediated recognition of h1n1 2009 pandemic viruses modified hiv envelope proteins with enhanced binding to neutralizing monoclonal antibodies human mannan-binding lectin inhibits the infection of influenza a virus without complement antiviral activity of tunicamycin on herpes simplex virus n-linked glycosylation in the hemagglutinin of influenza a viruses neuraminidase inhibitors as anti-influenza virus agents correction of pulmonary abnormalities in sftpdà/à mice requires the collagenous domain of surfactant protein d adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor evidence for n-glycan shielding of antigenic sites during evolution of human influenza a virus hemagglutinin rantes -28g delays and dc-sign -139c enhances aids progression in hiv type 1-infected japanese hemophiliacs point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants heparin-dependent attachment of respiratory syncytial virus (rsv) to host cells identification and functions of pattern-recognition receptors structural and functional analysis of the surface protein of human coronavirus oc43 dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection orthomyxoviridae: the viruses and their replication prevention of influenza pneumonitis by sialic acid-conjugated dendritic polymers altered t cell surface glycosylation in hiv-1 infection results in increased susceptibility to galectin-1-induced cell death the expanding horizons of asparagine-linked glycosylation quasispecies theory and the behavior of rna viruses treatment of hepatitis b virus-infected cells with alpha-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity mendelian resistance to human norovirus infections tunicamycin inhibits glycosylation and multiplication of sindbis and vesicular stomatitis viruses modes of paramyxovirus fusion: a henipavirus perspective cis expression of dc-sign allows for more efficient entry of human and simian immunodeficiency viruses via cd4 and a coreceptor viral piracy: hiv-1 targets dendritic cells for transmission surfactant protein d enhances clearance of influenza a virus from the lung in vivo absence of sp-a modulates innate and adaptive defense responses to pulmonary influenza infection surfactant protein-d enhances phagocytosis and pulmonary clearance of respiratory syncytial virus novel innate immune functions for galectin-1: galectin-1 inhibits cell fusion by nipah virus envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines discovery and development of gs 4104 (oseltamivir): an orally active influenza neuraminidase inhibitor surfactant protein-a-deficient mice display an exaggerated early inflammatory response to a beta-resistant strain of influenza a virus identification of the dc-sign-interactive domains on the envelope glycoprotein of hiv-1 crf07_bc oligosaccharide profiles of hiv-2 external envelope glycoprotein: dependence on host cells and virus isolates host-cell-specific glycosylation of hiv-2 envelope glycoprotein differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr analysis of genetic polymorphisms in ccr5, ccr2, stromal cell-derived factor-1, rantes, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in seronegative individuals repeatedly exposed to hiv-1 principles of structures of animal and plant lectins a yeast glycoprotein shows high-affinity binding to the broadly neutralizing human immunodeficiency virus antibody 2g12 and inhibits gp120 interactions with 2g12 and dc-sign surfactant protein d modulates hiv infection of both t-cells and dendritic cells sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection binding of human collectins (sp-a and mbp) to influenza virus adaptation of tick-borne encephalitis virus to bhk-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo the neck region of the c-type lectin dc-sign regulates its surface spatiotemporal organization and virus-binding capacity on antigen-presenting cells the core 2 beta-1,6-n-acetylglucosaminyltransferase-mucin encoded by bovine herpesvirus 4 was acquired from an ancestor of the african buffalo glycosyltransferases encoded by viruses the core 2 beta-1,6-n-acetylglucosaminyltransferase-m encoded by bovine herpesvirus 4 is not essential for virus replication despite contributing to post-translational modifications of structural proteins association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type 1 infection multivalent manno-glyconanoparticles inhibit dc-sign-mediated hiv-1 trans-infection of human t cells gold manno-glyconanoparticles: multivalent systems to block hiv-1 gp120 binding to the lectin dc-sign natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding influenza viruses differ in recognition of 4-o-acetyl substitution of sialic acid receptor determinant molecular mechanisms of serum resistance of human influenza h3n2 virus and their involvement in virus adaptation in a new host sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy recruitment of hiv and its receptors to dendritic cell-t cell junctions divergent roles for c-type lectins expressed by cells of the innate immune system ligand recognition by antigen-presenting cell c-type lectin receptors alpha-glucosidase inhibitors as potential broad based anti-viral agents galectin-1 promotes hiv-1 infectivity in macrophages through stabilization of viral adsorption alternative approaches to antiviral treatments: focusing on glycosylation as a target for antiviral therapy inhibition of hemagglutination activity of influenza a viruses by sp-a1 and sp-a2 variants expressed in cho cells dc-specific icam-3-grabbing nonintegrin mediates internalization of hiv-1 into human podocytes role of protein n-glycosylation in pathogenesis of human immunodeficiency virus type 1 dc-sign promotes exogenous mhc-i-restricted hiv-1 antigen presentation dendritic cells and hiv-specific cd4+ t cells: hiv antigen presentation, t-cell activation, and viral transfer extensive repertoire of membrane-bound and soluble dendritic cell-specific icam-3-grabbing nonintegrin 1 (dc-sign1) and dc-sign2 isoforms. inter-individual variation in expression of dc-sign transcripts rnai-directed inhibition of dc-sign by dendritic cells: prospects for hiv-1 therapy influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins post-translational modification of the myxoma-virus anti-inflammatory serpin serp-1 by a virally encoded sialyltransferase dendritic-cell-specific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses hiv-1 activates cdc42 and induces membrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation covert human immunodeficiency virus replication in dendritic cells and in dc-sign-expressing cells promotes long-term transmission to lymphocytes histo-blood group antigens act as attachment factors of rabbit hemorrhagic disease virus infection in a virus strain-dependent manner uterine epithelial cell regulation of dc-sign expression inhibits transmitted/founder hiv-1 trans infection by immature dendritic cells regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety c (2011) myeloid c-type lectin receptors in pathogen recognition and host defense galectin-1 acts as a soluble host factor that promotes hiv-1 infectivity through stabilization of virus attachment to host cells role of oligosaccharides in the processing and maturation of envelope glycoproteins of human immunodeficiency virus type 1 gp120: target for neutralizing hiv-1 antibodies inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles inhibition of influenza virus activity by multivalent glycoarchitectures with matched sizes cell-surface heparan sulfate proteoglycan mediates hiv-1 infection of t-cell lines the glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins cell surface expression of biologically active influenza c virus hef glycoprotein expressed from cdna methods in enzymology: o-glycosylation of proteins the interaction of hiv with dendritic cells: outcomes and pathways effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production dc-sign interactions with human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus basis for the potent inhibition of influenza virus infection by equine and guinea pig alpha 2-macroglobulin synthesis and sorting of proteoglycans nmr experiments reveal the molecular basis of receptor recognition by a calicivirus dc-sign on b lymphocytes is required for transmission of hiv-1 to t lymphocytes selection of predicted sirna as potential antiviral therapeutic agent against influenza virus a serum mannose-binding lectin mediates complement-dependent lysis of influenza virus-infected cells collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice glycosylation as a target for recognition of influenza viruses by the innate immune system loss of a single n-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice a role for carbohydrates in immune evasion in aids dc-sign (cd209) expression is il-4 dependent and is negatively regulated by ifn, tgf-beta, and anti-inflammatory agents anti-influenza drugs and neuraminidase inhibitors differential sensitivity of human, avian, and equine influenza a viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants influenza c virus uses 9-o-acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus protein glycosylation in the endoplasmic reticulum and the golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein a-gold and lectin-gold techniques binding of rabbit hemorrhagic disease virus to antigens of the abh histo-blood group family distinct glycoprotein inhibitors of influenza a virus in different animal sera alpha 2-macroglobulin is the major neutralizing inhibitor of influenza a virus in pig serum tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle a variant in the cd209 promoter is associated with severity of dengue disease signaling by myeloid c-type lectin receptors in immunity and homeostasis lactoferrin and surfactant protein a exhibit distinct binding specificity to f protein and differently modulate respiratory syncytial virus infection galectins in innate immunity: dual functions of host soluble beta-galactoside-binding lectins as damage-associated molecular patterns (damps) and as receptors for pathogen-associated molecular patterns (pamps) glycans, galectins, and hiv-1 infection inhibition of dc-sign-mediated hiv infection by a linear trimannoside mimic in a tetravalent presentation exploiting the defensive sugars of hiv-1 for drug and vaccine design isolation and characterization of sialate 9 (4)-o-acetylesterase from influenza c virus the s protein of bovine coronavirus is a hemagglutinin recognizing 9-o-acetylated sialic acid as a receptor determinant transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity mechanisms and principles of n-linked protein glycosylation the sialic acid binding activity of the s protein facilitates infection by porcine transmissible gastroenteritis coronavirus cd209 gene polymorphisms in south indian hiv and hiv-tb patients structural requirements for multimerization of the pathogen receptor dendritic cell-specific icam3-grabbing non-integrin (cd209) on the cell surface dendritic cells and transmission of hiv-1 norovirus and histo-blood group antigens receptor binding and membrane fusion in virus entry: the influenza hemagglutinin constitutive and induced expression of dc-sign on dendritic cell and macrophage subpopulations in situ and in vitro host-soluble galectin-1 promotes hiv-1 replication through a direct interaction with glycans of viral gp120 and host cd4 alpha2,6-linked sialic acid acts as a receptor for feline calicivirus dc-sign binds to hiv-1 glycoprotein 120 in a distinct but overlapping fashion compared with icam-2 and icam-3 a novel viral alpha2,3-sialyltransferase (v-st3gal i): transfer of sialic acid to fucosylated acceptors membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions n-linked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the 1918 pandemic and seasonal h1n1 influenza a viruses sialobiology of influenza: molecular mechanism of host range variation of influenza viruses origin and evolution of influenza virus hemagglutinin genes sialoglycoproteins that bind influenza a virus and resist viral neuraminidase in different animal sera c-type lectin dc-sign: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity norovirus-host interaction: implications for disease control and prevention specific sites of n-linked glycosylation on the hemagglutinin of h1n1 subtype influenza a virus determine sensitivity to inhibitors of the innate immune system and virulence in mice glycosylation of the hemagglutinin modulates the sensitivity of h3n2 influenza viruses to innate proteins in airway secretions and virulence in mice ganglioside-linked terminal sialic acid moieties on murine macrophages function as attachment receptors for murine noroviruses glycosphingolipids as receptors for non-enveloped viruses murine noroviruses bind glycolipid and glycoprotein attachment receptors in a strain-dependent manner introduction to glycobiology belshe rb & fang f (2009a) inhibition of neuraminidase inhibitor-resistant influenza virus by das181, a novel sialidase fusion protein novel pandemic influenza a(h1n1) viruses are potently inhibited by das181, a sialidase fusion protein das181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis phenotypic and genotypic characterization of influenza virus mutants selected with the sialidase fusion protein das181 glycosaminoglycan-binding ability is a feature of wild-type strains of herpes simplex virus type 1 the multiple facets of hiv attachment to dendritic cell lectins platelet activation suppresses hiv-1 infection of t cells effect of addition of new oligosaccharide chains to the globular head of influenza a/ h2n2 virus haemagglutinin on the intracellular transport and biological activities of the molecule diversity of receptors binding hiv on dendritic cell subsets immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells the m/gp(5) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner concepts and principles of o-linked glycosylation langerin functions as an antiviral receptor on langerhans cells innate signaling in hiv-1 infection of dendritic cells porcine surfactant protein d is n-glycosylated in its carbohydrate recognition domain and is assembled into differently charged oligomers porcine pulmonary collectins show distinct interactions with influenza a viruses: role of the n-linked oligosaccharides in the carbohydrate recognition domain interactions of influenza a virus with sialic acids present on porcine surfactant protein d specificity of dc-sign for mannose-and fucose-containing glycans effects of heparin on the entry of porcine reproductive and respiratory syndrome virus into alveolar macrophages a multipotential beta-1,6-n-acetylglucosaminyltransferase is encoded by bovine herpesvirus type 4 the carbohydrate recognition domain of collectins n-linked glycosylation attenuates h3n2 influenza viruses the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities the war against influenza: discovery and development of sialidase inhibitors anti-influenza drugs: the development of sialidase inhibitors interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics functional balance between haemagglutinin and neuraminidase in influenza virus infections functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells structural basis for receptor specificity of influenza b virus hemagglutinin crystal structure of unliganded influenza b virus hemagglutinin targeting the carbohydrates on hiv-1: interaction of oligomannose dendrons with human monoclonal antibody 2g12 and dc-sign glycans on influenza hemagglutinin affect receptor binding and immune response a/g polymorphism is associated with dengue hemorrhagic fever and correlated to dc-sign expression and immune augmentation glycan shielding of the influenza virus hemagglutinin contributes to immunopathology in mice asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems antibody neutralization and escape by hiv-1 influenza viruses: role of glycans in viral evolution and vaccine design cell-surface carbohydrate recognition by animal and viral lectins molecular architectures of trimeric siv and hiv-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure the complete genome sequence of shope (rabbit) fibroma virus differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary t-lymphocyte and macrophage cultures resistance of human immunodeficiency virus type 1 to the high-mannose binding agents cyanovirin n and concanavalin a dendritic-cell interactions with hiv: infection and viral dissemination antiviral effects of an iminosugar derivative on flavivirus infections inhibition of dc-sign-mediated transmission of human immunodeficiency virus type 1 by toll-like receptor 3 signalling in breast milk macrophages hiv traffics through a specialized, surface-accessible intracellular compartment during trans-infection of t cells by mature dendritic cells structures, biosynthesis, and functions of gangliosides-an overview complementation of pulmonary abnormalities in sp-d(à/à) mice with an sp-d/conglutinin fusion protein mass spectrometric characterization of the glycosylation pattern of hiv-gp120 expressed in cho cells the authors thank leslie bosseler for critical reading of the manuscript and assistance in creating the figures. the authors apologize to all colleagues whose work has not been cited due to space limitations. key: cord-291534-c6cjxq07 authors: gwyer findlay, emily; currie, silke m.; davidson, donald j. title: cationic host defence peptides: potential as antiviral therapeutics date: 2013-05-07 journal: biodrugs doi: 10.1007/s40259-013-0039-0 sha: doc_id: 291534 cord_uid: c6cjxq07 there is a pressing need to develop new antiviral treatments; of the 60 drugs currently available, half are aimed at hiv-1 and the remainder target only a further six viruses. this demand has led to the emergence of possible peptide therapies, with 15 currently in clinical trials. advancements in understanding the antiviral potential of naturally occurring host defence peptides highlights the potential of a whole new class of molecules to be considered as antiviral therapeutics. cationic host defence peptides, such as defensins and cathelicidins, are important components of innate immunity with antimicrobial and immunomodulatory capabilities. in recent years they have also been shown to be natural, broad-spectrum antivirals against both enveloped and non-enveloped viruses, including hiv-1, influenza virus, respiratory syncytial virus and herpes simplex virus. here we review the antiviral properties of several families of these host peptides and their potential to inform the design of novel therapeutics. abstract there is a pressing need to develop new antiviral treatments; of the 60 drugs currently available, half are aimed at hiv-1 and the remainder target only a further six viruses. this demand has led to the emergence of possible peptide therapies, with 15 currently in clinical trials. advancements in understanding the antiviral potential of naturally occurring host defence peptides highlights the potential of a whole new class of molecules to be considered as antiviral therapeutics. cationic host defence peptides, such as defensins and cathelicidins, are important components of innate immunity with antimicrobial and immunomodulatory capabilities. in recent years they have also been shown to be natural, broad-spectrum antivirals against both enveloped and non-enveloped viruses, including hiv-1, influenza virus, respiratory syncytial virus and herpes simplex virus. here we review the antiviral properties of several families of these host peptides and their potential to inform the design of novel therapeutics. viral diseases are a leading cause of morbidity and mortality worldwide, particularly of children [1] , and yet development of effective therapies is slow. in particular, progress is hampered by the fact that the majority of antiviral drugs are specific for only one virus. current approaches are expensive, require rapid identification of the virus before therapy and, at the initial stages of development, involve enormous redundancy of research effort. this also results in efforts being concentrated on a few viruses; of the 60 antiviral drugs that have so far been approved by the us food and drug administration (fda), almost half target hiv-1; the remaining half are used for the treatment of hepatitis b virus (hbv), herpes simplex virus (hsv), varicella-zoster virus (vzv), cytomegalovirus (cmv), influenza (iav) and hepatitis c virus (hcv) infections [2] . these factors, in combination with the rate of development of drug resistance, mean that there is an urgent need for new broader-spectrum intervention strategies. it is therefore exciting that in recent years a new class of antiviral therapeutic peptides are emerging, with 15 peptidebased intervention strategies against viruses currently in various stages of clinical trials [3] . peptide-based strategies are proposed to be cost-effective, with peptides having low molecular weights, rapid elimination following treatment, and low levels of side effects [3] . an exciting current area of advancement is in understanding the antiviral properties of naturally occurring cationic host defence peptides (chdps) and the capacity of this to inform the design of novel synthetic antiviral analogues. in this review we will give an overview of the antiviral activities of chdps and consider their potential in the development of broad-spectrum antiviral therapeutics. defence against infection is emphasised by their conservation across plants, insects, reptiles, birds and fungi [7] . in mammals the two major families of chdps are defensins and cathelicidins. defensins are cationic, amphipathic peptides generated from prepropeptides via proteolysis and are categorised within three subfamilies: a, b and h. defensins were first characterised as ''natural peptide antibiotics'' with the discovery of a-defensins from the granules of neutrophils [8] . the a-defensin family has been identified in a range of higher eukaryotes (including primates, mice, rats, guinea pigs and rabbits) and comprises six distinct peptides in humans (human neutrophil peptides (hnp) 1-4 and human defensins (hd) 5-6), expressed from five defa genes [9] . all have a triple-stranded b-sheet core stabilised by three intramolecular disulphide bonds, and are made first as a prepropeptide which is proteolytically cleaved to the active form [10] . in the case of hd5 and hd6 the key protease is trypsin. hnp1-4 are produced mainly by neutrophils, where they comprise 5-7 % of total neutrophil protein [11] , and neutrophil precursors in the bone marrow [12] . hnp1-3 are also described in nk cells, b cells, cd t cells, macrophages and immature dendritic cells [13] , but can be acquired from neutrophils [14] . the release of active hnps from neutrophil azurophilic granules can be induced by a range of stimuli, including chemokines, fc gamma receptor cross linking, pma and tlr stimulation [13] . in contrast, hd5 and 6 are expressed by paneth cells of the small intestine [15, 16] and epithelial cells of the female genital tract [12, 17, 18] . interestingly, although mice express a large number of intestinal a-defensins (cryptdins) [19] , in contrast to humans they do not express a-defensins in their neutrophils [20, 21] . a-defensins have well-described broad spectrum antimicrobial activity against both gram-positive and -negative organisms in vitro [22] , with cationicity and hydrophobicity being shown to be key determinants of these properties [9, 23] . their cationic charge is proposed to enable interaction with the net negative charge on the surface of the gram-negative bacteria and the teichoic acids of the gram-positive organisms, while their amphipathic structure enables insertion into and disruption of the bacterial membranes, leading to lysis of the cells. in addition, various a-defensins are described as having additional, non-microbicidal properties, including chemotaxis for effector cells of the innate and adaptive immune systems [24, 25] , inhibition of macrophage pro-inflammatory cytokines [26] , modulation of the intestinal microbiome [27] and the formation of protective peptide nanonets [28] . the b-defensin family contains more than 30 members in humans and more than 50 in mice, and are widely expressed across many species, in particular being the only defensins found in birds and with close homologues present in snakes, platypus and sea anemones [29] . they are also triple-stranded b-sheet proteins, but differ from the a-family members in the organisation and arrangement of the three disulphide bonds [10] . the most well characterised human b-defensins are human b-defensin (hbd) 1-3, expressed by epithelial cells [30] , monocytes, macrophages and macrophage-derived dc [31] . hbd1 is encoded by defb1 and is expressed constitutively [30] , whereas expression of hbd2 (defb4a) and hbd3 (defb103a) are up-regulated in response to various inflammatory stimuli, including microbes [32] , tlr and nod proteins [33] and pro-inflammatory cytokines [34] . b-defensins are also described as having broad-spectrum antimicrobial activity in vitro [7] , with potency varying in different family members. interestingly, the weak microbicidal properties of hbd1 were recently shown to be greatly enhanced upon reduction of its disulphide bonds [35] . although relatively mild phenotypes were found in mouse models deficient in defb1 [36, 37] , redundancy in this multi-gene family may be responsible for this observation. in addition b-defensins are described as having a range of immunomodulatory properties, including chemotaxis of cd4 ? memory t cells, macrophages and immature dendritic cells [38] , enhancement of wound healing [39] and the modulation of inflammatory cytokine responses (with the capacity both to promote and to suppress inflammation in different settings) [40] . h-defensins are circular octadecapeptides, the only circular peptides of mammalian origin [41] , formed by the splicing of two nonapeptides, each of which contributes three cysteines to a series of disulphide bonds in the mature peptide [42] . three h-defensins have been found in the leukocytes of rhesus macaques and named rtd1-3, but although six rna transcripts homologous to rtd are found in the human bone marrow they contain a premature stop codon preventing their expression [42, 43] . however, an artificially constructed peptide based on these pseudogenes, called retrocyclin, has been studied with respect to its function and therapeutic potential and shown to kill escherichia coli in the same way as a-defensins, by permeabilising its membrane [44] . the cathelicidin family is quite distinct from defensins; cathelicidins are defined by a conserved cathelin domain and with a variable c-terminal region, which is proteolytically cleaved to produce a mature functional peptide, with a range of structural forms in different family members [45] . in contrast to the extensive defensin family, humans (and mice, rats and rabbits) express a single cathelicidin, whereas multiple cathelicidins are found in other species (e.g. protegrins in pigs). the sole human cathelicidin, human cationic antimicrobial peptide of 18 kda (hcap-18; encoded by the camp gene), is cleaved by proteinase 3 into its active form, ll-37, which is a cationic, amphipathic peptide of 4.5 kda with an a-helical structure [46, 47] . hcap-18 is stored in neutrophil-specific granules, inducible in epithelial cells, macrophages and other leukocytes to a lesser extent, and detectable in a range of body fluids, including airway surface liquid, plasma, urine, breast milk and sweat [48] . it is up-regulated in response to infectious and inflammatory signals [49] and wounding [50] and its expression can be increased by vitamin d metabolites [51] and compounds such as butyrate [52] . ll-37 has well-documented antibacterial potential; however, when studied in the presence of physiological concentrations of cations or serum, ll-37 has high minimum inhibitory concentrations compared to levels described in vivo [6] . mice deficient in mcramp (encoded by the camp gene, the orthologue of camp) have increased susceptibility to infections in multiple systems, including the skin, intestinal tract and lung [53] [54] [55] . although these models clearly demonstrate the critical role for cathelicidin in host defence against infection, it remains unclear to what extent this is due to microbicidal or modulatory properties of the peptide. ll-37 has been shown to have a broad range of immunomodulatory and inflammomodulatory properties [6] . these include chemotactic activity for neutrophils, monocytes and t cells [56] , modulation of cytokine production [57] , effects on dendritic cell differentiation and function [58, 59] , promotion of wound healing [60] and angiogenesis [61] , and modulation of cell death [62, 63] . although the field of antimicrobial peptide research has been dominated by evaluation of the antibacterial activities of these peptides, early studies evaluating the antiviral potential of human a-defensins showed promise and have been followed by an increasing level of research interest (fig. 1 ). the first paper detailing an antiviral role was published 27 years ago, describing inhibition of a number of viruses including hsv types 1 and 2, cytomegalovirus and vesicular stomatitis virus by hnp1 in vitro [64] . in particular, it demonstrated direct antiviral activity of hnp1 against hsv-1 in a temperature-and ph-dependent manner, inhibited by serum, but interestingly less sensitive to the inhibitory effects of cations than the more broadly studied antibacterial properties. hnp1-4, hd5 and hd6 have subsequently been found to be active against hsv-2 (up to approximately 1 log decrease at 50 lg/ml) by preventing viral binding to either glycoprotein b (gb2) or heparan sulphate (the primary receptor for hsv) [65] . gb2 binding capacity was found to be primarily determined by peptide sequence rather than net cationic charge [66] , with lectin-like properties likely to be key to the glycoprotein binding functions [67] . interestingly, those defensins (hnp1-3 and hbd5) which bound viral envelope gb were also found to be effective at preventing infection if added after viral entry, even up to 8 h post-infection (with hnp1 or hd5), suggesting additional later stage effects on viral replication [65] . furthermore, cumulative effects were observed with repeated application of peptide, reducing infection with hsv by greater than 7 logs at 100 lg/ml. these features make a-defensins of considerable interest as potential exogenous vaginal microbicides, with a proof of concept study in mice demonstrating protection against hsv following gel-based application of hd5 [65] . a-defensins have been demonstrated to directly inactivate hiv-1, with the observation of reduced cytopathogenicity in a cd4 ? t cell line [68] . although confusion existed following the retraction of a paper proposing that adefensins were the key component of the cd8 antiviral factor caf [69, 70] , it is worth noting that the retraction related to the source of the a-defensins (probably ''imported'' into cd8 cells from co-cultured cells), and the anti-hiv-1 properties of the peptides were not called into question. indeed, the potential of a-defensins to treat hiv was reinforced when it was found that breast milk a-defensin concentration is significantly associated with a decreased risk of intrapartum and postnatal hiv transmission [71] . recent studies demonstrate that multiple steps in the entry of hiv-1 virus into cells are disrupted by hnp1 [72] . this peptide has been shown to bind to cd4 and to the env glycoprotein on the virus, thus inducing the down-regulation of cd4 and cxcr4 and blocking interaction of env with the co-receptors. by targeting particular conformations of env it also inhibited late fusion steps [72] . hnp1-3 can bind to cd4 and hiv-1 gp120 with high affinity; however, hnp4, which is a much weaker binder, is a more potent inhibitor, meaning this aspect of direct inhibition is not, currently, entirely clear [73] . mechanistic studies have shown that hnp1-3 can also inhibit steps following reverse transcription and integration by inhibiting pkc activity; pkc is important for hiv replication as it upregulates transcription through nf-jb activation and tat phosphorylation, as well as regulating fusion and assembly of the virions [74] . it is worth noting that in these studies the direct effects on the virus particles occurred only in the absence of serum; in its presence, these mechanisms were inhibited and effects are instead on the host cells, resulting in inhibited replication of the virus [69, [74] [75] [76] . defensins can also stimulate an antiviral state in cells by promoting secretion of chemokines [76] . in macrophages this upregulation of chemokines also contributes to inhibition of hiv through competition for receptors [76] . a potential issue with using defensins as a topical antiviral treatment is that, despite chdps (including defensins) being required for in vitro anti-hiv activity of vaginal fluid from healthy women, defensins may also cause immune activation and subsequent loss of cd4 ? t cells by apoptosis, and may indirectly enhance hiv transmission [77] . in particular, it is known that hnp2 and hbd2 are both chemotactic for dc and induce infiltration of dc into cultures of hpv-transformed keratinocytes and subsequent immune activation [78] . in addition, hd5 and 6, which are produced by cervico-vaginal epithelial cells [18] and present at up to 50 lg/ml in the vaginal fluid of healthy women [79] , enhance hiv infectivity in vitro by promoting the virus attachment to target cells [18, 79] . thus, the balance of these effects remains to be determined. a-defensins are also found in high concentrations in the inflamed lung, generating interest in their potential activities against respiratory viruses. hnp1-3 are the most abundant antimicrobial peptides in airway fluid [80] and are up-regulated further following infection or inflammation [81] as they are produced by both immigrating neutrophils and airway epithelial cells [82] . daher et al. [64] first described antiviral effects of hnp1 against the wsn strain of iav in vitro, showing a fairly modest approximately 0.5 log reduction at 50 lg/ml. this property of hnp1 and 2 was later confirmed in other strains of iav (with decreased infectivity of approximately 1 log at 10 lg/ml in a fluorescent focus assay of infectivity). the peptides were shown to have no activity in a haemagglutinin inhibition assay [83] , but to induce aggregation of iav [84] . interestingly hnp aggregation of the pr8 viral strain was much higher than that of the phil82 strain, which has greater surface glycosylation [84] [85] [86] , and neutralising activity of hnps was greater against pr8 than against phil82 [83] , suggesting the reduced carbohydrate attachments on the envelope proteins allow greater interaction with the defensins. however, direct effects of hnp1 on iav viral particles were found to have no impact on viral growth in infected cultures [87] . maximal antiviral effects in this study (1-3 logs at 5-25 lg/ml hnp1 in a range of cell lines) required interaction of hnp1 with the eukaryotic cells before infection and the continued presence of peptide in the culture system. nevertheless, in contrast to pretreatment of virus, pretreatment of the cells did induce some protection. this suggests the predominant mechanism of action is cell-mediated, and has been proposed to be a consequence of hnp-mediated inhibition of pkc activity (essential for endosomal trafficking of the iav) [87, 88] . the capacity of a-defensin to protect against iav was lost in its linearised analogue [87] . in addition to effects on iav infection of epithelial cells, hnp1 and 2 at approximately 40 lg/ml (but not hnp3, hbd2 or 3) can enhance the uptake of iav by neutrophils, following pre-incubation of either the cells or the virus with the defensins [84] . however, this modulation of neutrophil phagocytic capacity was also observed using bacteria, and was not virus specific. these properties of a-defensins suggest potential as templates for novel therapeutics. however, their ability to bind surfactant protein d (sp-d) may be of concern, having mixed competitive or co-operative, and iav straindependent effects on the antiviral activities of sp-d [83] . hnps (but not b-defensins) can bind and precipitate bronchoalveolar lavage (bal) fluid sp-d [83, 89] and may account for sp-d depletion from the lung in diseases with chronic neutrophilic inflammation. it was originally suggested that a-defensins had no activity against non-enveloped viruses, on the basis of the absence of effects against echovirus type ii and reovirus type 3 [64] . however, more recent work has demonstrated inhibition of non-enveloped viruses such as adenovirus, human papillomavirus (hpv) [90] [91] [92] and bk virus [93] . the mechanisms underpinning these antiviral effects against non-enveloped viruses appear to be distinct from those targeting enveloped viruses. a study of hpv (utilising pseudovirus particles) found normal binding, uncoating and internalisation in the presence of a-defensins; however, the peptides prevented the virion from escaping the endocytic vesicles [92] . the antiviral activity was observed using hnp1-4 (and the cathelicidin ll-37), was maximal using hd5, but was not observed using hd6. the sensitivity of adenoviruses to a-defensin-mediated neutralisation is serotype-dependent [90, 94] . hnp1 and hd5 have been shown to inhibit human adenovirus infection in both lung and conjunctival epithelial cells by inhibiting an early step in viral entry [90, 91, 95, 96] . two arginine residues on one face of hd5 were found to be critical for antiviral activity, with arg-28 necessary for killing of both adv and hpv and arg-9 for adv only [97] . viral aggregation is not sufficient for neutralisation and binding of a-defensin to the adenoviral virus capsid appears to be critical, preventing uncoating in the cell and hence entry of the viral genome into the nucleus [97, 98] . in contrast, the antiviral effects of hnp1 and hd5 on the bk polyomavirus appear to primarily relate to viral aggregation preventing receptor binding on the host cells [93] . [100] . similarly, human rhinovirus (hrv) replication in human bronchial epithelial cells induces nf-jb-dependent hbd2 and 3 expression (but not hbd1) [101, 102] , whereas respiratory syncytial virus (rsv) can induce hbd2 in an nf-jb-dependent, but ifn type 1-independent manner in human lung epithelial cells [103] . the extent to which these innate responses are functionally effective against the viral pathogens is starting to be elucidated (fig. 2 ). in contrast to the effects of a-defensins, only one of the bdefensins tested (hbd3) had appreciable effects against hsv [65] . hbd3 was found to inhibit hsv-2 infection of human cervical epithelial cells (by approximately 1 log at 10 lg/ml) by interfering with the viral binding and penetration processes by binding both gb and heparan sulphate. in contrast, hbd1 and hbd2 had low affinity to gb and cellular glycosaminoglycans, and were not able to reduce hsv-2 infectivity. however, only hd5 was applied in vivo in this study [65] . hiv-1 infection of epithelial cells induces hbd2 and 3 expression in vitro [100] and in buccal mucosal cells in vivo [104] , and both inhibit hiv transmission through multiple mechanisms [100, 105] . both down-regulate the co-receptor cxcr4 expression (but not ccr5) on the surface of cd4 ? t cells via an increase in internalisation [106] . in addition, there are both direct effects on the virions in a concentration-dependent manner [100] and on intracellular, post-viral entry inhibition [105] . however, in large-scale studies, copy number variation of total b-defensin gene number positively correlates with hiv load in ethiopian and tanzanian patients [107] . the authors suggest that the chemoattractant nature of bdefensins may bring the target th17 cells into mucosal sites where they can be readily infected by virus. other work, however, has recently shown that exposed but seronegative individuals have much higher hbd2 and 3 copy numbers in oral mucosa (but not vaginal mucosa) than healthy controls, indicating a role for these defensins in combating infection [108] . although not normally regarded as inducible, expression of hbd1 (but not hbd2, 3 or 4) has been observed in primary human blood-derived plasmacytoid dc and monocytes following infection with iav [109] . in contrast, an early decrease in expression of this peptide was found in iav-infected epithelial cell lines [109] . recombinant hbd1 was found to have antiviral effects in vitro (approximately 1.5 log decrease at 50 lg/ml). however, despite evidence of a significant defect in host defence against iav in defb1-deficient mice, this did not extend to differences in viral load, suggesting a primary role of immunomodulatory properties in vivo [109] . iav has been found to up-regulate expression of murine defensins mbd3 and mbd4 in upper and lower airways, as well as transcription of the genes encoding mbd1 and mbd2 in the lung [99] , also suggesting protective roles for these peptides. recombinant mbd2 [110] and mbd3 [111] protected mdck cells against infection with iav pr8 strain (up to approximately 1 log decrease at 100 lg/ml); this protection was effective during binding or internalisation of the virus, but not after viral entry. furthermore, repeated intranasal administration of recombinant mbd2 (2 mg/kg; optimal when premixed with virus before infection) or intravenous delivery of recombinant mbd3 (10 mg/kg) was found to be protective in murine lethal infection models [110, 111] . the latter study also suggested that the effects may relate to immunomodulatory properties, with systemic mbd3 treatment up-regulating ifn-c and il-12 and reducing levels of tnf. thus, although b-defensins can inhibit influenza virus infectivity (albeit less potently than the a-defensins or ll-37) [89] , immunomodulatory properties, perhaps also including up-regulation of iav uptake by neutrophils [84] , may prove to be key to their protective function against this virus in vivo and future therapeutic developments. in addition to effects on iav, evidence has been found for b-defensin activity against rsv, another important respiratory virus, for which no effective vaccine or antiviral treatments exist. in studies using the a549 human lung cell line, hbd2 was identified as a component of an nf-jbdependent, ifn-a/b-independent antiviral response [103] . epithelial cell hbd2 production was induced in response to rsv replication and also in response to the tnf secreted by infected epithelial cells. hbd2, but not hbd1, was found to protect against rsv infection (approximately 2 log decrease at 4 lg/ml), by blocking viral cellular entry [103] . destabilisation of the viral envelope was proposed to occur upon contact with soluble hbd2 in solution, or following exposure to plasma membrane-associated hbd2 during cell entry. in addition to lung epithelial cells, myeloid cells can produce high levels of tnf in response to rsv infection [112] . this has the potential to up-regulate hbd2 expression in the airway lumen and might consequently modulate protection against rsv infection and limit virus spread. interestingly mbd4 (but not mbd3; both considered homologues to hbd2) was found to be upregulated in vivo in the murine lung in response to rsv infection [103] . hbd3 has been proposed to have antiviral activity against vaccinia virus [113] . however, hnp1, hbd1 and hbd2 are unable to neutralise the virus [114] . pre-exposure of virus to synthetic hbd3 for 24 h decreased infection of the bsc-1 monkey kidney cell line, shown both by reduced levels of dna-dependent rna polymerase expression and plaque formation (approximately 1 log decrease at 10 lm) [113] , although the mechanism remains to be elucidated. up-regulation of hbd3 expression was observed in response to vaccinia virus infection in primary human keratinocytes, but this could be inhibited by il-4 and il-13. interestingly, these cytokines are associated with pathology in atopic dermatitis, a condition in which b-defensin expression is reduced [115] and patients are at risk of developing eczema vaccinatum caused by vaccinia virus. circular h-defensins were identified in the leukocytes and bone marrow of macaques (rhesus h-defensins; rtd) [116] and discovered to have effective antibacterial activity. humans were found to have at least six h-defensin genes (deft genes) [117] , but none produce a translated protein, owing to insertion of a premature stop codon. putative ancestral human h-defensins, named retrocyclins (rc), were developed and their antimicrobial properties were tested [117] . in addition to activity against pseudomonas aeruginosa, e. coli, listeria monocytogenes and staphylococcus aureus, antiviral potential has also been described (fig. 3) . both rhesus h-defensins and retrocyclins (including rtd3, rc1 and rc2) have been found to inhibit hsv-1 and hsv-2 infection of human cervical epithelial cell lines following pre-incubation of virus and peptide [66] . however, rc2 was found to have no direct virucidal properties [118] , but to be active irrespective of pre-incubation by blocking attachment and cell penetration of hsv [66] . this activity resulted from peptide binding to gb2, in a manner dependent on the presence of sialic acid and carbohydrate moieties in the glycoprotein's o-and n-linked glycans. prophylactic application of rc2 in a murine hsv-mediated ocular keratitis model demonstrated the capacity of rc2 to modestly reduce viral titres in vivo and reduce blepharitis, corneal vascularization and stromal disease. however, rc2 had no effect upon disease pathology when applied post-infection [118] . initial studies on retrocyclins found no direct inactivation of hiv-1, but demonstrated strong inhibition of proviral dna formation and protection of primary human cd4 ? t cells from t-and m-tropic hiv-1 strains in vitro [117, 119] . these observations led to a significant body of work evaluating retrocyclins as hiv treatments. rc1 has been characterised as a lectin, which protects peripheral blood [117] . this peptide prevents viral entry into cells [42, 119] , blocking formation of the 6-helix bundle required for fusion, by binding to hiv gp120 and cellular cd4 through interactions with their o-and nlinked sugars [120] . activity of many of the initial retrocyclins produced was inhibited by serum, minimising usefulness in serum-containing anatomical compartments [42] . however, analogues, each differing from rc1 by a single amino acid substitution, show greater potential as topical microbicides [121] . of these, rc100 was found to inhibit primary cd4 ? t cell infection by hiv-1 similarly to rc1, but was active in the presence of vaginal fluid, and rc101 is significantly more potent against hiv than rc1; both have potential as topical microbicides [122] . interestingly, the rate of escape mutations of rc100-treated hiv was low; treatment altered sites in hr1 and hr2 of gp410 but these only reduced susceptibility by 10-fold, compared to 10,000-20,000-fold for ccr5 blockade [122] . using an organ culture model, rc101 was found to block transmission of two strains of hiv-1 across cervical mucosa. antiviral activity was retained in the presence of semen and vaginal fluid, there was no cytotoxicity to cervical tissue and, importantly, rc101 did not induce a pro-inflammatory response, with no chemotactic activity for immune cells [123] . in addition, topical intra-vaginal rc101 application in pigtailed macaques was found to be safe and well tolerated, with peptide retained in the cervical and vaginal tissue for up to 4 days post-application, no changes in vaginal ph observed and minimal effects on commensal microbiota [124] . the application of retrocyclins to pathogenic respiratory viruses has also been evaluated. expression of recombinant rc2 in both mdck cells and chicken embryos has been shown to inhibit replication of an h5n1 influenza virus [125] . in addition, rc1, rc2 and rc101 all had antiviral activities in studies using mdck and a549 cell lines (approximately 1 log decrease at 20 lg/ml against iav phil82, but less effective against pr8 strain) [89] . the retrocyclins were more effective than hbd1 and hbd2, with potency equivalent to hnp1-3. this study noted the capacity of the rc peptides to induce aggregation of the virus and increase viral uptake by neutrophils and macrophages [89] . rc2 was also found to inhibit influenza a/x31 infection of cell lines in vitro by blocking the haemagglutinin-mediated fusion of viral and endosomal membranes [85] . the inhibition of hemifusion formation was shown to be a lectin property which involved cross linking and immobilising surface glycoproteins and blocking the protein displacement required to bridge the bilayers for fusion. rc2 was only effective when given before viral internalisation, but had antiviral properties even when only applied to the host cell membrane. interestingly, despite the capacity to bind sp-d, retrocyclins increased, rather than inhibited, the antiviral activity of sp-d [89] . this is in contrast to the a-defensins [83] , and promising for therapeutic use at mucosal sites. to further improve the antiviral activity of synthetic retrocyclins against influenza and simplify their structure, a series of analogues containing 13-14 amino acids, termed hapivirins and diprovirins, were designed [126] . some of these analogues, including hpv1, 8, 11-15, 17, 19 and dpv 13, 16, 1623 and 1632 proved to be more effective than rc2 and rc101 against iav phil82 (h3n2) and pr-8 (h1n1). mechanistically analogous to the retrocyclins, these peptides were found to be active before viral internalisation, induce viral aggregation, opsonise virus, and have an additive effect with sp-d. in addition, hapivirins and diprovirins were shown to inhibit iav-induced macrophage tnf production, adding immunomodulatory mechanism to their therapeutic potential. antiviral activity of retrocyclins has also been reported against sars virus, a coronavirus which infects the alveolar epithelial cells and induces rapid severe lung pathology [127, 128] . in a mouse model of sars infection, rtd-1 treatment increased survival from 25 to 100 %. interestingly, this was in the absence of any impact on viral titres. instead the cytokine profile of the infected animals was modified, with increased early (day 2) production of il-6 and gm-csf, but decreased il-1a, il-1b, il-6, mip1a, and il-12p40 by day 4. these data highlight the possibility that novel peptide therapeutics may be productively targeted at modulation of the inflammatory response to infection, rather than developed for virucidal properties per se. such an approach may prove to have broad-spectrum applicability and lends further support to the potential for these peptides as novel immunomodulatory antiviral agents. cathelicidins have been widely studied with regard to their antibacterial properties and broad array of immunomodulatory activities [6] . although known to be up-regulated in inflammation and released by neutrophils, their roles in defence against viral infection and properties as antiviral agents are less well understood (fig. 4 ). hcap-18 is expressed in human epididymal epithelium and is present at high concentrations in seminal plasma [129] . it is also expressed in cervico-vaginal secretions and upregulated in participants with bacterial sexually transmitted infections [130] . cervico-vaginal ll-37 levels in hivnegative individuals who were in hiv sero-discordant relationships were also found to be greatest in those whose hiv-positive partners had the highest viral load [131] . ll-37 has been shown to inhibit the replication of a range of hiv-1 isolates in primary cd4 ? t cells [132] . this occurred in a manner that was independent of change in expression of any hiv-1 receptors in these cells. a recent study demonstrated that ll-37 was capable of a dosedependent suppression of hiv reverse transcriptase activity [133] . this study also demonstrated that this function was retained by a 16 amino acid fragment of ll-37 (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) , with implications for production of smaller synthetic analogues. thus, epithelial expression of ll-37 has been proposed to contribute to local protection against hiv-1 infection. however, although the cationic peptide fraction of cervico-vaginal secretions was found to have hiv-1neutralising activity, which could be enhanced by addition of recombinant ll-37, this property did not correlate with levels of endogenous ll-37 detected [131] . in addition, in one study ll-37 levels were independently associated with increased hiv acquisition, although both observations might be the result of a high prevalence of sexually transmitted infections in these individuals [130] . therefore, the in vivo significance of ll-37 in hiv remains unclear. we recently demonstrated that ll-37 has antiviral effects against iav, both in vitro and in vivo [134] . both human and murine cathelicidins had antiviral activity when preincubated with influenza viruses in vitro [approximately 1 log decrease at 10 lg/ml against a/pr/8/34 (h1n1), but somewhat less effective against a/udorn/307/72 (h3n2)]. in addition, ll-37 has recently been shown to bind iav, without aggregating or affecting haemagglutination activity, and to have maximal effects in vitro when pre-incubated with virus (although delayed addition of peptide or treatment of the cells, with washing before infection also has some antiviral effects) [135] . surprisingly, ll-37 was found not to alter the binding or initial uptake of virus by cells, but peptide-mediated disruption of viral membranes (shown by electron microscopy) was proposed to affect viral propagation or survival downstream of this [135] . we demonstrated that aerosolised therapeutic administration of either ll-37 or mcramp (given every day for 1 week, with an additional pre-infection dose) provided protection against infection in a mouse model [134] . peptide-treated mice showed increased survival and decreased weight loss compared to control infected animals, and were similarly protected to those treated with zanamavir (a neuraminidase inhibitor currently used therapeutically in humans). the cathelicidin-treated mice showed some decrease in viral loads, but more striking reductions in lung cytokines (in particular gm-csf, il-1b, kc and ccl5), again suggesting the possibility of a key immunomodulatory roles in the antiviral efficacy of such peptides. scrambled control peptides did not have these effects but interestingly the d-enantiomers did, indicating the actions of ll-37 are not solely related to charge and are likely not reliant on specific receptor interactions. the mechanisms by which cathelicidins modulate inflammation in iav infection remain unclear, but may relate to the observation that ll-37 can modulate tolllike receptor signalling [136] . induction of rapid antiviral responses depends, at least in part, on tlr recognition of the viral genome, with ssrna and dsrna viruses recognised by tlr7/8 and tlr3 [137] . ll-37 complexed with self dna and rna has been shown to induce tlr7-, tlr8-and tlr9-dependent inflammatory responses to otherwise non-immunostimulatory nucleic acids [59, 138] . in addition, ll-37 has been proposed to enhance [139, 140] or inhibit [141] tlr3-dependent responses to viral rna or synthetic mimics. these modulatory properties may well prove to underpin the in vivo effects. the expression of ll-37/hcap18 by airway epithelial cells can be induced in vitro by infection with rsv [142] , in a manner that is significantly enhanced by the 1,25oh metabolite of vitamin d. in addition, a recent study found that median serum hcap-18 levels are significantly lower in children with rsv bronchiolitis than in children with bronchiolitis caused by human rhinovirus [143] . furthermore, rsv-infected children with hcap-18 levels lower than the median are more likely to be hospitalised for prolonged periods than those with hcap-18 levels above the median. these findings suggest an important antiviral role for ll-37 in host innate immune response against rsv. in keeping with this hypothesis, we have recently demonstrated that ll-37 exhibits effective, dose-dependent and timing-specific anti-rsv activity in vitro in a number of cell lines [144] . these data indicate that therapeutic use of cathelicidin or strategies to up-regulate cathelicidin expression in vivo (particularly during the winter when low vitamin d levels may lead to diminished cathelicidin expression) may be protective against rsv infections. individuals with atopic dermatitis (ad) have low levels of ll-37 expression and increased susceptibility to skin infections, in contrast to those with psoriasis, who have high levels of ll-37 expression and are less prone to skin infections, despite similar disruption to skin barrier function [145] . the low levels of ll-37 expression in ad are proposed to be important in the susceptibility to eczema vaccinatum, a disseminated viral skin infection that follows inoculation with vaccinia virus (vv). ll-37 expression was induced in response to vv in normal and psoriatic skin biopsies, but not those from ad skin [146] . both ll-37 and mcramp have been shown to have antiviral activity against vaccinia virus in vitro (approximately 1 log decrease at 25 lm) [114] . these peptides were shown to damage the integrity of the double-layered viral envelope [114] , by removing the outer membrane [147] . in addition, camp-/-mice were found to develop significantly more pox skin lesions following infection with vv, demonstrating the antiviral effects of cathelicidins in vivo [114] . interestingly these effects against vv were found to be specific to cathelicidins, with hnp1, hbd1 and hbd2 unable to neutralise the virus [114] . this implies that the different chdps may have different (if sometimes overlapping) targets in order to successfully deal with the enormously wide range of human pathogens. the antiviral activities of host defence peptides have been somewhat neglected until recently, in contrast to the study of their antibacterial effects. yet, as discussed in this review, a strong basis exists for future evaluation of the physiological roles of chdps as key components of innate antiviral host defences and to develop synthetic analogues as novel antiviral therapeutics. the precise mechanisms involved clearly vary in a peptide-and virus-specific manner, from direct effects on the viruses through to primarily immunomodulatory properties. these need to be examined in detail and tested in vivo against specific viral infections. such research is expected to reveal therapeutic strategies that range from simple administration of more potent antiviral and/or immunomodulatory analogues, to therapeutic measures to enhance natural levels of expression, replace down-regulated peptides or provide peptide at an earlier, critical ''tipping point'' stage in infection. although some peptides may prove to be effective when administered after infection is established, the optimal use of other peptide therapeutics may be prophylactic administration in high-risk populations (e.g. infants and elderly in an outbreak of a novel influenza strain for which a vaccine is not available). challenges remain in optimising peptide therapeutics to develop the shortest, cheapest analogues that are stable and function at lower concentrations, in physiological environments, without cytotoxic effects or the generation of resistance. in this regard, the h-defensins appear particularly exciting as potential topical anti-hiv agents. they are well tolerated, certain members of the family appear to function in the presence of serum, they rapidly and directly kill hiv-1 without inducing large-scale immune activation and escape variants are infrequent. engineering of peptides is a simple process, which enables development of more suitable compounds. the difference in the antiviral capabilities and rc 50 values of hnp1 and 3, despite them differing by only a single amino acid, hints at what is possible. the engineering of rc101 from rc1 (again a single amino acid change) leading to significantly higher anti-hiv-1 activity also suggests that further enhancements should be possible. likewise, linear, disulphide bond-free defensins have been generated [148] , which retain potent antimicrobial activity. the development of linear peptides would be significantly easier than recreating their tertiary structure. however, the stability of such peptides against protease degradation remains uncertain and loss of antiviral properties following linearisation has been described in some instances [18, 94] , highlighting the need for further research. thus, in an era of increasing concern about the resurgent threats of infectious diseases, a very limited repertoire of antiviral drugs and fears about the rapidity with which new viruses can spread globally, peptide therapeutics have potential that is clearly worth pursuing. acknowledgments djd is supported by a mrc senior non-clinical research fellowship (g1002046). smc is supported by a university of edinburgh college of medicine and veterinary medicine studentship. the authors have no conflicts of interest that are directly relevant to the content of this article. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. the exclusive right to any commercial use of the article is with springer. global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since antivirals: current state of the art avppred: collection and prediction of highly effective antiviral peptides cationic host defence 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and il-8) production by respiratory syncytial virus-infected human alveolar macrophages antiviral activity of human beta-defensin 3 against vaccinia virus selective killing of vaccinia virus by ll-37: implications for eczema vaccinatum cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes a cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins retrocyclin: a primate peptide that protects cells from infection by t-and m-tropic strains of hiv-1 evaluation of a theta-defensin in a murine model of herpes simplex virus type 1 keratitis. invest ophthalmol vis sci the theta-defensin, retrocyclin, inhibits hiv-1 entry theta-defensins prevent hiv-1 env-mediated fusion by binding gp41 and blocking 6-helix bundle formation rc-101, a retrocyclin-1 analogue with enhanced activity against primary hiv type 1 isolates hiv-1 adapts to a retrocyclin with cationic amino acid substitutions that reduce fusion efficiency of gp41 retrocyclin rc-101 blocks hiv-1 transmission across cervical mucosa in an organ culture the formulated microbicide rc-101 was safe and antivirally active following intravaginal application in pigtailed macaques retrocyclin 2: a new therapy against avian influenza h5n1 virus in vivo and vitro hapivirins and diprovirins: novel theta-defensin analogs with potent activity against influenza a virus host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease the human cationic antimicrobial protein (hcap-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa levels of innate immune factors in genital fluids: association of alpha defensins and ll-37 with genital infections and increased hiv acquisition hiv-neutralizing activity of cationic polypeptides in cervicovaginal secretions of women in hiv-serodiscordant relationships the antimicrobial peptide ll-37 inhibits hiv-1 replication effects of cathelicidin and its fragments on three key enzymes of hiv-1 antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin ll-37 the human cathelicidin ll-37 inhibits influenza a viruses through a mechanism distinct from that of surfactant protein d or defensins modulation of the tlr-mediated inflammatory response by the endogenous human host defense peptide ll-37 toll-like receptors and viruses: induction of innate antiviral immune responses self-rna-antimicrobial peptide complexes activate human dendritic cells through tlr7 and tlr8 ll37 and cationic peptides enhance tlr3 signaling by viral double-stranded rnas low concentrations of ll-37 alter il-8 production by keratinocytes and bronchial epithelial cells in response to proinflammatory stimuli antimicrobial peptides inhibit polyinosinic-polycytidylic acid-induced immune responses respiratory epithelial cells convert inactive vitamin d to its active form: potential effects on host defense serum cathelicidin level is associated with viral etiology and severity of bronchiolitis the human cathelicidin ll-37 has antiviral properties against respiratory syncytial virus antimicrobial peptides and the skin immune defense system cytokine milieu of atopic dermatitis skin subverts the innate immune response to vaccinia virus a carpet-based mechanism for direct antimicrobial peptide activity against vaccinia virus membranes antibacterial activity of human neutrophil defensin hnp-1 analogs without cysteines key: cord-322933-5xnxjqm5 authors: murugaiah, valarmathy; tsolaki, anthony g.; kishore, uday title: collectins: innate immune pattern recognition molecules date: 2020-03-10 journal: lectin in host defense against microbial infections doi: 10.1007/978-981-15-1580-4_4 sha: doc_id: 322933 cord_uid: 5xnxjqm5 collectins are collagen-containing c-type (calcium-dependent) lectins which are important pathogen pattern recognising innate immune molecules. their primary structure is characterised by an n-terminal, triple-helical collagenous region made up of gly-x-y repeats, an a-helical coiled-coil trimerising neck region, and a c-terminal c-type lectin or carbohydrate recognition domain (crd). further oligomerisation of this primary structure can give rise to more complex and multimeric structures that can be seen under electron microscope. collectins can be found in serum as well as in a range of tissues at the mucosal surfaces. mannanbinding lectin can activate the complement system while other members of the collectin family are extremely versatile in recognising a diverse range of pathogens via their crds and bring about effector functions designed at the clearance of invading pathogens. these mechanisms include opsonisation, enhancement of phagocytosis, triggering superoxidative burst and nitric oxide production. collectins can also potentiate the adaptive immune response via antigen presenting cells such as macrophages and dendritic cells through modulation of cytokines and chemokines, thus they can act as a link between innate and adaptive immunity. this chapter describes the structure-function relationships of collectins, their diverse functions, and their interaction with viruses, bacteria, fungi and parasites. used by malhotra et al. (1992) . they are known to mediate pathogen recognition through calcium-dependent carbohydrate recognition domains (crds). the following nine collectins have been identified to date: mannan-binding lectin (mbl), three bovine serum collectins, conglutinin, cl-43 and cl-46, lung surfactant proteins sp-a and sp-d, and more recently discovered collectins including, collectin kidney 1 (cl-k1, also called cl-11), collectin liver 1 (cl-l1, also called cl-10) and collectin placenta 1 (cl-p1 also called cl-12). the overall functions of collectins include microbial aggregation and neutralisation, opsonisation, complement activation, and modulation of inflammatory responses. collectins are oligomers of trimeric subunits, for most collectins, the subunits are homotrimers (made up of three identical polypeptides) but heterotrimers can be found for sp-a, which is made up of highly homologous spa-1 and spa-2 polypeptides. hetrotrimers can also form in the case of cl-10 and cl-11 ( fig. 4.1 ). the subunit of each collectin is composed of (i) a short n-terminal (7-28 amino acid residues) cysteine-rich domain, involved in multimerisation (by disulphide bridging); (ii) a collagen-like domain composed of gly-x-y triplets repeats, where x and y represent any amino acids; (iii) a short segment which can form coiled-coil helices, and (iv) , and mbl (c). representations of the trimeric "head" of collectins. these structures represent the 'neck', and the crds of three polypeptides which make up the trimeric subunit. the helix interacts with a neighbouring carbohydrate recognition domains (kishore et al. 2006; skjoedt et al 2012) the c-terminal globular c-type lectin domain, also called the crd (carbohydrate recognition domain) (uemura et al. 2006) (fig. 4.1) . the triple-helical collagen region provides significant rigidity and stability to the molecule (colley and baenziger 1987) . another structural feature of the collagenlike domain of collectins is that it can be o-glycosylated (colley and baenziger 1987) . both mbl and sp-a show an interruption of the gly-x-y triplet repeats, which introduces a bend in the otherwise straight triple helix. this enables the fully assembled multi-subunit structure to angle away from the central core, producing a structure resembling a bouquet of flowers (fig. 4 .2) (voss et al. 1991) . several distinct functions of the collagen domain of collectins have been reported. the collagen domains of sp-a and mbl are involved in receptor-mediated properties. a gekgep specific motif found within the collagen domain of mbl is suggested to bind c1q receptor (arora et al. 2001) , and mediates the enhancement of phagocytosis through c1qr (arora et al. 2001) . a similar motif is within the collagen domain of sp-a (white et al. 1985) , which is also involved in the interaction with c1q receptor (malhotra et al. 1992; malhotra et al. 1990) , and mediates phagocytosis of staphylococcus aureus by monocytes (geertsma et al. 1994) . furthermore, the collagen domain of mbl is shown to bind mbl-associated serum proteases, masp1, 2 and 3, which mediate complement activation via the lectin pathway (thiel et al. 1997; tan et al. 1996) . additionally, the positively charged collagen region found in the membrane bound cl-p1 is involved in the uptake of oxidised ldl particles (ohtani et al. 2001) . the cysteine residues found within the n-terminal domain (7-28 amino acids) form disulphide bonds between monomers, thereby, stabilising trimeric subunits as well as a larger multimers. it was believed that at least two cysteine residues are required at the n-terminal domain for the formation of multimers of trimeric subunits (brown-augsburger et al. 1996; mccormack et al. 1999; mccormack et al. 1997a, b) . however, in the case of cl-43, it is secreted as a single trimeric subunit, despite having two cysteine residues (rothmann et al. 1997; lim et al. 1994a, b) . therefore, other factors contribute to oligomerisation of trimeric subunits, in addition to the of n-terminal cysteine residues. the c-terminal region contains a coiled-coil trimerizing neck region (residues 112-130 in human mbl) ( fig. 4.1) , and the crd (residues 134-245 in human mbl) which folds up into an independent globular carbohydrate-binding structure for each polypeptide chain. each subunit is held together covalently through disulphide bonds, or non-covalently structured into oligomers of up to six subunits. c-type crds are connected to the collagen-like domain through the 'neck' region (24-28 amino acid residues) . furthermore, the neck region is involved in aligning the collagen chains. a broad carbohydrate specificity is required by collectins in order to recognise and bind a large repertoire of (pathogen-associated molecular patterns) pamps. such broad specificity is achieved by an open and flexible trough -like binding pocket found within the crds. the selection of ligands by this site depends on the positioning of vicinal hydroxyl groups of sugars, which form coordination bonds with a ligated calcium ion, hydrogen bonds and a polar van der waals contact (ng et al. 1996) . ligand specificity of collectins is divided into two main sub-classes (mannosebinding or galactose-binding type), which is based on a three amino acid residue motif found in the ca ++ ion binding site. the sequence 185-glu-pro-asn is associated with binding of mannose-like sugars, while the sequence 185-gln-pro-asp is associated with binding galactose-like sugars. the molecular differences based on which crds discriminate between mannose and galactose-type ligands depend on the orientation of c3 and c4 vicinal hydroxyl groups presented on monosaccharides. mannose-specific crds bind ligands in which hydroxyl groups at the c3 and c4 positions are in an equatorial orientation (mannose, glucose, glucosamine), while in galactose these vicinal hydroxyls are in an axial orientation (drickamer and taylor 2015) . inhibition studies using monosaccharides have shown that most likely, all the above described collectins, except cl-p1, prefer mannose ligands over galactose (ohtani et al. 2001; holmskov et al. 1994) . however, a wider range of binding specificity has been reported for mbl and lung surfactant proteins sp-a and sp-d, as these collectins are also capable of binding to nucleic acids (nadesalingam et al. 2003) , phospholipids (sano et al. 1999) , as well as non-glucosylated proteins. fucose, a hexose deoxy sugar is bound by mannose-specific crds in a different manner as it has equatorial hydroxyl groups placed on its c2 and c3 position of the sugar ring, not the c3 and c4 (weis et al. 1991a, b; ng et al. 1996; iobst and drickamer 1994) . computational docking studies have demonstrated that αd-glucose docks into the crd of sp-d via vicinal equatorial hydroxyl groups on the 2-and 3-position of its sugar ring (allen et al. 2001a, b) . although mbl affinity is reported to be very low for monosaccharide galactose, mbl crystallographic studies demonstrate that galactose is ligated in the mbl binding region via coordination bonds with hydroxyl groups placed at c1 and c2 position of the sugar ring (ng et al. 1996) . in addition to galactose and mannose, binding of collectins to a range of sugars has also been studied ; they exhibit preferences for certain sugar residues over others. for instance, despite sp-d being structurally similar to conglutinin, it displays a greater affinity for maltose, a glucose disaccharide, which is a weak ligand for conglutinin. sp-d is suggested to have a lower affinity for glcnac, which is the best ligand for conglutinin. moreover, binding of cl-43 to sugars is closely related to mbl, although the structure of cl-43 is closer to sp-d and conglutinin (lu et al. 2002) . the sugar-binding specificity of cl-11/cl-k1 has been investigated (venkatraman girija et al. 2015) . it has a larger recognition interface than mbl, and recognises predominantly mannose-rich structures, interacting with two sugar residues at a glycan terminal, rather than a single sugar. human mbl is synthesised by hepatocytes and secreted into the blood stream (sastry et al. 1991; ezekowitz et al. 1988; hansen et al. 2000) . initially, mbl was isolated from the liver of the rabbit, rat and chicken, where expression levels were detected in the soluble cytosol, rather than on the cell surface. two forms of mbl (mbl-a and mbl-c) were detected in rodents (hansen et al. 2000; drickamer et al. 1986 ), rabbits (kawasaki et al. 1978 kozutsumi et al. 1980 ) and rhesus monkeys (mogues et al. 1996) . however, only one form of mbl is present in humans and chimpanzees (mogues et al. 1996) . although the liver is the main production site of mbl-a and mbl-c in mice, mrna expression of mbl was also detected in various tissues (table 4 .1) (shushimita et al. 2015) . substantial expression levels of mbl-a and mbl-c were reported in kidney and intestine (table 4 .1). detection of mbl proteins in the small intestine suggests that mbl may have similar roles to secretory iga (reichhardt et al. 2012 ). the collectins sp-a and sp-d are primarily detected in the alveolar space of the lungs, and synthesised by alveolar type-ii cells (table 4 .1) (voorhout et al. 1992 , nayak et al. 2012 , and nonciliated bronchial epithelial cells, also known as clara cells (voorhout et al. 1992; crouch et al. 1992) . although the lung is the main site of sp-a and sp-d synthesis, presence of sp-d has also been reported at extrapulmonary sites. sp-d expression has been shown immunohistochemically in human trachea, brain, heart, kidneys, testis, salivary gland, placenta, prostate, small intestine, and pancreas (table 4 .1). a low expression level has been detected in spleen, uterus, adrenal gland and mammary glands (fisher and mason 1995; madsen et al. 2000; herías et al. 2007) . furthermore, immunoreactivity of sp-d has also been shown in the epithelial cells of both small and large ducts of the parotid gland, lacrimal and sweat glands, epithelial cells of intra-hepatic bile ducts and gall bladder, as well as esophagus, exocrine pancreatic ducts, and in the urinary tract (madsen et al. 2000; bräuer et al. 2007 ). in the case of sp-a, low levels are detected in small intestines from human and rat (table 4 .1) (lin et al. 2001 , van iwaarden et al. 1990 ). in addition to its presence in the murine uterus, very low sp-a expression is found in human prostate, amniotic fluid, thymus and salivary gland . sp-a and sp-d have also been localised in the fetal membranes, and choriodecidual layer of the late pregnancy uterus (miyamura et al. 1994) . as a result of pulmonary microbial infection, the protein levels of both sp-a and sp-d have been reported to increase in the alveolar compartment (atochina et al. 2001) . thus, the level of sp-d increases in response to allergen-induced eosinophilia (kasper et al. 2002) , suggesting that both sp-a and sp-d may function as acute phase reactants within the lungs. furthermore, hypoxia results in an increased concentration of both sp-a and sp-d in the alveolar compartment (white et al. 2001) . conglutinin, cl-46 and cl-43 are serum collectins identified in bovidae and synthesised in the liver . these collectins provide a first line of defense against microbial pathogens. cl-l1 mrna was detected in the liver, and studies using northern blot analysis have suggested that low levels occur in the placenta. although most collectins are secreted, cl-l1 was found in the cytosol of hepatocytes, which may suggest its interaction with intracellular ligands . the presence of cl-p1 was reported in vascular endothelial cells (table 4 .1); cl-p1 is suggested to be membrane bound, and it contains an intracellular domain (ohtani et al. 2001) . expression of mbl, sp-a and sp-d at the mucosal surfaces suggest the innate immune roles of these collectins against invading pathogens. during helicobacter pylori infection, an increased level of sp-d has been detected, suggesting the possible role of sp-d in the mucosal defense outside the lungs (murray et al. 2002) , eg. gastrointestinal tract. collectins are important soluble pattern-recognition receptors (prrs) of the humoral arm of the innate immune response. collectins are able to recognise and bind to a wide variety of microbes and are involved in their clearance and forming a central link to adaptive immunity against microbial infections. in this section, we will discuss the well-known collectins: mbl, sp-a and sp-d, as well as newly discovered collectins: liver collectin (cl-l1), kidney collectin (cl-k1), and placenta collectin (cl-p1). we will also briefly discuss bovine collectins, conglutinin, cl-43 and cl-46. microbes can be cleared by collectins via a number of mechanisms such as aggregation, opsonisation, phagocytosis, microbial growth inhibition, complement activation, as well as modulation of adaptive immunity. pulmonary surfactant is composed of 90% phospholipids and 10% proteins (made up of surfactant proteins, sp-a, sp-b, sp-c and sp-d. whilst, sp-b and sp-c are hydrophobic and essential for the physiology of the alveolar surfaces, sp-a and sp-d are hydrophilic and contribute to lung immunity. an early study showed that pulmonary surfactant enhanced the killing of staphylococcus aureus by alveolar macrophages (am), in vitro (laforce et al. 1973) . both gram-negative and grampositive bacteria are recognised by sp-a and sp-d, enhancing their phagocytosis by ams (fig. 4 .3) (pikaar et al. 1995) . for gram-negative bacteria, sp-a and sp-d both bind to lipopolysaccharide (lps) but differ in preferential targets on the molecule. sp-a binds to the lipid a moiety of rough lps (which lacks the o-antigen and shortened oligosaccharides) (van iwaarden et al. 1994) , and enhances phagocytosis of bacteria by am (kalina et al. 1995) , but not to smooth lps (which contains the o-antigen) (van iwaarden et al. 1994) . in contrast, sp-d binds strongly to smooth lps from escherichia coli and salmonella species but does not recognise the lipid a moiety or oligosaccharide deficient lps (kuan et al. 1992) . this indicates that sp-d preferentially targets the core terminal saccharides in the bacterial ligand, whilst sp-a prefers lipid a. sp-d has also able been shown to bind to rough lps via its trimeric carbohydrate recognition domain (crd), (targeting shortened oligosaccharides) and agglutinating e. coli (kuan et al. 1992) , and rough lps from klebsiella (lim et al. 1994a, b; kishore et al. 1996) . in addition to lps, sp-a is able to bind to capsular polysaccharides of klebsiella species, enhancing their phagocytosis by am (kabha et al. 1997) . however, bacterial peptidoglycan is not a ligand for sp-a (murakami et al. 2002) . sp-a and sp-d directly inhibit the growth of several gram-negative bacteria by increasing the membrane permeability of the bacterial cell wall (fig. 4 .3) . sp-a and sp-d also inhibit biosynthetic functions in strains of e. coli, k. pneumoniae and enterobacter aerogenes . similarly, sp-a inhibits the growth of p. aeruginosa by increasing membrane permeability (van iwaarden et al. 1994 ), but the bacterium can resist through quorum-sensing and the secretion of a flagellum-mediated exoprotease that degrades sp-a (kuang et al. 2011a). furthermore, sp-a downregulates tnf-α secretion via toll-like receptor 2/nf-κb mediated pathway, indicating its role in modulating inflammatory responses against bacterial ligands (murakami et al. 2002) . sp-a can bind to the outer membrane protein (omp) of haemophilus influenzae type a and to a lesser extent, type b (mcneely and coonrod, 1994) . sp-a can also aggregate and opsonise h. influenzae type a, facilitating killing by am (mcneely and coonrod 1994) . similarly, sp-a binds to the capsular polysaccharide of some strains of k. pneumoniae, agglutinating the bacteria and increase phagocytosis by macrophages (kabha et al. 1997) , and treatment with sp-a plus sp-b n (n-terminal saponin domain of sp-b) significantly reduced bacterial infection and enhanced neutrophil recruitment (coya et al. 2015) . sp-a has a bacteriostatic effect on mycoplasma pneumoniae via binding to di-saturated phosphatidylglycerols on the bacterial membrane (piboonpocanun et al. 2005) . sp-a can interact with mycobacterium tuberculosis putative adhesin apa glycoprotein on its surface (ragas et al. 2007 ). sp-d can also bind to gram-positive bacterial ligands such as lipoteichoic acid and peptidoglycan via its crd (van de wetering et al. 2001) and to lipoarabinomannan (lam) from m. tuberculosis and mycobacterium avium (ferguson et al. 1999; kudo et al. 2004) . sp-d is also able to interact with cell membrane lipids of m. pneumoniae (chiba et al. 2002) . it is intriguing that although both sp-a and sp-d bind and agglutinate m. tuberculosis, they have opposing effects on phagocytosis by macrophages. sp-a enhances phagocytosis via increased expression of mannose receptor on the host cell surface (beharka et al. 2002) , whilst sp-d inhibits phagocytosis by blocking the interaction of lam with macrophage mannose receptor, and not as a result of bacterial agglutination by sp-d (ferguson et al. 1999 . in a mouse model of tuberculosis infection, sp-a −/− , sp-d −/− , and sp-a/d −/− knockout mice still had the ability to phagocytose and clear m. tuberculosis when given a low-dose aerosol challenge of the pathogen, suggesting that both sp-a and sp-d could be redundant in this animal model (lemos et al. 2011) . similarly, both sp-a and sp-d can also bind to legionella pneumophila, but seem to inhibit intracellular bacterial growth in the macrophage (sawada et al. 2010) . sp-a and sp-d can also directly facilitate phagocytosis without the need for microbial binding, by up-regulating the expression of cell surface phagocytic receptors in macrophages, such as mannose receptor (beharka et al. 2002; kudo et al. 2004) . in sp-a −/− knockout mice, expression of mannose receptor is down-regulated, showing that sp-a is important in regulating the expression of this receptor (beharka et al. 2002) . similarly, sp-a is able to enhance phagocytosis of streptococcus pneumoniae by am, independent of its binding to the bacterium, via the increased expression of scavenger receptor a (sr-a) . interestingly, the vast majority of clinical strains of the opportunist pseudomonas aeruginosa secrete an elastase that degrades sp-a and facilitates evasion of opsonisation by the collectin during phagocytosis (kuang et al. 2011b) . sp-a and sp-d can play important roles in modulating the intracellular environment after phagocytosis by stimulating reactive oxygen and nitrogen intermediates facilitating the killing of intracellular pathogens. this is of particular note in mycobacteria, which are specialist intracellular bacteria. sp-a enhances the killing of intracellular mycobacterium bovis bcg by increasing nitric oxide (no) production, in addition to enhancing the release of inflammatory mediators such as tnf-α (weikert et al. 2000) . in contrast, in ifn-γ primed am, sp-a decreases no production in response to intracellular infection with m. tuberculosis and m. avium by inhibiting tnf-α secretion and nuclear factor-kappa b (nf-κb) activation (pasula et al. 1999; hussain et al. 2003) . sp-a can also enhance the intracellular killing of mycoplasma pulmonis via a no dependent mechanism (hickmandavis et al. 1998) . bacteria-derived cell-wall molecules such as lps and peptidoglycan are potent stimulators of inflammation and can also interact with pattern-recognition receptors (prrs) such as cd14 or toll-like receptors (tlr), via pathogen-associated molecular patterns (pamps), and activate downstream intracellular signalling. sp-a and sp-d can also directly bind to prrs (e.g. tlr and cd14) and thus can modulate the inflammatory response. sp-a and sp-d can alter lps interactions with cd14 via different mechanisms (sp-a via neck domain; sp-d via crd) (sano et al. 2000) . furthermore, via direct interaction with cd14, sp-a inhibits production of tnfα induced by smooth lps, but not rough lps in u937 macrophages (sano et al. 1999 ). in sp-a −/− knockout mice, tnf-α induced by smooth lps, significantly increased, compared to wild-type mice (borron et al. 2000) , whilst sp-a has also been shown to inhibit tnf-α induction by peptidoglycan via direct binding to tlr-2 (murakami et al. 2002) . thus, sp-a significantly decreases peptidoglycan or smooth lps-induced pro-inflammatory responses (via nf-κb activation). sp-a has no effect or increases the inflammatory response induced by rough lps. in tuberculosis, sp-a has pleiotropic effects being able to promote inflammation in the presence of infection and suppresses inflammation in uninfected macrophages, probably protecting uninfected lung areas from the deleterious effects of inflammation (gold et al. 2004) . in humans, sp-a exists in two isoforms, sp-a1 and sp-a2, which are encoded by distinct genes. fully assembled sp-a protein contains both gene products. a number of studies have described polymorphisms in these genes and the sp-d gene which may have a role in susceptibility to microbial infection, particularly tuberculosis. polymorphisms within and flanking the sp-a1 and sp-a2 genes have been described which indicate protection or susceptibility toward pulmonary tb in the populations studied in mexico, ethiopia, india and china (floros et al. 2000; madan et al. 2002; malik et al. 2006; vaid et al. 2006; yang et al. 2014) . two sp-a1 alleles (sftpa1 307a, sftpa1 776t) and two sp-a2 alleles (sftpa2 355c and sftpa2 751c) were associated with tuberculosis susceptibility in ethiopia (malik et al. 2006 ). the sftpa2 751a/c polymorphism and the haplotype 1a 3 in sp-a2, which both affect the amino acids in crd region of sp-a, may alter binding to m. tuberculosis and thus were found to be strongly linked with tuberculosis susceptibility (malik et al. 2006) . another study also found two polymorphisms (sp-a2 g1649c and sp-a2 a1660g) in the introns of sp-a1 that were associated with tuberculosis in an indian population, but none in the sp-a1 gene (madan et al. 2002) . in a chinese population, the polymorphism 1649g in the sp-a2 gene was strongly associated with tuberculosis, mirroring the findings in the ethiopian and indian populations (yang et al. 2014 ). the sp-a2 1649g leads to a transversion (proline to alanine), affecting the triple helical structure of sp-a (yang et al. 2014) . in sp-d, the polymorphism, g459a, is significantly associated with tuberculosis susceptibility in an indian population, but the molecular basis for susceptibility is not understood (vaid et al. 2006) . these observations illustrate the complexities of host-pathogen interactions in bacterial infection mediated by these collectins. mbl is the recognition subcomponent of the lectin pathway of the complement system and is present mostly in the serum. the structure of mbl is similar to that of sp-a, and in the presence of ca 2+ , it has been observed to target terminal sugars (e.g. d-mannose, l-fucose, and n-acetyl-d-glucosamine), on the surface of a number of gram-positive and gram-negative bacterial species (ip et al. 2009; lugo-villarino et al. 2011) . the binding of mbl to microbial surfaces can activate complement through mbl-associated serine proteases (masps), resulting in enhanced microbial clearance via opsonisation (c3 and c4 deposition) and complement-mediated lysis. however, mbl also has complement-independent activity such as inhibition of bacterial adhesion (jack et al. 2005 ) and opsonisation to enhance bacterial uptake (kuhlman et al. 1989; polotsky et al. 1997; jack et al. 2005) . strong in vitro binding of mbl to s. aureus, streptococcus pyogenes, listeria monocytogenes and nonencapsulated neisseria meningitidis has been described (levitz et al. 1993; van emmerik et al. 1994; neth et al. 2000) . moderate levels of mbl binding were observed in e. coli, haemophilus influenzae and klebsiella species, whilst no binding has been observed for p. aeruginosa, enterococcus species and streptococcus pneumoniae (levitz et al. 1993; van emmerik et al. 1994; neth et al. 2000) . bacterial pathogens have involved strategies to interfere with mbl binding and functions for survival, via the synthesis of a polysaccharide capsule and sialylation of lps ligands on the bacterial surface which reduces the binding of mbl (jack et al. 2005; krarup et al. 2005 ). this effectively masks or alters the bacterial ligands for mbl interaction. a number of studies have characterised the bacterial ligands for mbl. mbl is able to bind to peptidoglycan and lipoteichoic acid from s. aureus (polotsky et al. 1996; nadesalingam et al. 2005a, b) , lam from m. avium (polotsky et al. 1997 (jack et al. 1998; devyatyarova-johnson et al. 2000; jack et al. 2001; gulati et al. 2002) . m. meningitidis can also interfere with mbl binding through encapsulation (van emmerik et al. 1994) , whilst m. gonorrhoeae is not able to form capsules. encapsulation seems to be less robust at decreasing mbl binding than sialyation of los (jack et al. 1998) . bound mbl can activate complement and the ability of neisseria species to cascade complement all the way to c9 (membrane attack complex (mac)) is crucial for protection against infection, since they are otherwise poorly phagocytosed by neutrophils and macrophages when opsonised by c3 (ross and densen 1984) . mbl bound to the surface of neisseria is able to increase bacterial killing via increased complement activation (jack et al. 1998 (jack et al. , 2001 gulati et al. 2002) , and similar observations of bactericidal activity have been reported for e. coli and salmonella species (kawasaki et al. 1989; ihara et al. 1991) . for most other bacteria, complement activation to the c3 deposition stage is enough for protection via increased phagocytosis through opsonisation by complement products on the bacterial cell surface. mbl can increase c3b deposition on s. aureus (neth et al. 2002) , but this does not appear to result in increased complement activation (cunnion et al. 2001) . mbl targets wall teichoic acid in s. aureus and this interaction is particularly important in infants that have not developed adaptive immunity, leading to bacterial clearance via mbl-mediated complement activation (kurokawa et al. 2016 ). in addition to its complement-mediated activities, mbl is also has various intrinsic effects, being able to act as an opsonin independently and other direct effects. mbl enhances uptake and intracellular killing of salmonella by neutrophils and monocytes (kuhlman et al. 1989) , but this may also involve interaction with fibronectin (ghiran et al. 2000) . recently, mbl has also been shown to have a direct inhibitory effect on flagellar activity in pathogenic salmonella bacteria, impairing their motility (xu et al. 2016) . mbl can also increase uptake of mycobacteria by macrophages (polotsky et al. 1997 ) and n. meningitidis by neutrophils, monocytes and macrophages (jack et al. 2001) , but this uptake by neutrophils may not result in intracellular killing (drogari-apiranthitou et al. 1997) . mbl also appears to improve the efficiency of internalisation of bacteria bound to the macrophage plasma membrane (neth et al. 2002) . mbl co-interacts with tlr2 in sensing s. aureus and thus influencing the subsequent inflammatory response (nauta et al. 2003; ip et al. 2008) . mbl deficiency increases susceptibility to microbial infection even though the majority of mbl-deficient individuals are usually healthy (eisen and minchinton 2003) . the concentration of mbl in the plasma varies considerably in humans (0-10, 000 ng/ml) due to polymorphisms in the mbl gene (steffensen et al. 2000) . mbl deficiency is commonly observed in around 25% of caucasians (having low levels (<500 ng/ml)), which renders them susceptible to infection (valdimarsson et al. 2004 ). mbl-deficient mice are susceptible to s. aureus infection , whilst mbl deficiency increases susceptibility to postburn infection with p. aeruginosa (moller-kristensen et al. 2006) . a large cohort study has also found a strong association between mbl deficiency and meningococcal infection, and pneumococcal pneumonia, in patients undergoing chemotherapy (gaynor et al. 1995; kronborg et al. 2002; roy et al. 2002) . in contrast, normal or increased levels of mbl are linked to frequent infection with m. tuberculosis and m. leprae (garred et al. 1994 (garred et al. , 1997b , probably through complement-mediated phagocytosis of the pathogen. up to 30% of healthy individuals have polymorphisms linked to mbl deficiency and these, together with serum levels, have been associated with susceptibility to tuberculosis and other inflammatory diseases in some ethnic populations (takahashi and ezekowitz 2005; thiel et al. 2006; goyal et al. 2016 ). of the three more recently discovered collectins (cl-l1, cl-k1, cl-p1), cl-l1 and cl-p1 have been shown to have bacterial interactions. cl-k binds to e. coli, k. pneumoniae, p. aeruginosa and m. tuberculosis (keshi et al. 2006; hansen et al. 2010; troegeler et al. 2015) , whilst cl-p1 can bind to e. coli and s. aureus jang et al. 2009 ). both cl-l1 and cl-k1 can activate the complement lectin pathway as can the heteromeric form cl-lk, which interacts with the masps (henriksen et al. 2013) . cl-p1 can activate the alternative and classical pathways via its interaction with c-reactive protein (crp) (roy et al. 2016 ). there is limited data on the activity of cl-lk in vivo and in vitro, but due to average serum concentrations being below that of mbl (0.3 μg/ml vs. 1.5 μg/ml), pathogen recognition and clearance through complement activation is likely to have a minor role to play for these collectins. it is not clear whether these collectins can act directly as opsonins in a complement-independent manner. cl-l1 can bind d-mannose, nacetylglucosamine, d-galactose, l-fucose and d-fructose in a ca 2+ dependent manner axelgaard et al. 2013) . similarly, cl-k1 can also bind l-fucose, d-mannose and n-acetylmannosamine hansen et al. 2010 ). furthermore, cl-lk was recently demonstrated to be a prr for m. tuberculosis, being able to primarily target mannose-capped lipoarabinomannan (manlam), in a ca 2+ dependent manner, on the surface of the mycobacterium, but not to m. smegmatis due to the lack of mannose caps on its lam (troegeler et al. 2015) . mice deficient in cl-k1 did not show altered susceptibility to m. tuberculosis infection and cl-lk opsonised m. tuberculosis did not result in altered phagocytosis or intracellular survival of the pathogen in human macrophages (troegeler et al. 2015) . interestingly, the levels of cl-lk in serum of tuberculosis patients is reduced, compared to controls, correlating inversely to the immune response to m. tuberculosis and suggesting that it may be useful as a biomarker for the disease (troegeler et al. 2015) . conglutinin was the first mammalian collectin to be discovered and is found in bovidae, together with other lesser known collectins (cl-43 and cl-46) . conglutinin is similar in overall structure to sp-d and is able to bind to microbial surfaces in the presence of ca 2+ . conglutinin is secreted by the liver and found predominantly in bovine serum at an average concentration of 12 μg/ml . conglutinin has been shown to have anti-microbial properties. low serum levels of conglutinin have been associated with acute infections (e.g. pneumonia and metritis) and predisposition to respiratory infection (ingram and mitchell 1971; holmskov et al. 1998) . conglutinin is able to bind many microbes, including gram-negative bacteria such as e. coli and salmonella typhimurium (friis-christiansen et al. 1990; friis et al. 1991) , lps and peptidoglycan (wang et al. 1995) and gram-positive bacteria such as mycobacteria (dec et al. 2012; mehmood et al. 2019) . conglutinin is uniquely able to bind to ic3b, via the mannose sugars on the α-chain of ic3b (laursen et al. 1994 ). conglutinin is able to bind and agglutinate ic3b-coated erythrocytes (lachmann and muller-eberhard 1968; laursen et al. 1994) , and as well as e. coli, increasing the respiratory burst of phagocytes (friis et al. 1991 ). conglutinin has also been shown to be protective against bacterial infection in vivo, being able to increase the survival of mice experimentally infected with highly virulent strains of s. typhimurium (friis-christiansen et al. 1990) . a recombinant truncated form of conglutinin, composed of the α-helical neck region and the crd of conglutinin (wang et al. 1995) , was recently shown to bind to able to bind to the vaccine strain mycobacterium bovis bcg (probably via lam), and act as an anti-opsonin both in the presence and absence of complement deposition. thus, conglutinin can interfere with the uptake of the bacterium by thp-1 macrophages and alter their inflammatory response (mehmood et al. 2019 ). this suggests that conglutinin interfers with uptake of mycobacteria by macrophages via two important mechanisms: (1) blocking interaction of mycobacterial lam with mannose receptor, and (2) blocking ic3b interaction with complement receptors cr3 and cr4 (mehmood et al. 2019) . these data potentially have important implications for bovine tuberculosis. cl-43 and cl-46 are also bovine-unique collectins, but their role in the physiology and innate immunity against bacteria has not been fully studied. there is one report of cl-43 binding to e. coli strain k12, enhancing attachment to phagocytes . there are numerous studies that describe direct interaction of sp-a and sp-d with a range of viruses, enhancing their phagocytosis, as well as neutralising viral infection of host cells (fig. 4.3) . experiments on sp-a −/− and sp-d −/− knockout mice infected with influenza a virus (iav) suggest that both collectins are protective, but this is dependent on viral strain-specific factors, such as the nature of glycosylation in ha and na (levine et al. 2001 (levine et al. , 2002 hawgood et al. 2004) . also, mice lacking both sp-a and sp-d, have an iav infection phenotype almost identical to sp-d −/− mice (hawgood et al. 2004 ). moreover, sp-d, but not sp-a, enhanced the clearance of iav infection in the mouse lung (levine et al. 2001) . thus, these studies suggest that sp-d plays a more significant role than sp-a in the host innate immune response to infection with iav. sp-a binds to iav, neutralises the virus and inhibits the release of viral particles from infected cells, by targeting mannose residues of viral surface haemagglutinin (ha) or neuraminidase (na) benne et al. 1995) . sp-d strongly inhibits hemagglutination activity of iav, resulting in viral aggregation and neutralisation (hartshorn et al. 1994) . sp-d is also able to inhibit na activity, with inhibition being stopped in the presence of d-mannose (reading et al. 1997) . sp-d has a stronger inhibitory effect on na compared to sp-a (tecle et al. 2007 ). sp-d binds to mannosylated, n-linked sugars on viral ha and na via its crd, resulting in potent anti-iav infectivity (hartshorn et al. 1994 (hartshorn et al. , 2000 . sp-d was able to inhibit virus-induced ha activity, block the enzymatic activity of viral na, and neutralise the ability of seasonal h1n1 strains of iav to infect human respiratory epithelial cells (job et al. 2010) . however, in the same study, some pandemic h1n1 were found to be resistant to sp-d inhibition that correlated with the degree of n-glycosylation in the globular head of ha (job et al. 2010) . it has been shown that porcine sp-d has an increased ability to inhibit, not just seasonal iav strains, but also a number of pandemic and avian strains (van eijk et al. 2003; hillaire et al. 2012 ). this is important as pigs are a source of iav pandemic strains (h1n1) that can be transmitted to humans, so studying porcine sp-d could provide further insights into this host reservoir. a recombinant truncated form of sp-a (rfhsp-a) made up of α-helical neck and crd, promotes iav infection, replication, upregulation of viral factors (m1) in lung epithelial a549 cells and enhances the pro-inflammatory response (al-qahtani et al. 2019 ). this contrasts with full-length sp-a which inhibits iav infection and dampens the pro-inflammatory response, demonstrating that the full-length sp-a molecule is required for iav protection (al-qahtani et al. 2019). however, in a similar study, a recombinant truncated form of sp-d (rfhsp-d) was shown to inhibit iav entry, down-regulate viral factors (m1) and down-regulate the pro-inflammatory response (al-ahdal et al. 2018) . these opposing effects of rfhsp-a and rfhsp-d provide further insight into iav pathogenesis and the possible utility of rfhsp-d as a therapeutic molecule. in bronchoalveolar lavage (bal), sp-d enhances iav uptake and virus-induced respiratory burst by neutrophils , but other collectins (sp-a), mucins and gp-340 dampen sp-d's effect, and thus, significantly reduce the ability of sp-d to promote neutrophil oxidative response . therefore, the net effect of bal is to increase neutrophil uptake of iav while reducing the respiratory burst response to virus . sp-a is also able to bind to herpes simplex virus type 1 (hsv-1) via viral n-linked sugars and enhance phagocytosis of the virus by macrophages (van iwaarden et al. 1991; van iwaarden et al. 1992a, b) . the mechanism of binding of sp-a to hsv-1 is similar to binding to iav, involving interaction with the sialylated carbohydrate on the collectin's crd. sp-a also has an opsonin activity, increasing uptake of hsv-1 by am (van iwaarden et al. 1991) . similarly, sp-a binds to cytomegalovirus and enhances viral entry into rat lung cells (weyer et al. 2000) . it is unknown whether sp-d has any activity against other herpesviridae. sp-a is able to bind to respiratory syncytial virus (rsv) targeting the f2 subunit of the viral f antigen and is able neutralise the virus (ghildyal et al. 1999; sano et al. 1999 sano et al. , 2000 . children with severe rsv infection have reduced levels of sp-a and sp-d in bal samples compared to healthy controls (kerr and paton 1999). in sp-a −/− knockout mice, rsv infection was more severe than in sp-a +/+ mice and the addition of exogenous sp-a to sp-a −/− mice reduced viral load and inflammation, and enhanced rsv clearance (levine et al. 1999) . sp-d can bind to rsv protein g and is able to neutralise rsv infectivity in vitro (hickling et al. 1999) . interestingly, rsv itself can alter sp-a expression in human pulmonary epithelial cells, upon infection by interfering with protein translation (bruce et al. 2009 ). sp-a binds to human immunodeficiency virus 1 (hiv-1) via the viral envelope gp120 glycoprotein and inhibits direct infection of cd4 + t cells (gaiha et al. 2008 ). yet, in dendritic cells (dc), sp-a increases hiv uptake, through enhanced binding to gp120 and facilitates transfer of hiv from dc to cd4 + t cells (gaiha et al. 2008) . sp-d is also able to bind to hiv gp120 and inhibit viral infectivity (meschi et al. 2005) , whilst rfhsp-d was also able to bind to gp120 and prevent infection of jurkat t cells, u937 monocytic cells and pbmc, and significantly suppress the hiv-1 induced cytokine storm in these cells (pandit et al. 2014 ). interestingly, a direct protein-protein interaction between rfhsp-d and dc-sign (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) modulates the capture of hiv-1 and its transfer to cd4 + t cells, revealing a novel and distinct anti-viral mechanism against hiv-1 by sp-d (dodagatta-marri et al. 2017) . this same rfhsp-d has also been recently shown to restrict the transfer of hiv across the vaginal epithelial barrier, by altering the gene expression signature of the epithelium (pandit et al. 2019) . these recent studies demonstrate the therapeutic potential of rfhsp-d against hiv infection. elevated levels of serum sp-d have been reported in severe acute respiratory syndrome (sars) coronavirus infected patients (wu et al. 2009 ). sp-d is able to bind to the glycosylated spike protein (s-protein) on the sars coronavirus (leth-larsen et al. 2007 ). both sp-a and sp-d bind to coronavirus strain hcov-229e, and inhibit viral infection of human bronchial epithelial (16hbe) cells. whilst sp-a only modestly reduced infection in am, whereas sp-d had no effect (funk et al. 2012) . human and porcine sp-d can interact with ebola virus glycoprotein and enhance viral infection in pulmonary cells, suggesting that sp-d may enhance viral spread (favier et al. 2018) . sp-a has been shown to enhance clearance of pulmonary adenovirus infection and inhibit lung inflammation (harrod et al. 1999) . bovine sp-d is also able to bind to bovine rotaviruses via the vp7 glycoprotein and neutralise infectivity (reading et al. 1998 ). sp-d binds to the a27 protein of vaccinia virus. sp-d −/− knockout mice challenged with vaccinia virus resulted in increased mortality, compared to sp-d +/+ mice, suggesting that sp-d has a protective role against vaccinia infection (perino et al. 2013 ). mbl is able to interact with a number of viral pathogens and its effect is generally protective, although there are examples of negative as well as positive outcomes for infection as a result of mbl-mediated binding (fig. 4.4) . several studies have shown that mbl is a potent inhibitor of iav infection (hartley et al. 1992; hartshorn et al. 1993b; reading et al. 1995 reading et al. , 1997 . moreover, mbl also has the added ability to deposit complement on iav-infected cells (reading et al. 1995) . there are also elevated levels of mbl in the lung during iav infection, suggesting that it may be important for protection against iav pathogenesis (reading et al. 1997; fidler et al. 2009 ). mbl can inhibit viral hemagglutination and directly neutralise iav in either a complement-dependent or independent manner (hartshorn et al. 1993b; anders et al. 1994; kase et al. 1999) . mbl binds to iav ha and na, and without involving complement, neutralises the virus ). however, some iav strains are resistant to the effects of mbl which is dependent on the degree of glycosylation on the viral ha globular domain (reading et al. 1997; job et al. 2010; tokunaga et al. 2011) . furthermore, mbl −/− mice were more susceptible to infection from highly glycosylated viral strains of iav than wild-type mice (chang et al. 2010 ). however, pandemic strain h1n1 and avian influenza a h9n2 produced more severe disease (enhanced production of pro-inflammatory response) in wild-type mice compared to mbl −/− mice, suggesting that mbl may have a deleterious effect in some iav infections (ling et al. 2012) . mbl is able to neutralise hiv-1 in vitro by binding to the n-linked mannose glycans of viral gp120, and binding to hiv-1 infected cd4 + t cells and monocytes and inhibiting reverse transcriptase activity (ezekowitz et al. 1989; teodorof et al. 2014) . another study has also shown mbl can also bind to viral gp41 as well as gp120 (saifuddin et al. 2000) , whilst mbl also activates complement on gp120 binding (haurum et al. 1993) . studies have shown a tentative link between low levels of mbl and increased risk of hiv-1 transmission or progression to aids, but this remains contentious (garred et al. 1997a; takahashi and ezekowitz 2005; ballegaard et al. 2014 ). there has also been a report of a positive correlation between the rate of aids progression and mbl plasma concentration (mangano et al. 2008) . however, other studies have found no correlation between mbl levels and aids disease progression (nielsen et al. 1995; mcbride et al. 1998) . in general, sp-d is better able to inhibit hiv-1 than mbl, but as is the case for mbl, sp-d's inhibitory activity against hiv-1 is lower than what has been observed for iav (meschi et al. 2005 ). mbl has also been shown to contribute to hiv-1 pathogenesis, where mbl mediates enhancement of hiv-1 dissemination to the brain by soluble gp120, which is taken up by the cxcr4 receptor on neurones, and then intracellularly trafficked by mbl, thus resulting in the apoptosis of neuronal cells (bachis et al. 2006; teodorof et al. 2014) . epidemiological studies have revealed association of mbl with hepatitis b virus (hbv) and hepatitis c virus (hcv) infection and disease severity, based on genetic polymorphisms (thomas et al. 1996; matsushita et al. 1998; yuen et al. 1999; sasaki et al. 2000; hakozaki et al. 2002) . however, one study found no link between mbl polymorphisms and hbv infection (hohler et al. 1998) . mbl is able to bind to hcv envelope glycoproteins, e1 and e2, and is able to activate complement (via masp-2), resulting in the neutralisation of hcv particles (brown et al. 2010) . mbl probably binds to n-linked glycosylated forms or hbv surface antigen (hbsag) (brown et al. 2007 ), but it is unknown whether this interaction neutralises the infectivity of the virus. mbl is also able to bind to ebola virus via its envelope glycoprotein (gp), which contains high mannose glycan sites, and is able to inhibit the binding of ebola (and marburg) viruses to dc-sign, blocking attachment to host cells and also neutralise the virus through complement activation (ji et al. 2005) . furthermore, soluble gp is a key component of ebola viral pathogenesis and mbl was found to be able to negate gp activity and the virally induced cytokine storm (escudero-perez et al. 2014) , and thus mbl could be involved in protection against increased vascular permeability which is a characteristic of ebola haemorrhagic disease. nevertheless, high dose mbl therapy in a mouse model, where mice we given recombinant human mbl at levels greater than seven times above average human levels, survived otherwise fatal ebola viral infection and became resistant to reinfection (michelow et al. 2011 ). there is limited or circumstantial data on the interaction of mbl with a number of other viral pathogens. in mice, mbl appears to modulate the immune responses to hsv-2 ( gadjeva et al. 2004) , mbl deficiency seems to be linked with recurrent infections (gadjeva et al. 2004; seppanen et al. 2009 ). mbl also binds to flaviviruses such as dengue and west nile virus and is able to neutralise infection through complement-dependent and independent mechanisms (avirutnan et al. 2011 ; fuchs et al. 2011) . genetic polymorphism affecting mbl serum levels may also contribute to the pathogenesis and disease severity of dengue fever (avirutnan et al. 2011). for collectins cl-l1, cl-k1, cl-p1, there is limited data on their interaction with viruses. only cl-k1 has been shown to bind iav and partially decrease the infectivity of the virus (hansen et al. 2010; selman and hansen 2012) . the binding of cl-l1 and cl-p1 to viruses has not been reported. like sp-d, conglutinin binds to iav resulting in the inhibition of hemagglutination and infectivity of the virus (hartshorn et al. 1993a ). conglutinin binds via its crd to the high mannose sites on the viral ha. iav treated with conglutinin also boosted neutrophil respiratory burst (hartshorn et al. 1993a ). conglutinin, cl-43 and bovine sp-d have been reported to bind the bovine rotavirus nebraska calf diarrhoea virus, targeting its vp7 glycoprotein (reading et al. 1998) . binding resulted in hemagglutination and neuralisation of rotavirus, with cl-43 showing the highest activity against the virus; it is the first report of collectin activity against a nonenveloped virus (reading et al. 1998 ). however, conglutinin has a higher inhibitory activity against iav (strain hkx31) than bovine sp-d or cl-43 (reading et al. 1998) . conglutinin binds to hsv-2 and enhances infection in mice (fischer et al. 1994) . it is also able to bind to hiv-1 gp160 and inhibit interaction of the virus with the cd4 receptor (andersen et al. 1991) . interestingly, a collectin-like protein analogous, to bovine conglutinin, was purified from human serum (called conglutinin-like protein) and this was demonstrated to bind to hiv-1 gp120 via its crd and inhibit viral infectivity (ushijima et al. 1992 ). collectins are able to recognise and bind to a number of fungi, both primary and opportunistic fungal pathogens, at various stages in their life cycle. collectins can exhibit direct growth inhibition and enhance phagocytosis of fungi; in some cases, they can contribute to the fungal pathogenesis. both sp-a and sp-d can bind to the conidia of aspergillus fumigatus, via its β-(1-6)glucan carbohydrate structures on the fungal cell surface in a ca 2+ dependent manner (fig. 4. 3) (madan et al. 1997a; allen et al. 2001a, b) . sp-a and sp-d can cause inhibition of conidia infectivity via agglutination, enhancement of phagocytosis and intracellular killing of a. fumigatus conidia by neutrophils and am (madan et al. 1997a ). the fungal ligands of sp-a are 2 n-linked glycosylated glycoproteins (gp45 and gp55) isolated from culture filtrate and are also used for elisa diagnosis of allergic aspergillosis (madan et al. 1997b) . fungal melanin was recently determined to be the primary ligand for sp-d on the a. fumigatus conidia cell surface, and is able to facilitate fungal phagocytosis and modulate the anti-fungal immune response (wong et al. 2018) . utilising a mouse model of invasive pulmonary aspergillosis (ipa), sp-d, but not sp-a, was found to be protective against a normally fatal challenge of a. fumigatus conidia (madan et al. 2001a, b) . in this study, ipa mice-treated intranasally with purified human sp-d or rfhsp-d showed 60 and 80% survival respectively (madan et al. 2001a, b) . the basis of this therapeutic protection by sp-d and rfhsp-d was observed to be enhanced phagocytosis of conidia by macrophages and neutrophils, fungistatic effects on the growth of conidia and a dampening of pathogenic th2 cytokines (il-4 and il-5), whilst enhancing protective th1 cytokines (tnf-α and ifn-γ) (singh et al. 2009 ). sp-d −/− knockout mice are more susceptible to ipa (madan et al. 2010 ). however, sp-a −/− knockout mice demonstrate resistance to ipa, suggesting that sp-a may be involved in the pathogenesis of ipa (madan et al. 2010 ). both sp-a and sp-d also have a direct effect on histoplasma capsulatum, inhibiting its growth by increasing the permeability of the fungal membrane ). however, no aggregation of h. capsulatum was observed by sp-a or sp-d, and neither collectin altered the phagocytosis of the fungus or inhibited the growth of macrophage-infected h. capsulatum . sp-a is also able to bind to cryptococcus neoformans, both in its encapsulated and non-encapsulated yeast form, but this does not result in increased phagocytosis of the acapsular form (walenkamp et al. 1999 ). sp-a binding was ca 2+ -dependent and was inhibited by glucose and mannose, but not galactose (walenkamp et al. 1999 ). intranasal infection with c. neoformans gave the same survival outcome in sp-a −/− knockout mice and wild-type mice, suggesting that the fungus is resistant to sp-a mediated host defence mechanisms (giles et al. 2007) . a subsequent study found that sp-d increases the phagocytosis of hypocapsular c. neoformans by murine macrophages and that this facilitated fungal survival (geunes-boyer et al. 2009 ). other studies have also shown that sp-d can agglutinate c. neoformans and a. fumigatus (schelenz et al. 1995; madan et al. 1997a ). furthermore, sp-d can bind to both encapsulated and acapsular c. neoformans and can aggregate acapsular c. neoformans in particular (van de wetering et al. 2004a ). the cryptococcal capsular components glucuronoxylomannan (gxm) and mannoprotein 1 (mp1) are the ligands for sp-d (van de wetering et al. 2004a ). sp-d is able to facilitate c. neoformans infection further by protecting the fungus against oxidative stress allowing for disease progression in the mouse model of infection (geunes-boyer et al. 2012) . sp-d is also able to bind blastomyces dermatitidis, via β-glucan on its surface, and subsequently block interactions with β-glucan-receptors on am (lekkala et al. 2006 ). this study also showed a reduction in tnf-α, dampening the host inflammatory response and thus may facilitate disease progression (lekkala et al. 2006 ). sp-d and sp-a can also bind to coccidioides posadasii via its surface antigens. in a mouse model of infection, c. posadasii infection is able to suppress the expression of pulmonary sp-a and sp-d, possibly facilitating fungal disease progression and dissemination (awasthi et al. 2004 ). sp-d can also bind to candida albicans via its surface antigens and agglutinate the pathogen and directly inhibiting its growth without the requirement of macrophage dependent phagocytosis (van rozendaal et al. 2000) . similarly, sp-a is able to bind to c. albicans and interfere with attachment to am, inhibiting phagocytosis (rosseau et al. 1997) . sp-a is also able to dampen the pro-inflammatory response elicited by c. albicans by human am and monocytes, which may be important in regulating excessive inflammation in the lung during candida infection (rosseau et al. 1999) . in saccharomyces cerevisiae, sp-d is observed to bind to its surface, but not sp-a, whilst the fungal ligand for sp-d is yeast β-(1-6)-glucan (allen et al. 2001a, b) . the opportunistic fungus, pneumocystis is able to infect a number of mammals with each species of the fungus displaying strict host specificity. for example, p. carinii and p. wakefieldiae infect rats, p. murina infects mice, p. oryctolagi infects rabbits, and p. jirovecii infects humans. sp-a and sp-d are able to recognise and bind pneumocystis species via the major surface glycoprotein (msg; also known as gpa) of the fungus mccormack et al. 1997a, b) . msg contains an n-linked carbohydrate chain made up of glucose, mannose, and n-acetylglucosamine and is involved in attachment of the fungus to alveolar epithelium in pneumocystis pneumonia (zimmerman et al. 1992; vuk-pavlovic et al. 2001) . cruciform dodecamers and other large oligomers of sp-d have a higher affinity of binding to p. carinii than do smaller oligomeric versions of sp-d (vuk-pavlovic et al. 2001) . sp-d is also able to recognise pneumocystis cysts via surface β-glucans (vuk-pavlovic et al. 2001 ). however, sp-d binding does not appear to increase the phagocytosis of the fungus (mccormack et al. 1997a, b; vuk-pavlovic et al. 2001) . despite this, sp-d does aggregate p. carinii in large complexes that may restrict phagocytosis by macrophages and may allow for persistence of the fungus within the host lungs (vuk-pavlovic et al. 2001) . pneumocystis pneumonia does alter the expression of sp-a in the lungs (atochina et al. 2001) , with a threefold increase in the levels of sp-a and sp-d (phelps et al. 1996; aliouat et al. 1998; qu et al. 2001 ), but decreases total phospholipid content (atochina et al. 2001) . human sp-a enhances attachment of p. carinii to rat am in vitro (williams et al. 1996) . sp-a also reduces phagocytosis of p. carinii in human am in vitro (koziel et al. 1998) . these data suggest that increased levels of sp-a during pneumocystis pneumonia (phelps et al. 1996 ) may contribute to the pathogenesis through binding to the fungus and interfering with its am recognition (koziel et al. 1998 ). immunosuppressed sp-a −/− mice also have increased susceptibility to p. carinii infection (linke et al. 2001) , whilst removal of immunosuppression resulted in efficient clearance of the infection , showing that sp-a does not enhance p. carinii clearance, but does modulate the host immune response during the resolution of infection. sp-d modulates interaction of p. carinii with am and also aggregates p. carinii, impairing phagocytosis by am (yong et al. 2003) . in sp-d −/− mice, there was delayed clearance of p. carinii infection, increased inflammation and altered nitric oxide response (atochina et al. 2004) . similarly, in immunosuppressed mice, sp-d was found to enhance p. carinii infection (vuk-pavlovic et al. 2006 ). mbl has been reported to interact with various primary and opportunistic fungal pathogens. low serum levels of mbl have been linked to increased likelihood of fungal disease (mullighan et al. 2002; granell et al. 2006) . mbl is able to bind to a. fumigatus (neth et al. 2000) , b. dermatitidis (koneti et al. 2008 ), c. albicans (kitz et al. 1992 neth et al. 2000; ip and lau et al. 2004; van asbeck et al. 2008) , candida parapsilosis (van asbeck et al. 2008) , and c. neoformans (chaka et al. 1997; van asbeck et al. 2008 ). the ligands for mbl binding to c. albicans and c. neoformans are mannan and mannoprotein, respectively (chaka et al. 1997; ip and lau, 2004) , whilst 1,3-β-glucan and mannose are the mbl ligands on b. dermatitidis and a. fumigatus, respectively (neth et al. 2000; koneti et al. 2008) . mbl is able to bind a. fumigatus conidia showing aggregation, enhancing phagocytosis, and complement deposition ). however, mbl binding of conidia did not always result in the killing of a. fumigatus by phagocytes (madan et al. 2005a, b; kaur et al. 2007 ). moreover, mbl may be less important in this context, since it is mainly a serum protein and may not be in significant levels in the lung. nevertheless, genetic polymorphisms in the mbl gene have been shown to be associated with severe aspergillosis (crosdale et al. 2001; vaid et al. 2007) . similarly, mbl deficiency is a risk factor for aspergillosis in immunocompromised patients, cancer patients and transplant recipients. in the mouse model of infection, mbl deficiency does not necessarily affect the survival of mice infected with a. fumigatus conidia, due to redundancy since mice having two copies of the mbl gene (mbl1 and mbl2), encoding mbl-a and mbl-c proteins in mouse serum (hogaboam et al. 2004) . however, treatment with recombinant mbl does enhance survival in ipa mice ). thus, the role of mbl in a. fumigatus infection may also depend on the route of infection and the level of immunosuppression of the host. mbl interaction with b. dermatitidis has only been studied in the mouse system. both mbl mouse proteins (mbl-a and mbl-c) bind to b. dermatitidis yeast cells (koneti et al. 2008) . inhibition of macrophage response to b. dermatitidis is also mediated by mbl, binding to 1,3-β-glucan ligand on b. dermatitidis, and thus inhibiting 1,3-β-glucan interaction with dectin-1 receptor on macrophages and also decreasing tnf-α production kimberg and brown 2008) . moreover, macrophage production of g-csf, ifn-γ, mcp-1, and rantes were significantly inhibited by mbl in response to b. dermatitidis, but not il-6 (brummer et al. 2007) . mbl can bind to c. albicans yeast and pseudohyphae and to c. parapsilosis yeast cells (denton and disalvo 1964; sugar and picard 1988; brummer et al. 2005; van asbeck et al. 2008) . mbl is able to aggregate c. albicans resulting in its growth inhibition and complement deposition of c4b and c3b on its surface via masps (ip and lau 2004; van asbeck et al. 2008) . similar levels of mbl-mediated complement deposition were also observed for c. parapsilosis (van asbeck et al. 2008 ). however, the binding of mbl to c. albicans may inhibit its phagocytosis by macrophages or dendritic cells (zimmerman et al. 1992; schelenz et al. 1995; chaka et al. 1997; vuk-pavlovic et al. 2001; ip and lau 2004; van de wetering et al. 2004a) . mbl seems to inhibit candida-induced macrophage responses in thp-1 cells through tlr-2 and tlr-4, suggesting that c. albicans modifies tlr signalling pathways in the macrophage (wang et al. 2013) . however, in the case of neutrophils, mbl enhances the phagocytosis of both c. albicans and c. parapsilosis yeast cells (van asbeck et al. 2008) . mbl greatly facilitates complement-mediated uptake of c. albicans via cr1 receptor in neutrophils and this results in the stimulation of reactive oxygen species by intracellular dectin-1, which recognises the phagocytosed fungal β-1,3 glucan (li et al. 2012) . the binding of mbl with c. albicans yeast also increases tnf-α production by monocytes in vitro (ghezzi et al. 1998 ) and in vivo (lillegard et al. 2006) . double knockout (mbl-a and mbl-c) mice were found to be more susceptible to systemic infection with c. albicans compared to wild-type mice (held et al. 2008) . vaginal candidiasis is an important mycosis in women. mbl protein is present in vaginal secretions (pellis et al. 2005) ; mbl levels seem to increase in vulvovaginal candidiasis. however, mbl levels were found to be lower in women with recurrent vulvovaginal candidiasis, because of polymorphisms in their mbl gene (babula et al. 2003; liu et al. 2006; giraldo et al. 2007; donders et al. 2008; milanese et al. 2008 ). the precise role of mbl in candidiasis remains to be fully explored. mbl can bind to acapsular c. neoformans yeast cells (chaka et al. 1997) , but this does not cause aggregation (eisen et al. 2008) . however, mbl binding to acapsular c. neoformans did facilitate complement deposition and enhancement of fungal phagocytosis by neutrophils (van asbeck et al. 2008) . furthermore, tnf-α production was induced in peripheral blood mononuclear cells by c. neoformans mannoprotein and this effect was enhanced by mbl (chaka et al. 1997) . it is unknown whether mbl binds to h. capsulatum or p. carinii. it is unlikely that mbl binds to h. capsulatum, since the cell wall contains 1,3-α-glucan (rappleye et al. 2007 ); however, in p. carinii, the cell surface of cyst forms does contain β-1,3-glucan (williams et al. 1996) , which may bind mbl. in coccidioides species, it is also unknown whether mbl interaction occurs, but patients with active coccidioidomycosis have been shown to have low serum mbl levels, compared to healthy individuals previously infected with coccidioides, and that low levels of mbl were associated with polymorphisms in their mbl gene (corredor et al. 1999 ). very few studies have investigated the interaction of these minor collectins with fungal species. cl-k1 can bind c. albicans (selman and hansen 2012) and cellular extracts (mannan) of s. cerevisiae (keshi et al. 2006; selman and hansen 2012) . cl-p1 has also been reported to bind to s. cerevisiae and mediate phagocytosis of yeast-derived zymosan, suggesting that cl-p1 mediates phagocytosis for fungi in the vascular endothelium jang et al. 2009 ). interestingly, cp-p1 also partially binds to a. fumigatus, via its crd, and in association with properdin, can activate the complement alternative pathway, resulting in c3b deposition and formation of the membrane attack complex (ma et al. 2015) . this shows a novel mechanism of triggering the alternative pathway of complement (ma et al. 2015) . there are no reports of cl-l1 interaction with fungi. there are also limited reports of the binding of bovine-unique collectins to fungi. cl-43 is able to bind to acapsular c. neoformans in vitro in a ca 2+ -dependent manner (schelenz et al. 1995) , and immobilised yeast mannan . conglutinin is able to bind to glycoproteins and polysaccharides derived from s. cerevisiae (n-acetylglucosamine, mannose, mannan) (loveless et al. 1989; lim and holmskov 1996) . an area of collectin that is yet to be fully explored is the interaction of collectins with protozoal and helminth pathogens, which are responsible for some of the most important global infections. there are limited studies and these are mostly based on genetic polymorphisms in collectin genes that are associated with predisposition or severity of these diseases. there is a limited number of functional studies on the role of collectins in protozoal and helminth infections. increases in levels of sp-d were observed in serum, renal and cerebral tissues in mice experimentally infected with plasmodium berghei, compared to control mice (cahayani et al. 2016) . low mbl serum levels and genetic polymorphisms in the mbl gene have been associated with more severe malaria, particularly in children (luty et al. 1998; holmberg et al. 2008) . mbl can bind to p. falciparum protein extracts, but it does not appear to inhibit the parasite directly (klabunde et al. 2002) . mbl does not opsonise p. falciparum, but it can bind to p. falciparum-infected erythrocytes, recognising the 78-kda glucose-regulated stress glycoprotein of the parasite . mbl binding to p. falciparum merozoite adhesins have also been reported, having the ability to activate complement (korir et al. 2014) . the complement lectin pathway can be activated by trypanosoma and leishmania (cestari et al. 2013) . mbl binds to glycosylated antigens on trypanosoma cruzi, on the surface of metacyclic trypomastigotes, resulting in complement activation (cestari idos et al. 2009 ). in a mouse model of t. cruzi infection, mbl influences host resistance and pathology (rothfuchs et al. 2012) . in some strains of t. cruzi, mbl mediates resistance to complement lysis of the parasite and enhances invasion of host cells (evans-osses et al. 2014) . mbl also binds to major cell surface glycoconjugates (lipophosphoglycans) on leishmania parasites, triggering lectin pathway activation and promastigote lysis (green et al. 1994; ambrosio and de messias-reason 2005) . certain genotypes of the mbl2 gene were also predictive for the risk for developing visceral leishmaniasis and other clinical complications in infections with leishmania chagasi (alonso et al. 2007) . similarly, there was a strong correlation found between serum levels of mbl and the probability of developing visceral leishmaniasis (santos et al. 2001 ). monocytes challenged with mbl-opsonised l. chagasi promastigotes secreted higher levels of tnf-α and il-6 than controls, suggesting that mbl may play an important role in pathogenesis (santos et al. 2001) . in helminth infections, mbl binds to the surface glycoproteins of schistosoma mansoni cercariae and adult worms and is able activate the lectin pathway (klabunde et al. 2000) . curiously, no differences in serum mbl levels were observed between patients infected with schistosoma and in healthy controls (klabunde et al. 2000) . another study has shown that high mbl serum levels are associated with protection in schistosomiasis (antony et al. 2013) . interestingly, high levels of mbl and cl-k1 were inversely correlated with urogenital infections with s. haematobium (antony et al. 2015b) . although cl-k1 has not been shown to bind directly to the parasitic worm, it was observed to be a risk factor for urinary schistosomiasis (antony et al. 2015a). furthermore, concomitantly elevated il-6 levels were also observed in urinary schistosomiasis cases compared to controls that correlated with mbl levels (antony et al. 2015b). similar findings linking il-6 and mbl have also been described in patients with visceral leishmaniasis (santos et al. 2001; antony et al. 2015b) . sp-d has also been shown to bind to fucosylated glycoconjugates (α-1-3 linked fucose) on the surface of s. mansoni larval stages, although the significance of this interaction remains unclear (van de wetering et al. 2004b, c) . however, a recent study has suggested that sp-d is essential for protection against helminth infection, using the experimental model nematode nippostrongylus brasiliensis (thawer et al. 2016) . n. brasiliensis infection of sp-d −/− knockout mice resulted in severe susceptibility to parasitic disease, whilst treatment with rfhsp-d enhanced parasite clearance and anti-parasitic immune responses (thawer et al. 2016) . sp-d was determined to bind to n. brasiliensis larvae via its crd, and to enhance their killing by am (thawer et al. 2016 ). a considerable number of in vitro and in vivo studies have focused on the immunomodulatory functions of collectins and their contribution to the host defense system. through activation of complement, and production of pro-inflammatory cytokines, mbl makes a major impact on the generation and regulation of the immune-mediated inflammatory response. allergen-mediated activation of the complement lectin pathway has been demonstrated (varga et al. 2003) . allergen extracts (parietaria (pa) and house dust (hd) mite) were shown to bind purified mbl, and trigger the lectin complement pathway. differences in plasma mbl levels may affect the degree of complement activation in different individuals, thus, susceptibility to allergic diseases. significantly elevated serum mbl levels were observed in pediatric mild-asthma patients, suggesting the possible role of mbl in the pathogenesis of asthma by contributing to airway inflammation, or increasing the risk of asthma development (uguz et al. 2005) . enhanced levels of serum mbl also correlate with an increased peripheral blood eosinophils in these individuals. it is also suggested that oxidative stress increases the mbl synthesis, and triggers complement activity. mbl can initiate complement activation following oxidative stress in asthma (collard et al. 2000; nadeem et al. 2003; uguz et al. 2005) , and aggravate inflammation. significantly increased mbl levels and mbl pathway was also detected in patients with bronchial asthma, rhinitis and allergic bronchopulmonary aspergillosis (abpa) (kaur et al. 2005) . a higher level of plasma mbl is likely to contribute to activation of lectin pathway, and an increased severity, including enhanced blood eosinophil counts. in addition, production of mbl in the liver is suggested to increase by up to three fold in response to environmental stimuli. therefore, higher levels of plasma mbl in allergic patients, compared to the non-allergic patients, may result from elevated hepatic synthesis caused by allergen exposure. furthermore, the circulating level of mouse mbl-a was also measured in aspergillus fumigatus allergen-sensitised and non-sensitised mice. increased level of mmbl-a was observed following allergic sensitisation, suggesting that challenging these mice with allergen may contribute to a higher level of mbl in sensitised mice as well as allergic patients (kaur et al. 2005) . earlier in vivo studies using mouse mbls have reported the likely role of mbl-a as a mediator of inflammation (santos et al. 2001; takahashi et al. 2002) . moreover, a substantial decline in the airway hyperresposiveness to a. fumigatus conidia was seen in mbl-a-deficient mice (mbl-a −/− ) when compared to mbl-a +/+ control mice, which suggest the possible role of mbl-a and its ability to trigger progression of airway hyper-responsiveness (hogaboam et al. 2004) . since levels of plasma mbl are genetically determined, it is of interest to study the genetic polymorphisms in mbl in relation to allergic susceptibility. in order to address the correlation between polymorphisms in the mbl gene and the progression of atopic diseases, nagy et al. found a contribution of variant mbl alleles to the susceptibility to acute or chronic chlamydia pneumoniae infection in asthmatic children . another study that focused on the genetic association of mbl related single nucleotide polymorphisms (snps) with allergic patients , reported the identification of g1011a, an intronic snp found in the mbl gene, and presence of 1011a allele of snp g1011a to be associated with an enhanced level of plasma mbl., snp g1011a has also been suggested to play a role in regulating mbl expression. additional polymorphisms were found at positions 550 (h/l variants) and 221 (x/y variants) in the promoter region of the mbl gene, which associated with high mbl levels in the plasma. 1011a allele was also associated with bronchial asthmatic patients with allergic rhinitis and abpa, which positively correlated with allergic markers, including high peripheral blood eosinophil counts, and reduced levels of forced expiratory volume at timed interval of 1 s (fev1) in these patients. however, no structural snps have been observed within the mbl gene in these allergic patients. as carbohydrate recognition immune molecules, both sp-a and sp-d have been shown to interact with gp55 and gp45 of a. fumigatus in a calcium and carbohydrate specific dependent manner (madan et al. 1997b) . both these collagenous molecules inhibit specific ige binding to these glycoproteins, and block allergen triggered histamine release from human basophils isolated from derp-and a. fumigatus-sensitised patients (madan et al. 1997a, b) . dodecameric forms of human sp-d mediate binding, aggregation, and phagocytosis of starch granules, containing grass pollen allergens from dactylis glomerata and phleum pratense via alveolar macrophages (erpenbeck et al. 2005) . sp-d can suppress proliferation of pbmcs isolated from children with derp-sensitive asthma (wang et al. 1998) , and suppress secretions of il-2 by pbmcs (borron et al. 1998) . suppressive effects of sp-a on the production and release of il-8 by eosinophils were also reported, which is stimulated by ionomycin in a concentration-dependent manner (cheng et al. 1998 ). since ige cross-linking, release of histamine and pmbcs proliferation are crucial immunological factors contributing to the development of asthmatic symptoms, both sp-a and sp-d are crucial immune modulators in resisting allergenic challenge, as well as suppressing substantial hypersensitivity reactions in the lungs (kishore et al. 2002) . intranasal administration of sp-d or rfhsp-d caused reduced levels of peripheral and pulmonary eosinophilia, and the effect persisted up to 16 days in the abpa mice. these observations therefore indicate the potential of sp-d as a therapeutic agent (kishore et al. 2002; madan et al.2001a; 2005a, b) . in addition, protective role of rhfsp-d has also reported in murine model of derp allergens-induced pulmonary hypersensitivity (singh et al. 2003) . shifting of th2 to a th1 following sp-d treatments appeared to be crucial to the protective mechanism, since, ifn-γ gamma is suggested to inhibit differentiation of th2 in response to il-4 (elser et al. 2002) . additionally, production of nitric oxide was significantly inhibited when derp mice derived alveolar macrophages are pre-incubated with rfhsp-d, and resulted in low levels of tnf-α in the rfhsp-d treated derp mice (liu et al. 2005) . culturing alveolar macrophages with allergen and sp-d has induced an increased production of il-10, il-12, and ifn-γ, indicating a positive correlation between macrophages and sp-d triggered inhibition of airway inflammation and airway hyper-responsiveness (ahr) (takeda et al. 2003) . a study by madan et al. has focused on the susceptibility of sp-a −/− and sp-d −/− mice to challenge with a. fumigatus allergen compared to wild-type mice (madan et al. 2005a, b) . intrinsic hypereosinophilia and seven fold increase in il-5 and il-13 levels were seen in both sp-a −/− and sp-d −/− mice. however, lower levels of ifn-γ to il-4 ratio in the lungs were observed, suggesting the possible th2 basis of immune response. treating these mice with exogenous intranasal sp-a and sp-d resulted in reversal of th2 polarisation. sp-d −/− mice was reported to be more susceptible to a. fumigatus allergen-induced pulmonary hypersensitivity when compared to wildtype mice. however, resistant to sensitisation was seen with sp-a −/− mice. intranasal administration of sp-d or rfhsp-d led to rescue of the sensitised sp-d −/− mice, while sp-a −/− mice demonstrated an enhanced levels of il-5 and il-13, causing greater pulmonary eosinophilia. genetic polymorphisms in the collagen region of sp-a2 (sp-a2 g1649c and sp-a2 a1660g) may also increase susceptibility to allergic bronchopulmonary aspergillosis (abpa) (saxena et al. 2003 ). the collectins are structurally related to the complement protein c1q (having a collagenous region and similar overall tertiary structure. a common receptor for sp-a, mbl and c1q was described in 1990 (malhotra et al. 1990) (fig. 4.5) , as collagen region binding cc1qr. this was subsequently identified as calreticulin (~56kda). two other candidate receptors were subsequently proposed: fig. 4 .5 collectin receptors on immune cells. collectins have been shown to bind a number of receptors and putative receptors, which lead to immunomodulatory responses. binding of collectins to toll-like receptor 2 (tlr-2), tlr-4, sp-a receptor 210 (sp-r210), cd91-calreticulin, and signal inhibitory regulatory protein-α (sirp-α) alters production of pro-inflammatory mediators. for example, sp-a and sp-d binds to sirp-α via their collagenous tails, and stimulates pro-inflammatory chemokine production via calreticulin/cd91 interaction. furthermore, bacterium bound collectins induces the conformational changes of calreticulin/cd91 interaction, which then activates p38-mitogen-activated protein kinase (mapk) signalling pathway, leading to transcriptional activation of nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells), and expression of pro-inflammatory cykines, including tumour necrosis factor alpha (tnf-α). sirpα is abundantly expressed in macrophages, and ligation of sirp-α by lung collectins are crucial in preventing damage to the airways caused by the production of pro-inflammatory responses. thereby, phosphorylated cytoplasmic region of sirp-α recruits shp-1 (src homology region 2 domain-containing phosphatase-1), which in turn dephosphorylates protein substrates involved in mediating physiological effects. thus, the interaction between sirp-α and shp-1 negatively regulates p38-map kinase signalling, and stimulates nf-κb activity, and cells become resistant to tnf-mediated effects, such as apoptosis (1) c1qrp (cd93) (c1q receptor associated with phagocytosis stimulated by c1q, mbl or sp-a): but this has subsequently been shown not to bind any of these ligands. it may be an adhesion receptor (mcgreal et al. 2002; norsworthy et al. 2004 ). (2) cr1, the complement c3b receptor, does interact with c1q and mbl, but functional aspects are not yet widely explored (jacquet et al. 2018 ). (3) calreticulin remains the main candidate as a receptor/adapter involved in phagocytosis mediated by c1q and collectins (ogden et al. 2001; vandivier et al. 2002) . calreticulin bound to the cell surface cd91 mediates uptake of apoptotic cells to which c1q, mbl, sp-a and sp-d are bound. it also mediates uptake of microorganisms targeted by the collectins. sp-a and sp-d can interact with phagocytic receptors and are able to influence receptor-mediated uptake of bacteria (lawson and reid 2000) . sp-a enhances the phagocytosis of s. aureus by monocytes but does not induce intracellular killing or the production of reactive oxygen intermediates (roi) (geertsma et al. 1994) . sp-a is also able to enhance the uptake of m. tuberculosis and m. avium by macrophages via the increased expression of mannose receptor (gaynor et al. 1995; kudo et al. 2004) , whilst sp-a enhances the scavenger receptor a (sr-a)-mediated uptake of streptococcus pneumoniae by am . sp-a is also reported to bind to a 210-kda sp-a receptor (spr210) in u937 macrophages and rat am and mediate uptake of mycobacterium bovis bacillus calmette-guérin (bcg) via this receptor (chroneos et al. 1996; weikert et al. 1997) ; in rat macrophages, this led to enhanced mycobacterial killing and an increase in the production of inflammatory mediators, tnf-α and nitric oxide (weikert et al. 2000) . sp-a and sp-d can also bind to the major lps receptor, cd14 that is present on alveolar macrophages. sp-a binds to the peptide portion of cd14, whilst sp-d interacts with the glycan of the receptor (sano et al. 2000) . sp-a modulates lps-induced cellular responses by direct interaction with cd14 (sano et al. 1999) , serving as an important mechanism for the recognition and clearance of this endotoxin. as noted above, sp-a and sp-d can interact with the versatile protein calreticulin (malhotra et al. 1990 ). when sp-a and sp-d bind to surface target ligands such as lps or apoptotic cells, multiple collagen regions are presented on the surface, which interact with the calreticulin -cd91 receptor complex (gardai et al. 2003) . this can then lead to the promotion of phagocytosis and initiation of cell signalling pathways leading to the production of pro-inflammatory cytokines and priming of adaptive immunity (ogden et al. 2001; vandivier et al. 2002; gardai et al. 2003) . in contrast, sp-a and sp-d can also suppress inflammatory responses by binding to signal regulating protein α (sirp-α) on macrophages and epithelia via their crd region, and this leads to a signalling pathway that blocks pro-inflammatory mediator production ( fig. 4 .5) (table 4 .2) (gardai et al. 2003) . therefore, the orientation of binding of sp-a and sp-d (collagen or lectin (crd)) domains to host receptors calreticulin-cd91 or sirp-α, respectively, have opposing effects and illustrate a dual function of these collectins, which could be, (1) protection of the naïve lung via maintenance of immune homeostasis and (2) protection via the triggering of inflammation to clear pathogens, allergens, necrotic and apoptotic cells. another receptor, gp-340 has also been found to bind to sp-d and sp-a (holmskov et al. 1997a; tino and wright 1999) , but any microbial interaction has been described for influenza virus (hartshorn et al. 2006a, b) and not bacteria, whilst binding of sp-a to gp-340 stimulates macrophage chemotaxis (tino and wright 1999) . calreticulin (cc1qr) is a collectin binding protein of 56 kda, and is known to bind c1q, mbl, sp-a, conglutinin and cl-43 (fig. 4 .5) (table 4 .2) (malhotra et al. 1993 ). this interaction is independent of calcium ions, and ionic in nature. it is mediated by the collagen domain 'c' of the collectins (cc1qr). the c1q binding site has regulation of phospholipid secretion and cytokine production, phagocytosis, and inhibition of t-cell proliferation been mapped to the s-domain of calreticulin (stuart et al. 1996) . conglutinin and cl-43 also bind c1qr and calreticulin (dec and wernicki 2006) . sp-a receptor 210 (sp-r210) is a 210 kda oligomeric molecule that earlier has been purified from the macrophage cell line u937 by affinity chromatography (fig. 4 .5 (table 4 .2) (chroneos et al. 1996) . sp-r210 is another candidate receptor for sp-a, and also found in type ii cells and alveolar macrophages. the direct interaction between sp-r210 and sp-a occurs through the collagen region of sp-a. antibodies raised against sp-r210 inhibit sp-a binding to alveolar type ii cells and alveolar macrophages, thus, prevent sp-a-mediated uptake of mycobacterium bovis, and block sp-a-bacillus calmette-guerin complexes to phagocytes. furthermore, sp-r210 inhibited sp-a-mediated phospholipid secretion by alveolar type ii cells (weikert et al. 1997 ). additionally, a study by yang et al. reported sp-r210 as a cell surface myosin 18a (yang et al. 2005) , and expressed in multiple isoforms. gp340 is a 340 kda sp-d binding glycoprotein purified from lung bronchoalvelar lavage of alveolar proteinosis patients , and belongs to the scavenger receptor superfamily, consisting of multiple scavenger receptor type b domains (holmskov et al. 1999) . furthermore, gp340 has been shown to be identical to salivary agglutinin, and its binding interaction with streptococcus mutans and helicobacter pylori has been reported (ligtenberg et al. 2001) . the direct binding of sp-d to gp340 occurs in a calcium dependent manner, and is inhibited by edta. the interaction is not affected by the presence of maltose, suggesting that the binding is a protein-protein interaction via the crd region of sp-d rather than a lectin-carbohydrate interaction. similar binding pattern was observed between gp340 and rfhsp-d, composed of trimeric neck region and crd . gp340 exists in a soluble form, and acts as an adaptor for sp-a and sp-d, but how gp340 is anchored in the membrane and the mechanism of binding to cell surface has not been elucidated fully yet (table 4 .2). sp-d can bind to human neutrophil defensins (hnps) via its neck and crd region (hartshorn et al. 2006a, b) . the interaction between sp-d and hnps can trigger anti-viral activity in the bal fluid. hd6, human β-defensins, and human neutrophil peptide (hnp)-4 bind sp-d with weaker affinity, while hnp-1-3 bind sp-d with high affinity and trigger inhibition of sp-d mediated anti-viral activity (doss et al. 2009 ). additionally, sp-d binds human decorin from amniotic fluid in a calcium dependent manner via sulphated n-acetyl galactosamine moiety of decorin (nadesalingam et al. 2003) . rfhsp-d interacts with dermatan sulphate moiety of decorin, and core protein of decorin interacts with sp-d via the collagen-like region. the interaction between lung collectins (sp-a and sp-d) and native as well as recombinant cd14 has been reported (table 4 .2) (sano et al. 1999) . cd14 is a soluble receptor for lps, and the neck domain of sp-d has been shown to bind the leucine-rich peptide portion of cd14, whilst the carbohydrate moiety of cd14 interacts with the lectin domain of sp-d (sano et al. 1999) . thus, both these surfactant proteins appear to modulate the cellular response to smooth and rough lps by interaction with cd14. (sano et al. 1999) . furthermore, the association of sp-a and sp-d with toll-like receptors (tlr), and or the tlr-associated molecule, could be one of the mechanisms by which they function as modulators of inflammation an inflammatory mediators (sano et al. 1999) . studies have reported direct involvement of sp-a in tlr2 signalling, and inhibition of downstream gene activation (murakami et al. 2002) . sp-a interacts directly with tlr4 and myeloid differentiation factor 2 (md-2), which are known critical signalling receptors for lps (billod et al. 2016) . thus, binding of sp-a with extracellular domain of tlr4 and md-2 was revealed in a calcium-dependent manner, involving the crd region. additionally, sp-a has attenuated cell surface binding of smooth lps, and induced nf-κb activation in cells expressing tlr4 and md-2 (murakami et al. 2002) . sp-d does not have significant effect on tlr4 expression, but it down-regulates the tlr4-mediated signalling against lps (henning et al. 2008 ). thus, sp-a's ability to dampen tlr2 and tlr4 signalling is associated with decrease in the phosphorylation of ikappabalpha, a key regulator of nf-κb activity, and nuclear translocation of p65, resulting in reduced tnf-α secretion in response to tlr ligands. furthermore, the same study also reported diminished phosphorylation of akt, an essential regulator of nf-κb and potentially mapks. therefore, there is a critical role for sp-a in modulating lung inflammatory reactions by regulating macrophage-mediated tlr4 activity. as noted above, calreticulin was identified as a common receptor for c1q, mbl and sp-a (malhotra et al. 1990) . calreticulin is mainly an intracellular protein but it is present on cell surfaces bound to cd91 and, thus, acts as an adaptor or co-receptor while binding to collagenous region of these collectins (fig. 4 .5) ( table 4 .2) (ogden et al. 2001; vandivier et al. 2002) . uptake of apoptotic cells by phagocytes mediated by c1q or mbl binding to calreticulin-cd91 complex was revealed (vandivier et al. 2002) . hla class i heavy chain (arosa et al. 1999 ), or cd59 have been shown to act as a calreticulin-binding proteins on cells which do not express cd91, allowing c1q or mbl coated particles to adhere to the cells. although a number of collectin receptors have been identified, there is still a need to fully elucidate how collectins stimulate phagocytes, and mediate phagocytosis, as well as other signalling transduction pathways. more studies on the structural aspects of receptors are needed, especially how these receptors are anchored in the membrane of immune and non-immune cells and, which co-receptors and signalling pathways are involved. in conclusion, collectins appear to play important roles in controlling lung allergy, inflammation and hypersensitivity, in addition to dealing with a wide variety of pathogens at pulmonary and extra-pulmonary sites. they act against pulmonary allergens through their ability to resist allergen challenge by interfering with allergen triggered ige interaction, degranulation of mast/basophils, cellular infiltration, and polarisation of helper th response. their roles have been also implicated in altering profiles of pro-inflammatory cytokines and chemokines as a result of infection, and allergen challenge. further research is needed to characterise specific collectin receptors that are crucial for collectin functions other than phagocytosis. drickamer k, dordal ms, reynolds l (1986) mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. complete primary structures and homology with pulmonary surfactant apoprotein. j biol chem 261 (15) entry inhibition and modulation of pro-inflammatory immune response against influenza a virus by a recombinant truncated surfactant protein d surfactant changes during experimental pneumocystosis are related to pneumocystis development polysaccharide recognition by surfactant protein d: novel 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donor source and sialylation complement activation upon binding of mannan-binding protein to hiv envelope glycoproteins pulmonary collectins modulate strain-specific influenza a virus infection and host responses increased susceptibility of complement factor b/c2 double knockout mice and mannan-binding lectin knockout mice to systemic infection with candida albicans pulmonary surfactant protein a regulates tlr expression and activity in human macrophages heteromeric complexes of native collectin kidney 1 and collectin liver 1 are found in the circulation with masps and activate the complement system expression sites of the collectin sp-d suggest its importance in first line host defence: power of combining in situ hybridisation, rt-pcr and immunohistochemistry a recombinant trimeric surfactant protein d carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo surfactant protein a mediates mycoplasmacidal activity of alveolar macrophages the number and position of n-linked glycosylation sites in the hemagglutinin determine differential recognition of seasonal and 2009 pandemic h1n1 influenza virus by porcine surfactant protein d mannose-binding lectin deficiency alters the development of fungal asthma: effects on airway response, inflammation, and cytokine profile no association between mannose-binding lectin alleles and susceptibility to chronic hepatitis b virus infection in german patients mannose-binding lectin variant associated with severe malaria in young african children collectins: collagenous c-type lectins of the innate immune defense system affinity and kinetic analysis of the bovine plasma c-type lectin collectin-43 (cl-43) interacting with mannan isolation and characterization of a new member of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-d binding molecule the plasma levels of conglutinin are heritable in cattle and low levels predispose to infection cloning of gp-340, a putative opsonin receptor for lung surfactant protein d collectins-soluble proteins containing collagenous regions and lectin domains-and their roles in innate immunity surfactant protein a decreases nitric oxide production by macrophages in a tumor necrosis factor-alpha-dependent mechanism major component of ra-reactive factor, a complement-activating bactericidal protein, in mouse serum conglutinin level in dairy cattle: changes associated with disease binding of sugar ligands to ca 2+ -dependent animal lectins. ii. generation of high-affinity galactose binding by site-directed mutagenesis role of mannose-binding lectin in the innate defense against candida albicans: enhancement of complement activation, but lack of opsonic function, in phagocytosis by human dendritic cells mannose-binding lectin enhances toll-like receptors 2 and 6 signaling from the phagosome mannose-binding lectin and innate immunity activation of complement by mannose-binding lectin on isogenic mutants of neisseria meningitidis serogroup b mannose-binding lectin accelerates complement activation and increases serum killing of neisseria meningitidis serogroup c mannose-binding lectin enhances phagocytosis and killing of neisseria meningitidis by human macrophages c1q and mannosebinding lectin interact with cr1 in the same region on ccp24-25 modules scavenger receptor collectin placenta 1 (cl-p1) predominantly mediates zymosan phagocytosis by human vascular endothelial cells mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization pandemic h1n1 influenza a viruses are resistant to the antiviral activities of innate immune proteins of the collectin and pentraxin superfamilies sp-a enhances phagocytosis of klebsiella by interaction with capsular polysaccharides and alveolar macrophages interaction of surfactant protein a with bacterial lipopolysaccharide may affect some biological functions human mannan-binding lectin inhibits the infection of influenza a virus without complement increased surfactant protein d in rat airway goblet and clara cells during ovalbumin-induced allergic airway inflammation plasma mannan-binding lectin levels and activity are increased in allergic patients elevated levels of mannan-binding lectin (mbl) and eosinophilia in patients of bronchial asthma with allergic rhinitis and allergic bronchopulmonary aspergillosis associate with a novel intronic polymorphism in mbl protective role of mannan-binding lectin in a murine model of invasive pulmonary aspergillosis pulmonary collectins enhance phagocytosis of mycobacterium avium through increased activity of mannose receptor the human mannose-binding protein functions as an opsonin the staphylococcal surface-glycopolymer wall teichoic acid (wta) is crucial for complement activation and immunological defense against staphylococcus aureus infection pulmonary surfactant protein a augments the phagocytosis of streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor a the demonstration in human serum of "conglutinogenactivating factor" and its effect on the third component of complement inactivation of staphylococci by alveolar macrophages with preliminary observations on the importance of alveolar lining material bovine conglutinin binds to an oligosaccharide determinant presented by ic3b, but not by c3, c3b or c3c the roles of surfactant proteins a and d in innate immunity effect of lung surfactant collectins on bronchoalveolar macrophage interaction with blastomyces dermatitidis: inhibition of tumor necrosis factor alpha production by surfactant protein d dispensability of surfactant proteins a and d in immune control of mycobacterium tuberculosis infection following aerosol challenge of mice the sars coronavirus spike glycoprotein is selectively recognized by lung surfactant protein d and activates macrophages surfactant protein-a enhances respiratory syncytial virus clearance in vivo surfactant protein d enhances clearance of influenza a virus from the lung in vivo absence of sp-a modulates innate and adaptive defense responses to pulmonary influenza infection effect of mannose-binding protein on binding of cryptococcus neoformans to human phagocytes mbl-mediated opsonophagocytosis of candida albicans by human neutrophils is coupled with intracellular dectin-1-triggered ros production human salivary agglutinin binds to lung surfactant protein-d and is identical with scavenger receptor protein gp-340 recognition of candida albicans by mannan-binding lectin in vitro and in vivo expression of the carbohydrate recognition domain of bovine conglutinin and demonstration of its binding to ic3b and yeast mannan primary structure of bovine collectin-43 (cl-43). comparison with conglutinin and lung surfactant protein-d expression of the carbohydrate recognition domain of lung surfactant protein d and demonstration of its binding to lipopolysaccharides of gram-negative bacteria surfactant protein-d modulates interaction of pneumocystis carinii with alveolar macrophages both human sp-a1 and sp-a2 genes are expressed in small and large intestine mannose-binding lectin contributes to deleterious inflammatory response in pandemic h1n1 and avian h9n2 infection immunosuppressed surfactant protein a-deficient mice have increased susceptibility to pneumocystis carinii infection efficient resolution of pneumocystis murina infection in surfactant protein a-deficient mice following withdrawal of corticosteroidinduced immunosuppression therapeutic effect of surfactant protein d in allergic inflammation of mite-sensitized mice mannose-binding lectin and vulvovaginal candidiasis bovine serum conglutinin is a lectin which binds non-reducing terminal n-acetylglucosamine, mannose and fucose residues collectins and ficolins: sugar pattern recognition molecules of the mammalian innate immune system c-type lectins with a sweet spot for mycobacterium tuberculosis mannose-binding lectin plasma levels and gene polymorphisms in plasmodium falciparum malaria soluble collectin-12 (cl-12) is a pattern recognition molecule initiating complement activation via the alternative pathway binding of pulmonary surfactant proteins a and d to aspergillus fumigatus conidia enhances phagocytosis and killing by human neutrophils and alveolar macrophages lung surfactant proteins a and d can inhibit specific ige binding to the allergens of aspergillus fumigatus and block allergen-induced histamine release from human basophils surfactant proteins a and d protect mice against pulmonary hypersensitivity induced by aspergillus fumigatus antigens and allergens protective role of lung surfactant protein d in a murine model of invasive pulmonary aspergillosis association of polymorphisms in the collagen region of human sp-a1 and sp-a2 genes with pulmonary tuberculosis in indian population susceptibility of mice genetically deficient in the surfactant protein (sp)-a or sp-d gene to pulmonary hypersensitivity induced by antigens and allergens of aspergillus fumigatus susceptibility of mice genetically deficient in the surfactant protein (sp)-a or sp-d gene to pulmonary hypersensitivity induced by antigens and allergens of aspergillus fumigatus susceptibility of mice genetically deficient in sp-a or sp-d gene to invasive pulmonary aspergillosis localization of lung surfactant protein d on mucosal surfaces in human tissues expression and localization of lung surfactant protein a in human tissues human leukocyte c1q receptor binds other soluble proteins with collagen domains interaction of c1q receptor with lung surfactant protein a structure and homology of human c1q receptor (collectin receptor) binding of human collectins (sp-a and mbp) to influenza virus variants of the sftpa1 and sftpa2 genes and susceptibility to tuberculosis in ethiopia detrimental effects of mannose-binding lectin (mbl2) promoter genotype xa/xa on hiv-1 vertical transmission and aids progression association of mannose-binding lectin gene haplotype lxpa and lypb with interferon-resistant hepatitis c virus infection in japanese patients mannose-binding protein in hiv-seropositive patients does not contribute to disease progression or bacterial infections the cys6 intermolecular disulfide bond and the collagen-like region of rat sp-a play critical roles in interactions with alveolar type ii cells and surfactant lipids the carbohydrate recognition domain of surfactant protein a mediates binding to the major surface glycoprotein of pneumocystis carinii deletion mapping of n-terminal domains of surfactant protein a. the n-terminal segment is required for phospholipid aggregation and specific inhibition of surfactant secretion macrophageindependent fungicidal action of the pulmonary collectins human c1qrp is identical with cd93 and the mni-11 antigen but does not bind c1q aggregation and opsonization of type a but not type b hemophilus influenzae by surfactant protein a complement dependent and independent interaction between bovine conglutinin and mycobacterium bovis bcg: implications in bovine tuberculosis surfactant protein d binds to human immunodeficiency virus (hiv) envelope protein gp120 and inhibits hiv replication high-dose mannosebinding lectin therapy for ebola virus infection mbl2 genetic screening in patients with recurrent vaginal infections surfactant proteins a (sp-a) and d (sp-d): levels in human amniotic fluid and localization in the fetal membranes characterization of two mannose-binding protein cdnas from rhesus monkey (macaca mulatta): structure and evolutionary implications deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with pseudomonas aeruginosa mannose-binding lectin gene polymorphisms are associated with major infection following allogeneic hemopoietic stem cell transplantation surfactant protein a inhibits peptidoglycan-induced tumor necrosis factor-alpha secretion in u937 cells and alveolar macrophages by direct interaction with toll-like receptor 2 expression of surfactant protein d in the human gastric mucosa and during helicobacter pylori infection increased oxidative stress and altered levels of antioxidants in asthma identification and characterization of a novel interaction between pulmonary surfactant protein d and decorin collectin surfactant protein d binds antibodies and interlinks innate and adaptive immune systems mannose-binding lectin recognizes peptidoglycan via the n-acetyl glucosamine moiety, and inhibits ligand-induced pro-inflammatory effect and promotes chemokine production by macrophages the development of asthma in children infected with chlamydia pneumoniae is dependent on the modifying effect of mannosebinding lectin mannose-binding lectin engagement with late apoptotic and necrotic cells an insight into the diverse roles of surfactant proteins, sp-a and sp-d in innate and adaptive immunity mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition enhancement of complement activation and opsonophagocytosis by complexes of mannose-binding lectin with mannose-binding lectinassociated serine protease after binding to staphylococcus aureus structural analysis of monosaccharide recognition by rat liver mannose-binding protein ca 2+ -dependent structural changes in c-type mannosebinding proteins the level of the serum opsonin, mannan-binding protein in hiv-1 antibody-positive patients murine cd93 (c1qrp) contributes to the removal of apoptotic cells in vivo but is not required for c1q-mediated enhancement of phagocytosis c1q and mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells molecular cloning of a novel human collectin from liver (cl-l1) the membrane-type collectin cl-p1 is a scavenger receptor on vascular endothelial cells surfactant protein d interacts with pneumocystis carinii and mediates organism adherence to alveolar macrophages surfactant protein d inhibits hiv-1 infection of target cells via interference with gp120-cd4 interaction and modulates proinflammatory cytokine production surfactant protein d reverses the gene signature of transepithelial hiv-1 passage and restricts the viral transfer across the vaginal barrier surfactant protein a suppresses reactive nitrogen intermediates by alveolar macrophages in response to mycobacterium tuberculosis mannose binding lectin and c3 act as recognition molecules for infectious agents in the vagina protective effect of surfactant protein d in pulmonary vaccinia virus infection: implication of a27 viral protein surfactant protein-a levels increase during pneumocystis carinii pneumonia in the rat surfactant protein a binds mycoplasma pneumoniae with high affinity and attenuates its growth by recognition of disaturated phosphatidylglycerols opsonic activities of surfactant proteins a and d in phagocytosis of gram-negative bacteria by alveolar macrophages interactions of human mannose-binding protein with lipoteichoic acids interaction of human mannose-binding protein with mycobacterium avium alteration of surfactant proteins a and d in bronchoalveolar lavage fluid of pneumocystis carinii pneumonia the mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary c-type lectin surfactant protein a histoplasma capsulatum alpha-(1,3)-glucan blocks innate immune recognition by the beta-glucan receptor a serum mannose-binding lectin mediates complement-dependent lysis of influenza virus-infected cells collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice antiviral activity of bovine collectins against rotaviruses the salivary scavenger and agglutinin binds mbl and regulates the lectin pathway of complement in solution and on surfaces complement deficiency states and infection: epidemiology, pathogenesis and consequences of neisserial and other infections in an immune deficiency phagocytosis of viable candida albicans by alveolar macrophages: lack of opsonin function of surfactant protein a surfactant protein a down-regulates proinflammatory cytokine production evoked by candida albicans in human alveolar macrophages and monocytes mannose-binding lectin regulates host resistance and pathology during experimental infection with trypanosoma cruzi structural characterization of bovine collectin-43 mbl genotype and risk of invasive pneumococcal disease: a casecontrol study collectin cl-p1 utilizes c-reactive protein for complement activation interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 pulmonary surfactant protein a modulates the cellular response to smooth and rough lipopolysaccharides by interaction with cd14 surfactant proteins a and d bind cd14 by different mechanisms mannan-binding lectin enhances susceptibility to visceral leishmaniasis mannose-binding lectin polymorphisms in patients with hepatitis c virus infection molecular characterization of the mouse mannose-binding proteins. the mannose-binding protein a but not c is an acute phase reactant pulmonary collectins protect macrophages against pore-forming activity of legionella pneumophila and suppress its intracellular growth association of polymorphisms in the collagen region of sp-a2 with increased levels of total ige antibodies and eosinophilia in patients with allergic bronchopulmonary aspergillosis binding of host collectins to the pathogenic yeast cryptococcus neoformans: human surfactant protein d acts as an agglutinin for acapsular yeast cells structure and function of collectin liver 1 (cl-l1) and collectin 11 (cl-11, cl-k1) mannose-binding lectin 2 gene polymorphism in recurrent herpes simplex virus 2 infection mannose-binding lectin-deficient mice are susceptible to infection with staphylococcus aureus mannanbinding lectin is involved in the protection against renal ischemia/reperfusion injury by dietary restriction protective effects of a recombinant fragment of human surfactant protein d in a murine model of pulmonary hypersensitivity induced by dust mite allergens therapeutic effects of recombinant forms of full-length and truncated human surfactant protein d in a murine model of invasive pulmonary aspergillosis crystal structure and functional characterization of the complement regulator mannosebinding lectin (mbl)/ficolin-associated protein-1 (map-1) detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (mbl) gene by polymerase chain reaction with sequence-specific primers localisation of the c1q binding site within c1q receptor/calreticulin experimental blastomycosis pneumonia in mice by infection with conidia mycobacterial antigen 85 complex (ag85) as a target for ficolins and mannose-binding lectin the role of the mannose-binding lectin in innate immunity lack of mannose-binding lectin-a enhances survival in a mouse model of acute septic peritonitis surfactant protein d regulates airway function and allergic inflammation through modulation of macrophage function improvements on the purification of mannan-binding lectin and demonstration of its ca( 2+ )-independent association with a c1s-like serine protease inhibition of influenza viral neuraminidase activity by collectins intracellular mannose binding lectin mediates subcellular trafficking of hiv-1 gp120 in neurons surfactant protein-d is essential for immunity to helminth infection a second serine protease associated with mannan-binding lectin that activates complement clinical manifestations of mannan-binding lectin deficiency mutation of gene of mannose-binding protein associated with chronic hepatitis b viral infection glycoprotein-340 binds surfactant protein-a (sp-a) and stimulates alveolar macrophage migration in an sp-a-independent manner the pandemic (h1n1) 2009 influenza virus is resistant to mannose-binding lectin collectin cl-lk is a novel soluble pattern recognition receptor for mycobacterium tuberculosis surfactant protein a without the interruption of gly-x-y repeats loses a kink of oligomeric structure and exhibits impaired phospholipid liposome aggregation ability mannose-binding lectin levels in children with asthma inhibition of human immunodeficiency virus-1 infection by human conglutinin-like protein: in vitro studies association of sp-d, mnl and i-nos genetic variants with pulmonary tuberculosis distinct alleles of mannose-binding lectin (mbl) and surfactant proteins a (sp-a) in patients with chronic cavitary pulmonary aspergillosis and allergic bronchopulmonary aspergillosis human plasma-derived mannose-binding lectin: a phase i safety and pharmacokinetic study mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells characteristics of surfactant protein a and d binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of gram-positive bacteria collectins: players of the innate immune system aggregation of cryptococcus neoformans by surfactant protein d is inhibited by its capsular component glucuronoxylomannan surfactant protein d binding to terminal alpha1-3-linked fucose residues and to schistosoma mansoni porcine pulmonary collectins show distinct interactions with influenza a viruses: role of the n-linked oligosaccharides in the carbohydrate recognition domain binding of mannan-binding protein to various bacterial pathogens of meningitis pulmonary surfactant protein a enhances the host-defense mechanism of rat alveolar macrophages surfactant protein a is opsonin in phagocytosis of herpes simplex virus type 1 by rat alveolar macrophages rat surfactant protein d enhances the production of oxygen radicals by rat alveolar macrophages binding of surfactant protein a (sp-a) to herpes simplex virus type 1-infected cells is mediated by the carbohydrate moiety of sp-a binding of surfactant protein a to the lipid a moiety of bacterial lipopolysaccharides role of pulmonary surfactant protein d in innate defense against candida albicans role of surfactant proteins a, d, and c1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and cd91 as a common collectin receptor complex studies on the mechanisms of allergen-induced activation of the classical and lectin pathways of complement molecular basis of sugar recognition by collectin-k1 and the effects of mutations associated with 3mc syndrome immunocytochemical localization of surfactant protein d (sp-d) in type ii cells, clara cells, and alveolar macrophages of rat lung structural comparison of recombinant pulmonary surfactant protein sp-a derived from two human coding sequences: implications for the chain composition of natural human sp-a carbohydrate recognition domain of surfactant protein d mediates interactions with pneumocystis carinii glycoprotein a surfactant protein d enhances pneumocystis infection in immune-suppressed mice pulmonary surfactant protein a binds to cryptococcus neoformans without promoting phagocytosis a recombinant polypeptide, composed of the alpha-helical neck region and the carbohydrate recognition domain of conglutinin, self-associates to give a functionally intact homotrimer inhibitory effect of pulmonary surfactant proteins a and d on allergen-induced lymphocyte proliferation and histamine release in children with asthma mannan-binding lectin inhibits candida albicans-induced cellular responses in pma-activated thp-1 cells through toll-like receptor 2 and toll-like receptor 4 sp-a enhances uptake of bacillus calmette-guerin by macrophages through a specific sp-a receptor surfactant protein a enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway drickamer k (1991a) physical characterization and crystallization of the carbohydrate-recognition domain of a mannose-binding protein from rat structure of the calciumdependent lectin domain from a rat mannose-binding protein determined by mad phasing surfactant protein a binding to cytomegalovirus proteins enhances virus entry into rat lung cells isolation and characterization of the human pulmonary surfactant apoprotein gene elevated expression of surfactant proteins in newborn rats during adaptation to hyperoxia respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza a virus human surfactant protein a enhances attachment of pneumocystis carinii to rat alveolar macrophages fungal melanin stimulates surfactant protein d-mediated opsonization of and host immune response to aspergillus fumigatus spores surfactant proteins a and d inhibit the growth of gram-negative bacteria by increasing membrane permeability elevated plasma surfactant protein d (sp-d) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific igg antibody in sars patients mannose-binding lectin inhibits the motility of pathogenic salmonella by affecting the driving forces of motility and the chemotactic response identification of the surfactant protein a receptor 210 as the unconventional myosin 18a correlation analysis between single nucleotide polymorphisms of pulmonary surfactant protein a gene and pulmonary tuberculosis in the han population in china surfactant protein dmediated aggregation of pneumocystis carinii impairs phagocytosis by alveolar macrophages ) h progression of liver disease in chronic hepatitis b infection 120-kd surface glycoprotein of pneumocystis carinii is a ligand for surfactant protein a key: cord-028887-eseo7lyh authors: li, chong; culhane, marie r.; cheeran, maxim; galina pantoja, lucina; jansen, micah l.; amodie, deborah; mellencamp, martha a.; torremorell, montserrat title: exploring heterologous prime-boost vaccination approaches to enhance influenza control in pigs date: 2020-07-09 journal: vet res doi: 10.1186/s13567-020-00810-z sha: doc_id: 28887 cord_uid: eseo7lyh influenza a viruses evolve rapidly to escape host immunity. in swine, this viral evolution has resulted in the emergence of multiple h1 and h3 influenza a virus (iav) lineages in the united states (us) pig populations. the heterologous prime-boost vaccination strategy is a promising way to deal with diverse iav infection in multiple animal models. however, whether or not this vaccination strategy is applicable to us swine to impart immunity against infection from north american strains of iav is still unknown. we performed a vaccination-challenge study to evaluate the protective efficacy of using multivalent inactivated vaccine and/or a live attenuated iav vaccine (laiv) in pigs following multiple prime-boost vaccination protocols against a simultaneous h1n1 and h3n2 iav infection. our data show that pigs in the heterologous prime-boost vaccination group had more favorable outcomes consistent with a better response against virus challenge than non-vaccinated pigs. additionally, delivering a multivalent heterologous inactivated vaccine boost to pigs following a single laiv administration was also beneficial. we concluded the heterologous prime boost vaccination strategy may potentiate responses to suboptimal immunogens and holds the potential applicability to control iav in the north american swine industry. however, more studies are needed to validate the application of this vaccination approach under field conditions. influenza a viruses are important zoonotic pathogens and one of the most prevalent causes of respiratory disease. swine influenza a virus (iav) causes respiratory disease in pigs worldwide and is considered a significant player in the porcine respiratory disease complex together with other viruses and bacteria such as porcine reproductive and respiratory syndrome virus (prrsv) and mycoplasma hyopneumoniae (m. hyopneumoniae) [1] . influenza causes important economic losses in the swine industry with estimates ranging between $3 and $10/pig [2] . h1n1, h3n2, and h1n2 are the major subtypes of iav found in pigs around the world and there is significant genetic diversity within those subtypes [3] . influenza a virus is an enveloped rna virus with a genome consisting of 8 negative-sense rna segments. its genetic diversity is driven mostly by two mechanisms: antigenic drift which is the result of mutations in antigenic sites due to the poor proofreading ability of the rna polymerase, and antigenic shift, or reassortment, which is the exchange of gene segments between distinct viruses resulting in new strains with a gene combination distinct from the parental strains. these viral evolution mechanisms are responsible for the emergence of multiple novel distinct h1 and h3 iav lineages in pigs open access *correspondence: torr0033@umn.edu 1 college of veterinary medicine, university of minnesota, st. paul, mn 55108, usa full list of author information is available at the end of the article during the last 20 years [4] . between 2009 and 2016, 74 genome patterns were documented for the h1 subtype from us swine alone [5] . the high diversity of iav makes control of the disease using vaccination difficult. the differences of prevailing lineages between different continents and regions require the iav vaccines for swine to be produced locally and contain distinct strains for each region. currently, vaccination is the primary measure to control iav infection in pigs. the success of vaccination control measures is largely attributed to the ability of the vaccine to stimulate the host immune system and elicit high levels of humoral and cell meditated immune response. the protection imparted by the humoral immune response comes not only through the secretion of anti-hemagglutinin antibodies that neutralize the virus but also from the production of antibodies that stimulate complement system and subsequent antibodydependent cellular cytotoxicity (adcc) which further inhibits virus replication [6] . cellular immunity also plays a vital role in virus elimination by activating cytotoxic t cells and macrophages to lyse infected cells and destroy ingested microbes [7] . even though much progress has been made to improve the efficacy of recombinant subunit, vector and dna vaccines against iav and these have promising results as demonstrated in experimental studies, whole-cell inactivated vaccines (wiv) remain the most commonly used licensed vaccines to control iav in pigs. wiv are widely used in sows and gilts to induce serum antibodies against the viral hemagglutinin and enhance transfer of passive immunity to newborn piglets. more than 60 percent of large breeding herds in the u.s vaccinated gilts against iav before or at entry into the herds by using commercial or autogenous wiv [8] . furthermore, over one-half of large breeding herds vaccinated sows during the last 4 weeks of gestation [8] . in addition, a live attenuated influenza vaccine (laiv) became commercially available in the us in 2018 [9] . this vaccine, a multivalent laiv with an ns1 truncated protein, has been purported to elicit both humoral and cell-mediated immune responses in pigs 1 day of age and older [10] . however, there is less overall information regarding the use of laiv. laiv are usually administrated intranasally to piglets to bypass or minimize the limiting effect of maternal antibodies. due to antigenic drift and shift, vaccines for iav need to be updated on a regular basis to provide sufficient protection. however, updating the vaccine requires significant time to determine the vaccine composition, testing, distribution, and administration [1] . the delayed time, almost 8 months for human vaccines, is enough for new strains to emerge and undergo antigenic drift and shift, which renders the immunization too late to have substantial impact on the next influenza outbreak [11] . in the case of vaccines for animals, this time may be even longer [12] . moreover, there is no systematic worldwide surveillance program for iav in pigs and iav vaccines for swine are updated far less frequently than humans [13] . therefore, it is much harder to develop vaccines that incorporate strains that are appropriately matched with the circulating strains in a farm or region and provide complete protection against iav for pigs. for these reasons, broadening the immune response is key to providing more complete protection and one way to do this is to adopt a heterologous prime-boost vaccination strategy. heterologous prime-boost vaccination refers to the delivery of antigens with overlapping nucleotides or same antigenic inserts expressed by different vectors or delivery systems for primary and boost vaccination [14, 15] . previous research with h5n1 influenza virus in poultry, h1n1 in mice, and h3n2 in ferrets has resulted in the desired broad and long-lasting immune response using the prime-boost approach [16] [17] [18] . the benefit of heterologous prime-boost vaccination has also been evaluated in controlled experiments with pigs and the desired broader protection against h3n2 iav was demonstrated [19] . whether the heterologous primeboost strategy is applicable to us swine infected with north american strains of iav using currently available multivalent iav vaccines for pigs is still unknown. therefore, we evaluated the protective efficacy of heterologous prime-boost vaccination with wiv and laiv vaccines in a swine model against h1 and h3 iav co-infection. the outcomes of this study may be used to develop better vaccination protocols and new control strategies for iav in pigs. this project consisted of two separate studies ( table 1 ). the first study included only pigs vaccinated with commercial (com) or autogenous (aut) wiv and had five different treatment groups: com/com, aut/aut, aut/com, com/aut and no vaccination but challenged (no vac/cha). the second study included pigs vaccinated with the laiv and two treatment groups: laiv/com and laiv/none (none = no boost vaccination). a combined total of ninety, 3-week-old pigs were enrolled in the project. pigs originated from a farm seronegative to iav, prrsv and m. hyopneumoniae and the farm was monitored for these pathogens regularly. all pigs tested negative for antibodies against the iav nucleoprotein (herdchek, idexx elisa) and negative for iav rna in nasal swabs using a matrix gene real-time rt-pcr (rrt-pcr) [20] upon arrival to the university of minnesota bsl-2 animal isolation units. pigs were randomly allocated into 8 treatment groups as depicted in table 1 and figure 1a . a microchip was implanted intramuscularly in the neck of each pig (lifechip ® , destron fearing, south saint paul, mn) to monitor body temperature. sixty of the pigs were vaccinated against influenza using different prime-boost vaccination protocols at 3 and/or 6 weeks of age. the vaccines were administered according to their labels. the com and aut vaccines were administrated intramuscularly (im) with 2 ml per dose, while, the laiv was administrated as a single 1 ml dose intranasally (in) for each pig. both, sixteen control pigs (include no vac/cha and no vac/no cha groups) and fourteen seeder pigs received two administrations of a saline solution intramuscularly at 3 and 6 weeks of age. the seeder pigs were challenged with either an h1 or h3 iav at 8 weeks of age and served as infection sources to the vaccinated pigs and no vac/ cha pigs. each seeder pig was inoculated intratracheally and intranasally with a 2 ml dose of 1 × 10^6 tcid 50 /ml table 1 description of vaccination protocols applied to pigs by treatment groups in the two separate studies. i.m: intramuscular; i.n: intranasal; iav: influenza a virus. a three pigs from the no vac/no cha group were necropsied prior to challenge and the remaining 3 pigs were necropsied at the termination of the study (7 dpc challenge virus (1 ml intratracheally and 1 ml intranasally). when seeder pigs were confirmed iav positive in their nasal secretions by rrt-pcr, two seeder pigs (one h1 seeder and one h3 seeder) were commingled with the pigs in each room. there were five rooms in total for the study 1 with each room containing ten contact pigs (two from each wiv treatment group), and two seeder pigs for a total of 12 pigs per room ( figure 1b ). similarly, the two rooms in the second study had twelve total pigs per room with five pigs from laiv/com, five pigs from laiv/none and two seeder pigs ( figure 1c ). six pigs from no vac/no cha group served as unvaccinated negative controls and were kept in a separate room. three of the no vac/no cha pigs were euthanized at 8 weeks of age (0 days post-contact (dpc)) for histopathological evaluation and the remaining of the 3 pigs were euthanized at 9 weeks of age (7 dpc). all the vaccines used in this study were licensed vaccines. antibiotic-antimycotic (gibco, life technologies, grand island, ny, usa)) and following a common laboratory virus isolation protocol and incubated at 37 °c, 5% co 2 for 1 h [22] . after that, fresh medium was added and the cells were further incubated for at least 72 h until at least 80% cytopathogenic effect was observed. the media containing virus were harvested and centrifuged (1500 rpm, 10 min) and viruses were titrated on mdck cells based on established protocols [23] . the virus titer for h1n1 challenge strain was 1 × 10^6.5 tcid 50 /ml and the titer for h3n2 challenge strain was 1 × 10^7.5 tcid 50 / ml. the harvested viruses were aliquoted and stored at − 80 °c. blood samples were collected on arrival to the animal isolation units, 1 week after boost vaccination and at challenge. the pigs were manually restrained and 2 to 6 ml of blood was collected from the cranial vena cava using 19 to 21 gauge needles and blood collection tubes (bd vacutainer ® sst ™ , franklin lakes, nj, usa). nasal swabs were collected from pigs on arrival, before challenge and at days 2, 4, 5 and 6 after contact challenge with the seeder pigs using swabs (bd bbl ™ culture swabs ™ , sparks, md, usa) inserted 2-3 cm into the pig's nostrils and gently rotated. the nasal swabs were stored refrigerated until processing which took place within 48 h of collection. all pigs were euthanized and necropsied at 7 days postcontact (dpc) with the seeder pigs by an overdose of intravenously injected pentobarbital (fatal-plus, vortech pharmaceuticals ltd, dearborn, mi, usa). the percentage of gross lung lesions for each pig was analyzed and scored by a pathologist blinded to the treatments based on previously published protocols [24, 25] . bronchoalveolar lavage fluid (balf) was collected from each pig using a saline solution as described previously [26] (gibco, life technologies, grand island, ny, usa). an iav matrix gene real-time rt-pcr (rrt-pcr) was used to detect iav shedding in all the nasal swabs and balf samples [27] . iav nucleic acid was extracted from the nasal swabs and balf using the magmax-96 viral isolation kit (applied biosystems, life technologies, carlsbad, ca, usa) based on the methods previously described [28] . the iav rrt-pcr was performed using the agpath-id one-step rt-pcr kit (applied biosystems, life technologies, carlsbad, ca, usa) following the established protocols [20] . a sample was considered positive by rrt-pcr if the cycle threshold (ct) value was 38 or lower and, due to the semiquantitative nature of the rrt-pcr test, the lower the ct value, the higher the amount of viral rna detected in the sample. virus isolation was attempted on all positive nasal swabs and balf samples on mdck cells and titrated by calculating the tcid 50 per ml [23] . a sample was considered positive by virus titration if the virus titer was 1 × 10^1.75 or higher. to evaluate which strain the infected pigs were shedding, the nasal swabs and balf samples with ct value less than or equal to 38 (based on the matrix rt-pcr described above) were selected for next generation sequencing. one step reverse transcription-pcr amplification was performed on extracted rna from selected samples by using superscript iii one-step rt-pcr system with high fidelity platinum taq dna polymerase (invitrogen, life technologies, usa) with degenerate primers (10um mbtuni-12 m and mbtuni-13) [29] . the pcr product was visually verified by gel electrophoresis, the quality and quantity of rt-pcr product was checked by nanodrop 1000 (thermo fisher scientific). the pcr product was then cleaned up by qiagen qiaquick pcr purification kit (qiagen, usa). the sequencing library was prepared by using the nextera dna xt sample preparation kit (illumina, san diego, ca, usa) and quantified by using the quant-it ™ picogreen ™ dsdna assay kit (invitrogen). the barcoded libraries were pooled in equimolar concentrations and sequenced in multiplex for 150 bp paired-end on illumina nextseq mid-output mode (130 m) at the university of minnesota genomics center (umgc). the raw contigs released from umgc underwent quality assessment by fast-qc [30] and then trimmed by trimmomatic [31] to remove the adapter, barcode and low-quality sequences. the trimmed contigs were de novo assembled by shovill [32] and the consensus sequences were annotated by flan [33] which is a web based influenza annotation tool available at ncbi influenza virus resource. blood samples collected prior to challenge were tested using the hemagglutination inhibition (hi) assay according to methods previously described [34] . before the hi assay, the serum was separated from the blood cells and the serum was treated with three parts receptor destroying enzyme (rde; denka seiken, tokyo, japan) for 18 h at 37 °c, followed by heat inactivation at 56 °c for 30 min. after the sera cooled down to room temperature, 20% of turkey red blood cells were added and hemadsorbed for at least 30 min to remove nonspecific hemagglutinin inhibitors and natural serum agglutinins. the hi assay was performed using turkey red blood cells and the challenge strains (a/swine/minnesota/pah-618/2011 h1n1 and a/swine/minnesota/080470/2015 h3n2) as antigens. hi antibody titer was calculated as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. the elispot analysis of ifn-γ secreting cells was performed on peripheral blood mononuclear cells (pbmc) collected 1 week after boost vaccination on pigs and on lymph nodes collected at necropsy. pbmcs were isolated from heparinized whole blood collected from pigs in treatment groups com/com, aut/aut, laiv/com, laiv/none and no vac/no cha prior to challenge [35] . lymph nodes were collected during necropsy from all vaccinated pigs. the lymph nodes were crushed to separate the cells from the tissue layers and passed through a 70-µm mesh nylon membrane (bd falcon 352350). after treating them with ack lysis buffer (thermo fisher scientific, grand island, ny, usa) and washing with hank's balanced salt solution (hbss; bd falcon, franklin lakes, nj, usa), the lymph node cells were ready to proceed for elispot assay. an elispot assay to determine ifn-γ secreting cells specific for h1 and h3 challenge strains was performed as described [36] . briefly, 96-well elispot filter plates (maips4510, millipore corp., ma, usa) were coated overnight with porcine ifn-γ capture antibody (r&d systems, minneapolis, mn, usa). freshly isolated pbmc cells (5 × 10^5 cells per well) or lymph node cells (1 × 10^6 cells per well) were loaded into duplicate wells and the stimulants (heat-inactivated h1 and h3 challenge strains) were also added into corresponding wells. after 18 h incubation (37 °c, 5% co 2 ), the porcine ifn-γ detection antibody (r&d systems, minneapolis, mn, usa) was added to each well and the ifn-γ secretion cells were visualized using the streptavidin-ap and nbt/bcip for membranes (r&d systems, minneapolis, mn, usa) and counted with an elispot plate reader. non-specific spots detected in wells coated with mockinfected rpmi (gibco, life technologies, grand island, ny, usa) medium were subtracted from the counts of influenza-specific ifn-γ secretion cells. statistical analyses were performed by comparing results of treatment groups receiving wiv (study 1) and separate analyses for comparing results from laiv groups (study 2). the data from no vac/no cha pigs was summarized but not statistically analyzed. quantitative rrt-pcr, virus titers from nasal swabs, weight, body temperature and influenza-specific antibody responses were analyzed by a generalized linear mixed model (glmm) approach for repeated measures. virus titers, hi titers, and the elispot reads were log-transformed while percent lung lesions were transformed using arcsine square-roots prior to analysis. using the r software (version 3.4.1), transformed and non-transformed data were analyzed with a model that considered the fixed effects of treatment, day, the interaction of treatmentby-day, the random effects of room and the residual error. day was considered the repeated factor. treatment and treatment-by-day were assessed at the 5% level. if the treatment-by-day interaction was significant then treatment comparisons were assessed for each study day separately. percent lung lesions, elispot counts and balf were analyzed using a glmm approach with a model that considered the fixed effects of treatment and the random effects of room and the residual error. binary data were analyzed with r software (version 3.4.1) by fisher's exact test. comparisons of lsmeans for all continuous variables were performed by the tukey's multiple method at the 5% level of significance. lsmeans from transformed data were back-transformed (geometric means) after analyses. no pigs displayed any clinical signs of respiratory disease such as coughing or nasal discharge from the start of the study to necropsy. the average daily weight gain (adg) from challenge to necropsy for the different treatment groups was measured in grams and is summarized in figure 2a . no statistically significant differences in adg were observed among pigs receiving the wiv combinations (p = 0.3062). the adg for com/com pigs was 342.3 ± 147.8 g, 377.2 ± 76.7 g for aut/aut pigs, 567.7 ± 76.7 g for aut/com pigs, 524.8 ± 75.7 g for com/aut pigs, and 439.1 ± 43.6 g for no vac/cha pigs. also, there was no statistical difference in adg for pigs receiving the live-attenuated vaccines. the adg for laiv/com pigs was 430.9 ± 36.6 g and 293 ± 104.5 g for laiv/none pigs (p = 0.2297). the no vac/no cha pigs had an adg of 1106.3 ± 243.5 g. the mean body temperatures for each treatment group shown as mean (± sem) are summarized in figure 2b . we considered fever present if body temperature was greater than 40 °c with no pigs having fever prior to the challenge. three of 10 com/com pigs, 3/10 aut/ aut pigs, 3/10 aut/com pigs, 5/10 com/aut pigs and 1/10 no vac/cha pigs had fever after challenge. in contrast, none of the pigs receiving the laiv vaccine (laiv/com and laiv/none) and none of the pigs from no vac/no cha had fever during the study. there were no significant differences in body temperatures between any of the groups at any time-point (additional file 2). there were mild gross lung lesions in all challenged pigs in all treatment groups. the range of gross lung lesions for all vaccinated pigs ranged from 0 to 13%, the average gross lung lesions of pigs from each group were less than 5% and no significant differences were detected between treatment groups ( figure 2c ). the nasal mucosa and lungs are major targets for virus replication in the upper and lower respiratory tract of pigs. before commingling with vaccinated pigs, the seeder pigs were confirmed iav positive by rrt-pcr on nasal swabs at day 2 post-challenge (ct value range 21.15 to 34.19) . to determine the presence and amount of virus in the respiratory tract, we collected nasal swabs at 2, 4, 5 and 6 days post-contact with the seeder pigs and balf samples at necropsy from all pigs. the detailed infection kinetics of the individual pigs is shown in additional file 3. no iav rna was detected in any of the no vac/ no cha pigs from either balf samples or nasal swabs. based on the balf samples collected from pigs receiving wiv, the least amount of virus post-contact was detected in the heterologous treatment group aut/ com (table 3) . only a small quantity of iav rna was detected in the balf sample from a single pig in this treatment group (ct value = 34.31). the amount of viral rna detected and the number of iav rrt-pcr positive pigs in homologous treatment group aut/aut was similar to the heterologous treatment group com/aut. the wiv group with the highest amount of viral rna detected was the homologous treatment group, com/ com. nevertheless, com/com pigs still had fewer positive pigs and less virus detected in balf samples compared with the pigs from no vac/cha group. for the pigs receiving laiv, all pigs in laiv/none treatment group were positive by rrt-pcr and only half of the pigs in the laiv/com treatment group were rrt-pcr-positive, with the average ct value for balf samples from laiv/none pigs significantly lower, e.g. more iav viral rna detected, than the laiv/com pigs (p < 0.0001). there was less viral rna detectable by rrt-pcr in the nasal cavities of pigs receiving wiv combinations and, similarly, the number of pigs positive (positive rate, pr), was also lower. for the laiv treatment groups, the laiv/com treatment had less infected pigs and significantly higher ct values in both balf samples and nasal swabs at necropsy (table 3) . we performed next-generation sequencing on all nasal swabs and balf samples that tested positive by rrt-pcr to determine the h1 and/or h3 gene segment in each sample (table 3) . among the balf samples collected from wiv pigs, no h1 or h3 genes were detected in the heterologous aut/com pigs. there were only h3 sequences detected in the balf of 2 out of 10 pigs from the heterologous com/aut group. for the homologous wiv pigs, both ha sequences were detected in a balf sample of 1 out of 10 aut/aut pigs. based on the nasal swabs collected from wiv pigs, the ha sequences detected from heterologous aut/com, com/aut and homologous com/com pigs belonged to the h3 subtype. in contrast, only h1 sequences were detected in nasal swabs from homologous aut/aut pigs. in addition, both h1 and h3 sequences were found in nasal swabs of no vac/cha pigs. among laiv administrated pigs, both h1 and h3 gene sequences were detected in pigs from laiv/none and laiv/com group no matter on nasal swabs or balf samples. furthermore, we observed the co-infection of both challenge strains happened on multiple pigs from no vac/cha and laiv/none group either based on results from nasal swabs or balf samples (additional file 4). to better understand the shedding and transmission of the challenge viruses in the vaccinated pigs' lungs and nasal cavities, we performed virus titration on all the balf samples and nasal swabs collected at 2, 4 and 6 days post-contact with the seeder pigs to determine the extent of virus shedding. among the wiv treatment groups, no virus was isolated from any balf samples from heterologous prime-boost treatment pig groups com/aut and aut/com ( figure 3a ). only one pig in the homologous aut/aut treatment group was shedding a low quantity of virus (1.75 tcid 50 /ml). however, in the homologous com/com, virus was isolated in the balf of 4/10 pigs and at higher virus concentrations than in other inactivated vaccinated groups. for the laiv treatment groups, virus was isolated from balf samples of 8/10 pigs in laiv/none group and only 2 figure 3b ). pigs in no vac/cha group had significantly higher amounts of nasal virus shedding than all the wiv treatment groups on day 2 and day 4 post-contact. for the pigs from laiv groups, the heterologous laiv/ com treatment had significantly lower virus shedding from the nasal cavities at all selected time-points compared to that from pigs in laiv/none group. to quantify the antibody titers against h1 and h3 challenge strains induced by different vaccine primeboost combinations, the hi test was performed on the sera collected from each pig prior to challenge. consistent with the virus shedding results, the heterologous aut/com group had the highest antibody titers against both the h1 and h3 challenge strains (figures 4a and b) . also, pigs in all four wiv groups had significantly higher hi antibody levels against both h1 and h3 challenge strains than pigs from the no vac/ cha group. anti-h1 titers were significantly higher in heterologous aut/com than in the homologous prime-boost groups (e.g. com/com and aut/aut). pigs from aut/com groups had significantly higher antibody levels against the h3 challenge strain than pigs from com/com and com/aut groups. the laiv only group had no serum antibody responses detected against either h1 or h3 challenge strains. in contrast, pigs in the laiv/com treatment group had significantly higher antibody responses against both h1 and h3 challenge strains. we compared the frequency of ifn-γ secreting cells between groups and assessed the antigen-specific cd8 t cell response in pigs. the cells of lymph nodes that were collected from pigs during the necropsy were stimulated with the challenge h1 or h3 viruses and the numbers of virus-specific ifn-γ secreting cells were determined by elispot plate reader (figures 4c and d) . the h1 and h3 specific ifn-γ secreting cells were undetectable in no vac/no cha pigs. for the h1 response in the pigs in the wiv treatment groups, the ifn-γ secreting cell counts in no vac/ cha pigs were significantly higher than homologous prime-boost wiv treatment groups (com/com and aut/aut) and pigs in all four of the wiv treatment groups had similar counts of h1 ifn-γ secreting cells. the h3 ifn-γ secreting cell response was at comparable levels for all pigs in the wiv treatment groups and did not differ significantly from the no vac/ cha group. for pigs in the laiv treatment groups, the com boost vaccination preceded by laiv prime increased the ifn-γ secreting cells against h1 and h3 challenge strains, but it did not differ significantly from pigs in the treatment group receiving a single laiv vaccination. we also tested the frequency of virus-specific ifn-γ secreting cells in pbmc samples collected at 1 week after boost vaccination from pigs in selected groups (com/com, aut/aut, laiv/ com and laiv/none) and obtained similar results between the wiv administration groups (com/com and aut/aut). however, we observed significantly increased number of h1 and h3 specific ifn-γ secreting cells in pigs from laiv/com group than pigs in laiv/none group (additional file 5). the advantage of heterologous prime-boost vaccination in protecting pigs and reducing influenza infections has been illustrated in a previous study [19] . to test whether this vaccination approach is applicable to commercial u.s. pig farms, we mimicked field conditions by using a seeder pig infection model where seeder pigs were infected with either an h1 or h3 virus and commingled with the vaccinated pigs to serve as challenge. the vaccinated pigs received different licensed multivalent vaccine combinations as prime and boost doses. our results suggested that the heterologous aut/com prime/boost vaccine combination resulted in lower numbers of infected pigs than a wiv homologous prime/boost vaccine combination when compared to no vac/cha pigs. among the laiv groups, lower infection levels were also observed in pigs receiving heterologous laiv/com prime/boost vaccination compared to a single administration of laiv. since the ability of vaccination to induce an immune response in pigs is an important indicator for vaccine efficacy, we also examined the humoral and cell-mediated immune responses for all vaccinated pigs receiving the different vaccine administrations. we found the heterologous prime-boost vaccination protocols may have expanded the antibody response to both h1 and h3 challenge strains as demonstrated by the higher hi titers especially in pigs from aut/com and laiv/com treatment groups. however, as expected we did not observe a significantly enhanced cell-meditated immunity in the wiv groups [37] . overall the pigs in the treatment group that received the heterologous aut/com vaccination had more favorable outcomes consistent with a better response against the virus challenge than non-vaccinated pigs and compared to all the other wiv groups. the responses seen in the heterologous laiv/com treatment group were also more favorable than those responses observed compared to the treatment group receiving the single dose of laiv vaccine. compared with traditional vaccination approaches, the goal of a heterologous prime-boost vaccination approach is two-fold in that there is not only an increase in the titer and longevity of the immune responses but also an expansion of the scope of immune responses elicited through humoral and cell-mediated immune responses [15] . as a result, heterologous prime-boost approaches have been applied against a wide range of pathogens and even complex diseases such as malaria or tuberculosis [38, 39] . the heterologous prime-boost vaccination approach was first employed in a macaque model against the simian immunodeficiency virus in the 1990s and displayed one of the most promising protection results in the early human immunodeficiency virus vaccine development effort [40] . similar strategies to prevent influenza infections have been documented [17] . applying heterologous prime-boost influenza regimens are thought to elicit higher levels of anti-hemagglutinin stalk antibodies which typically exhibit much broader and neutralizing activity than antibodies that bind to conventional antigenic sites on the hemagglutinin head [41] . therefore, the heterologous prime-boost strategy may potentiate responses to suboptimal immunogens and may elicit broadly cross-reactive responses that could eliminate the need for additional vaccinations. in our study, the heterologous aut/com and laiv/com groups appeared to have the best immune responses among their comparison groups. although we did not measure antibodies against the stalk part of the ha protein, hi titers were highest for aut/com in wiv groups and laiv/com in laiv groups against both h1 and h3 challenge viruses, which resulted in less virus detected in pigs within these groups. we also quantified the t cell immune response for the vaccinated pigs using elispot to evaluate the antigen-specific cell-mediated immune responses. even though we did not observe a significant difference between different treatment groups, there were increased h1-specific ifn-gamma secreting cells in lymph nodes examined from pigs in the no vac/cha group. this may reflect differences in iav exposure of pigs in the different treatment groups to the seeder pigs or individual differences in the ability of the pigs to respond to nonspecific challenges as exemplified by the higher counts of ifn-γ secretion cells in pigs in the no vac/cha group. it is also plausible that the higher counts are due to the intense reactivity of pigs' natural defenses against severe influenza infection [42] . the amount of virus shedding is one of the key factors used to demonstrate the extent of protection against the virus challenge afforded by vaccination. since the iav can replicate in both the upper and lower respiratory tract and the lung is the target organ that correlates with disease, we performed virus detection and sequencing on nasal swabs and balf samples to evaluate not only the virus shed in nasal secretions and the amount of virus detected in the lower respiratory tract but also assessed lung pathology. diverse infection patterns of each subtype viral population were observed in pigs from homologous and heterologous wiv groups, and in multiple pigs from no vac/cha and laiv/none groups shedding both challenge viruses through the lungs and nasal cavities. in our study, we used a seeder pig model that intended to mimic transmission via nose-to-nose contact instead of direct intranasal or intratracheal inoculation of pigs with high quantities of challenge virus as these direct inoculation methods are not an accurate reflection of what occurs naturally [43, 44] . furthermore, direct intratracheal challenge is a consistent factor in vaccination/challenge studies wherein enhanced respiratory tract pathology is observed [45, 46] . meanwhile, high dose intranasal challenge could cause high virus nasal shedding at the next day after challenge which represents a problem for vaccine evaluation, especially when evaluating t cell-inducing vaccines which usually have poor ability to prevent the virus entry into cells [44] . pigs from the laiv vaccine treatment groups were not housed in the same rooms as pigs in the wiv treatment groups because the laiv vaccine is shed for a short period of time after administration (https ://www.ingel vacpr ovenz a.com/addit ional -data). as a result, direct comparisons between pigs in the wiv treatment groups to pigs in the laiv treatment groups could not be made. all pigs receiving laiv vaccine were confirmed iav rrt-pcr negative on nasal swabs collected prior to challenge and the hemagglutination sequences obtained from the iav positive samples all belonged to the challenge viruses. thus, we can conclude that the virus shed after challenge is the challenge strain rather than the vaccine strain. we found the heterologous laiv/com pigs shed reduced levels of virus in both the upper and lower respiratory tract compared with laiv/none pigs. among the wiv groups, the heterologous aut/com vaccination may be said to offer the best protection since no virus was isolated from any balf samples or nasal swabs from pigs in this group. even though we did not detect significant differences in virus shedding with the com/ aut group, this group had lower hi titers compared to the aut/com pigs. although the limited number of pigs in each treatment group may have reduced the sensitivity of our study, thereby impairing our ability to detect differences between vaccination protocols, there remain questions regarding the order of the vaccine used in heterologous prime-boost vaccinations and whether the benefits imparted by vaccination are dependent on which vaccine is used as the prime and which vaccine is used as the boost. which vaccine is first and which is second may be irrelevant. if, perhaps, order does affect heterologous prime-boost vaccination performance, then this order "phenomenon" seen in our study and also present in other influenza vaccination studies [19] may be explained by the assumption that the heterologous prime-boost favors the antibody response against the first encountered antigen(s) without impairing the immune response to the second encountered antigen(s), since in our study, the ha protein of the aut vaccine strains shared the highest amino acid identity with the ha of the challenge viruses. moreover, this is also supported by the concept of "back-boosting" which refers to the tendency of the immune system during second influenza exposures to boost the titers of antibodies against the previous encountered vaccine strains [47, 48] . the variability of ha homology between the different vaccines and the challenge strains could also lead to differences on results between homologous wiv groups. however, it was not the main aim of the study to compare the differences between the homologous vaccinated groups but rather to compare as a whole the differences between homologous and heterologous vaccinated groups. nevertheless, the key question remains whether this assumption applies to any heterologous prime-boost scenario; thus, the order and delivery of multivalent vaccine combinations is still an area that needs further investigation. the seeder pig model, while closely mimicking on-farm exposure to influenza, is often challenging to perform. one difficulty was the variability encountered in virus shedding from seeder pigs for a duration of sufficient length to uniformly expose in-contact pigs. however, the effects on the overall study results due to this were mitigated given that each room contained animals from all wiv vaccination treatment groups, thereby minimizing the impact of the seeder and/or room effect. the distribution of pigs from all treatments in each of the rooms was a strength of the study and, though increasing the study complexity, was helpful in minimizing the seeder and/or room effect. control of iav in pigs is complicated, and thorough evaluation of available vaccines for pigs in a controlled vaccine/challenge experiment that mimics field conditions is necessary. to accomplish this, we chose virus challenge strains that represent the iav subtypes and clades most commonly detected in u.s. swine iav surveillance [49] , used a seeder-pig model to simultaneously exposed pigs to both iav challenge strains, and vaccinated pigs with vaccines that are commercially licensed and ready-to-use in u.s. pig populations. in summary, we demonstrated the advantage of applying a heterologous vaccine combination using inactivated vaccines against simultaneous exposure to two iav subtypes. meanwhile, delivering a whole inactivated vaccine boost to pigs following a single priming administration of laiv also significantly improved protection that may be imparted by vaccines, as was evident in decreased virus detected and increased immune response. the potential to apply this vaccination strategy to pigs in the united states is appealing. more studies are still needed to validate the concept of heterologous primeboost to control iav 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thank the following people at the college of veterinary medicine, university of minnesota: jorge garrido mantilla, gustavo lopez, carlos vilalta and erika vasquez for their help in sampling the pigs. the authors also thank my yang for her laboratory assistance and data organization, and venkatramana d krishna for his help on the elispot methodology. additional file 3. virus detection via rrt-pcr on nasal swabs and balf samples from each individual pig before and after contact.additional file 4. hemagglutinin sequences detected by next generation sequencing from nasal swabs and balf from individual pigs after contact with infected seeder pigs.additional file 5. the number of ifn-γ specific h1 (a) and h3 (b) secreting cells in pigs from peripheral blood mononuclear cells (pbmc) collected at necropsy by treatment group. the number of ifn-γ secreting cells in each treatment group are summarized as mean ± sd. the spot counts for individual pigs are displayed as data points. the asterisks denote the significant difference (p < 0.05) of the number of ifn-γ secreting cell spots between groups. the dark blue, green and dark red bars and/or data points represent the pigs from the whole inactivate vaccine comparisons, negative control and live attenuate vaccine comparisons, respectively. authors' contributions mt, mc, mam and lgp conceived the study. mc and mt designed and supervised the experiments. cl performed the experiments. da and cl performed the statistical analysis. all the authors reviewed and interpreted the data. cl wrote the first draft of the manuscript. all authors contributed to the paper revision, had full access to the data. all authors read and approved the final manuscript. the authors gratefully acknowledged the funding from zoetis. all data generated or analyzed during this study are included in this published article. all protocols and procedures were approved by the university of minnesota institutional animal care and use committee (protocol id: 1712-35407a) and the institutional biosafety committee (protocol id: 1508-32918h). not applicable. lg., m.j., d.a. and m.m. are employed by zoetis at the time of study. all other authors declare no competing interests. key: cord-307148-k1uo3fxm authors: bradshaw, patrick c.; seeds, william a.; miller, alexandra c.; mahajan, vikrant r.; curtis, william m. title: covid-19: proposing a ketone-based metabolic therapy as a treatment to blunt the cytokine storm date: 2020-09-09 journal: oxid med cell longev doi: 10.1155/2020/6401341 sha: doc_id: 307148 cord_uid: k1uo3fxm human sars-cov-2 infection is characterized by a high mortality rate due to some patients developing a large innate immune response associated with a cytokine storm and acute respiratory distress syndrome (ards). this is characterized at the molecular level by decreased energy metabolism, altered redox state, oxidative damage, and cell death. therapies that increase levels of (r)-beta-hydroxybutyrate (r-bhb), such as the ketogenic diet or consuming exogenous ketones, should restore altered energy metabolism and redox state. r-bhb activates anti-inflammatory gpr109a signaling and inhibits the nlrp3 inflammasome and histone deacetylases, while a ketogenic diet has been shown to protect mice from influenza virus infection through a protective γδ t cell response and by increasing electron transport chain gene expression to restore energy metabolism. during a virus-induced cytokine storm, metabolic flexibility is compromised due to increased levels of reactive oxygen species (ros) and reactive nitrogen species (rns) that damage, downregulate, or inactivate many enzymes of central metabolism including the pyruvate dehydrogenase complex (pdc). this leads to an energy and redox crisis that decreases b and t cell proliferation and results in increased cytokine production and cell death. it is hypothesized that a moderately high-fat diet together with exogenous ketone supplementation at the first signs of respiratory distress will increase mitochondrial metabolism by bypassing the block at pdc. r-bhb-mediated restoration of nucleotide coenzyme ratios and redox state should decrease ros and rns to blunt the innate immune response and the associated cytokine storm, allowing the proliferation of cells responsible for adaptive immunity. limitations of the proposed therapy include the following: it is unknown if human immune and lung cell functions are enhanced by ketosis, the risk of ketoacidosis must be assessed prior to initiating treatment, and permissive dietary fat and carbohydrate levels for exogenous ketones to boost immune function are not yet established. the third limitation could be addressed by studies with influenza-infected mice. a clinical study is warranted where covid-19 patients consume a permissive diet combined with ketone ester to raise blood ketone levels to 1 to 2 mm with measured outcomes of symptom severity, length of infection, and case fatality rate. there are tremendous demands on governments and the private sector to solve the covid-19 crisis with an effective and timely vaccine or therapy. as time passes, the demand for information grows pertaining to how healthy lifestyle and nutrition may play a role in protection against the detrimental outcomes of the sars-cov-2 virus. in this review, the intricate and detailed interplay among nutrition, metabolism, and the tightly controlled immune system is highlighted. the data suggest that exogenous ketones can increase cell efficiency and metabolic flexibility to provide significant immune modulation. however, challenges remain in identifying the exact dietary macronutrient combinations that will best influence the immune system. it is important for researchers and clinicians to consider metabolic strategies when attempting to identify novel preventative measures for viral infection, as these therapies can support the patient's immune system while showing minimal toxicities. the mechanisms through which exogenous ketones improve energy and redox metabolism and blunt inflammation likely apply not only to covid-19 but to any viral or bacterial infection where excessive cytokine production can lead to multiple organ failure and mortality. there are many types of metabolic therapies. however, therapies that increase r-bhb levels, including the consumption of a ketogenic diet or different forms of exogenous ketones, will be the focus of this review. others have also suggested that increasing systemic ketone levels may aid host defenses against respiratory viral infection, in part, by decreasing inflammation [1, 2] , including a recent comprehensive review [3] , while a clinical trial of the effects of a ketogenic diet on intubated sars-cov-2 patients has recently been registered (nct04358835). induces the innate and acquired immune responses. sars-cov-2 infects many cell types including type ii alveolar epithelial cells (aec ii) in the lungs [4] , where this leads to respiratory infection. aec ii either divide to maintain their levels or differentiate into aec type i, which provide the surface area for the vast majority of gas exchange in the lungs [5] . other important functions of aec ii include the secretion of surfactants, superoxide dismutase 3 (sod3) [6] , and type i (α/β) and type iii (λ) interferons [7] to protect airway function. due to these functions, aec ii have high energy requirements and rely heavily on fatty acid oxidation for energy production [8] . the partial loss of these functions during infection facilitates viral spread and disrupts the immune response and tissue repair. nearly all nucleated cells, including aec ii, can recognize the presence of viruses and initiate an innate immune response to recruit phagocytic cells to the infection. rna viruses such as sars-cov-2 are primarily recognized by cytosolic retinoic acid-inducible gene i-like receptors (rlrs), rig-1, and melanoma differentiation-associated gene 5 (mda5). the endosomal toll-like receptors (tlrs), tlr7/8 and tlr3, also play a role [9] . excessive signaling through these endosomal tlrs can cause inflammatory pathology [10] . in a cytokine storm, the number of phagocytic cells, including macrophages and neutrophils, increases along with the levels of proinflammatory cytokines, while the numbers of b and t lymphocytes, mediators of the adaptive immune response, decline [11] . this results in a failure to clear the virus and facilitates a runaway positive feedback loop that increases the numbers of cytokine-secreting innate immune cells. this cytokine storm is emerging as a major contributor to acute respiratory distress syndrome (ards), multiple organ dysfunction, and patient death in covid-19 [12, 13] . figure 1 summarizes the molecular pathologies that occur during sars-cov-2 infection that lead to a cytokine storm and ards, while figure 2 summarizes how metabolic therapy with ketone ester and a moderately high-fat diet may intervene in the disease process to protect against pathology. 1.2. metabolic therapy. central metabolism is controlled by four major nucleotide coenzyme couples: adp/atp, nad + /nadh, nadp + /nadph, and acetyl-coa/coa [14] . the prominent role these couples play in central metabolism is highlighted in figure 3 . metabolic therapy aimed at restoring these ratios is often used as an adjunct to more targeted therapies [15] . the ketogenic diet as a treatment for childhood epilepsy has drawn focus to (r)-beta-hydroxybutyrate (r-bhb) as a metabolic therapy. recently, exogenous ketones, which are various formulations of bhb, acetoacetate, or their precursors, have made it possible to raise blood r-bhb levels and alter the ratios of the controlling coenzyme couples without implementing a ketogenic diet [16] . (r)-3-hydroxybutyl (r)-3-hydroxybutyrate, a type of ketone ester, is one of the several forms of exogenous ketones that increase systemic r-bhb levels. r-bhb-derived metabolites restore flux through the citric acid (krebs) cycle and oxidative phosphorylation when viral-induced changes in enzyme activity prevent glucose [17] or fatty acids [18, 19] from fueling these pathways. increasing r-bhb levels has been shown to normalize adp/atp, nad + /nadh, nadp + /nadph, and acetyl-coa/coa ratios in diseased tissue [16] . r-bhb has multiple anti-inflammatory signaling roles and functions as an epigenetic modifier to stimulate a program of gene expression that alters metabolism to restore cellular redox function. the focus of metabolic therapy is on the restoration of the coenzyme ratios that largely control metabolic flux through central metabolic pathways. function in humans. in a study of blood cytokine levels in well-trained cyclists who compete in multiday races, the levels of tnf-α, il-6, il-2, and ifn-γ were raised following intense exercise, indicating increased inflammation, whereas the level of il-1β was unchanged [21] . on the last day of an eighteen-day trial, cyclists given daily ketone ester (r)-3hydroxybutyl (r)-3-hydroxybutyrate showed a 15% higher mean power output and 25% increase in the cd4 + /cd8 + (t helper cells/cytotoxic t cells) ratio than controls [22] . an increased cd4 + /cd8 + ratio is associated with increased immune function [23] , and this ratio declines with aging as immune system function declines [24] . induced by ionizing radiation in model systems. the same ketone ester used in the cycling studies has also been used in radiation mitigation studies. cytokines are central to the pathophysiology of covid-19; while some are beneficial, others are detrimental (il-1β, il-6, and tnf-α), at least in the context of the cytokine storm [25] [26] [27] [28] . exposure to acute doses of radiation results in tissue damage and an activation of cytokine cascades [26] . several pharmaceutical approaches are being studied to prevent or decrease radiation-induced tissue damage and the cascade of harmful cytokines [29] . there is interest in using this radiation countermeasure strategy as a model for a viral-induced cytokine storm. ir has been shown to increase the expression of the following cytokines and growth factors including il-4, il-5, il-10 [30] , tgf-β, il-12, il-18 [31] , type i interferons, il-1α, il-1β, il-6, gm-csf, and tnf-α [32, 33] . maximal cytokine production occurs between 4 and 24 hours following exposure to short-term radiation [34, 35] . the balance of proinflammatory and anti-inflammatory cytokines synthesized is critical in the determination of outcomes [36] with several factors figure 1 : mechanisms that lead to acute respiratory distress syndrome (ards) and mortality following sars-cov-2 infection are shown. the cells of the innate immune response secrete increasing amounts of cytokines. the cells that normally protect against a cytokine storm lose this ability leading to a runaway positive feedback loop of cytokine production. abbreviations: 11β-hsd1 and 11β-hsd2: 11β-hydroxysteroid dehydrogenase types 1 and 2; ace2: angiotensin-converting enzyme 2; aec i and aec ii: alveolar epithelial cell types i and ii; dcs: dendritic cells; foxo1: forkhead box o1 transcription factor; foxo3: forkhead box o3 transcription factor; hif-1α: hypoxia-inducible factor 1 alpha; ifn: interferon; il-6: interleukin-6; irf3: ifn-regulatory factor 3; nad(h): nicotinamide adenine dinucleotide; nadp(h): nicotinamide adenine dinucleotide phosphate; onoo -: peroxynitrite; pgc1-α: pparg coactivator 1-alpha; pdk1 and pdk4: pyruvate dehydrogenase kinases 1 and 4; rlr: retinoic acid-inducible gene i-like receptors; rns: reactive nitrogen species; ros: reactive oxygen species; sod: superoxide dismutase; tgf-β: transforming growth factor-β; tnf-α: tumor necrosis factor-alpha. 3 oxidative medicine and cellular longevity altering the profiles of the cytokines produced including the specific animal species and tissue studied, the magnitude of the radiation received, and whether whole animals, portions of animals, or only cells were exposed [30] [31] [32] [33] . chronic exposure to very low-dose nonacute radiation can induce hormesis and alter the levels of several cytokines to improve tissue responses [30, 35] . further studies would be needed to determine the mechanism of these acute versus subacute radiation cytokine responses. human polymorphisms in cytokine genes have been shown to be responsible for the differences in the extent of pathology that occurs following radiation damage [31] . limited ir studies with acute radiation have demonstrated that ketone ester was able to decrease chromosomal damage in mice and increase survival in cells [37] . ongoing studies are directly measuring the effects of ketone ester on animal survival following radiation and the effects on the radiation-induced cytokine storm. there are several other therapies being tested against a radiationinduced cytokine storm that could be considered for the treatment of covid-19 as well [38] . targets to treat a cytokine storm. several planned or recently initiated studies are targeting cytokines or their receptors in an attempt to blunt the cytokine storm of covid-19. for example, the il-6 receptor monoclonal antibody (mab) antagonist tocilizumab has been identified as a strong candidate for treatment [39] . other potential treatments to limit cytokine signaling include sarilumab (il-6r mab antagonist), anakinra (il-1r recombinant protein antagonist) [40] , and emapalumab (ifn-γ mab). some cytokines, such as type i interferons, may be beneficial to reduce the cytokine storm. sars-cov-2 was shown to be quite susceptible to treatment with type i interferons in vitro [41] figure 3 : the major pathways of central metabolism and reactions that alter the ratios of coenzyme couples are shown. the mitochondrial matrix and the cytoplasm have independent coenzyme couple ratios. atp synthesized in the mitochondrial matrix is exported to the cytoplasm in exchange for adp by the adenine nucleotide translocase present in the inner mitochondrial membrane. acetyl-coa synthesized in the mitochondrial matrix must also be exported to the cytoplasm to provide two carbon units for fatty acid synthesis and protein acetylation. however, acetyl-coa cannot cross the mitochondrial inner membrane. therefore, the transfer of acetyl units across the inner mitochondrial membrane is accomplished using the citrate-pyruvate shuttle or the citrate-malate shuttle. these shuttles alter coenzyme levels and use inner membrane carrier proteins for the transport of citrate, pyruvate, and malate. the net result of the citratepyruvate shuttle on coenzyme levels is the use of energy from atp hydrolysis and nadh oxidation to reduce nadp + to nadph. there is also a citrate-alpha-ketoglutarate shuttle system that has the net effect of using atp hydrolysis to increase nadph in the cytoplasm instead of increasing nadh or nadph in the mitochondrial matrix. synthesizing fatty acids in the cytoplasm and reducing antioxidants require nadph. the malate-aspartate shuttle transfers reducing equivalents from nadh between the cytoplasm and the mitochondrial matrix. the glycerol 3-phosphate shuttle transfers reducing equivalents from cytoplasmic nadh to the mitochondrial etc. glucose is catabolized by three pathways including the hexosamine biosynthesis pathway that synthesizes uridine diphosphate (udp) nacetylglucosamine, glycolysis that reduces nad + to nadh and synthesizes atp from adp and p i , and the pentose phosphate pathway (ppp) that reduces nadp + to nadph and synthesizes ribose sugars for nucleotide synthesis. when the pyruvate dehydrogenase complex (pdc) is inhibited, lactate is synthesized from pyruvate to recycle nad + from nadh so glycolysis can continue. however, the lactate is exported from the cell and may contribute to lactic acidosis and multiorgan failure [20] . r-bhb decreases the reliance of cells on glycolysis leading to reduced cellular lactate export. abbreviations: glut1 and glut3: glucose transporters 1 and 3; mct: monocarboxylate transporter; nnt: nicotinamide nucleotide transhydrogenase; pdc: pyruvate dehydrogenase complex. 5 oxidative medicine and cellular longevity 7, which protects lymphocyte function, has been proposed as a therapy to treat lymphopenia that contributes to the cytokine storm [42] . therefore, clinical studies to determine the effects of metabolic therapy with exogenous ketones in combination with one of these more targeted therapies should be considered for patients with severe sars-cov-2 infection. mechanism to blunt the cytokine storm 2.1. increased ros levels stimulate inflammasome activity and cytokine production. reactive oxygen species (ros) and reactive nitrogen species (rns) levels increase in the lungs by at least two different mechanisms during the viralinduced cytokine storm. first, viral rna binding to tlrs leads to decreased expression of mitochondrial electron transport chain (etc) genes, which increases mitochondrial superoxide production [43] [44] [45] . second, phagocytic cells are recruited to the lungs and, together with resident lung phagocytes, are activated through tlr7 and rig-1 to increase nadph oxidase 2 (nox2) activity [46, 47] , increasing the production of both intracellular and extracellular ros and rns to kill pathogens. however, host cells can be damaged as a byproduct of this response, especially in a cytokine storm. a review on the redox biology of respiratory viral infections has recently been published [48] . the increased ros production from virus-induced tlr signaling [47] , rig-1 signaling [46] , and altered mitochondrial function [49] leads to the activation of several transcriptional regulators such as nf-κb, ifn-regulatory factor 3 (irf3), and stat1 to increase the production of cytokines, including tnf-α, il-6, and il-8, from aec and macrophages [50] . the enhanced nf-κb activity also leads to the activation of the nlrp3 inflammasome that increases il-1β and il-18 production [51] . the transcription of nf-κb is a necessary step in the two-stage model of nlrp3 activation [52] . storm. in the lungs, catalase and extracellular superoxide dismutase 3 (sod3) [6] are synthesized at high levels by aec ii [53, 54] . in addition to its normal peroxisomal localization, catalase is secreted to the extracellular space by alveolar macrophages [55, 56] through a mechanism that is distinct from the classical secretory pathway [56, 57] . older covid-19 patients, who have a higher risk of mortality from the disease [58] , were shown to express much less sod3 from their aec ii than younger patients [59] , suggesting an important role of sod3 in protecting against the cytokine storm. these antioxidant enzymes reduce the concentration of toxic superoxide and hydrogen peroxide in extracellular fluids preventing oxidative damage to extracellular structures. in this regard, nox2 has been shown to synthesize superoxide and release it into the luminal extracellular space mostly from aec i [9, 60, 61] . consistent with its important antioxidant function, polymorphisms in sod3 are associated with reduced lung function and chronic obstructive pulmonary disease (copd) [62] . exogenous administration of catalase has been shown to mitigate respiratory viral infections. intranasal catalase protected against respiratory syncytial virus (rsv) infection [63] , a virus that can induce a cytokine storm [64] . catalase treatment led to a significant reduction in the levels of the cytokines il-1α, tnf-α, and il-9 and the chemokines cscl1, ccl2, and ccl5 [63] . during the early stages of other types of respiratory infections, increased ros activates the nuclear factor erythroid 2-related factor 2 (nfe2l2 commonly called nrf2) transcriptional regulator to induce antioxidant genes such as sod3 and catalase to protect against the ros-induced proinflammatory gene expression and subsequent cytokine storm. however, rsv infection leads to the proteolytic degradation of nrf2, preventing the protective antioxidant response [6] to facilitate the cytokine storm. in addition, catalase can be inactivated by high levels of ros and rns in the lungs [65] . influenza a virus (iav) is another virus that induces a cytokine storm [66] . in mice infected with iav, intranasal administration of the mitochondrialtargeted antioxidant, mitotempo, quenched etc-derived ros in the lungs to decrease proinflammatory gene expression, cytokine storm, and consequent mortality [67] . a similar study found that intranasal administration of a nox2 inhibitor had similar protective effects against iav infection in mice [68, 69] . therefore, both mitochondria-and nadph oxidase-mediated increases in ros contribute to iav mortality. the role of nadph oxidase in respiratory virus infections has been reviewed [9] . phagocytes. phagocytes release proteolytic enzymes, ros, and rns into their phagosomes to mediate the killing of endocytosed pathogens. the phagosomal oxidative burst requires cytoplasmic nadph as a coenzyme for membrane-bound nadph oxidase to produce phagosomal superoxide ( figure 4 ). the iron-dependent metalloprotein myeloperoxidase (mpo) is a component of primary granules that fuse with the phagosome [70] . mpo catalyzes the synthesis of toxic hypochlorous acid from hydrogen peroxide and chloride ion. two gases, nitric oxide and co 2 , are critical for the synthesis of toxic carbonate radicals. for this to occur, first the superoxide radical must bind to the nitric oxide radical to form peroxynitrite, and then, peroxynitrite reacts with carbon dioxide to form nitrosoperoxycarbonate that degrades to the carbonate radical. peroxynitrous acid, having a pka of 6.8, forms physiologically when peroxynitrite binds to a proton and is a major source of hydroxyl radicals [71] . as co 2 levels increase, the half-life of peroxynitrite decreases from roughly a second down to several milliseconds [72] . the ros and rns that are produced during viral infection take on specific roles as sentinels, messengers, and oxidizing agents that determine the activity of many classes of proteins including transcription factors [71] . hydroxyl radicals are short lived and function primarily as oxidizing agents. superoxide is negatively charged and does not diffuse directly across lipid bilayers, but it has been shown to be transported by proteinaceous channels from the mitochondria to the cytoplasm [73, 74] . hydrogen peroxide is transported by aquaporins across cellular membranes. the concentration 6 oxidative medicine and cellular longevity of hydrogen peroxide in the cytoplasm is generally indicative of the health of mitochondria, but transient increases can be the result of signaling events. nitric oxide is a free radical that passes through membranes and can potentially signal to nearby cells in its relatively short half-life. peroxynitrous acid can also cross cellular membranes. the half-lives and diffusion limits of different types of ros are shown in figure 5 . the importance of this diffusion of ros and rns between cells is that cells that lack the receptor for the virus can attempt to mount an appropriate response to the infection. virus-induced ros production and cytokine storm induce energy dysfunction and redox imbalance in host cells in the lungs by altering the adp/atp, nad + /nadh, and nadp + /nadph ratios that control central metabolism. (1) ros produced by the mitochondrial etc damages proximal etc proteins resulting in decreased electron flux, which increases the cellular adp/atp (less atp) (2) the decreased etc flux also decreases the cellular nad + /nadh (more nadh) as the rate of nadh hydrolysis by etc complex i slows ( 3) the increased superoxide produced from the etc is converted by superoxide dismutase 1 (sod1) and sod2 to hydrogen peroxide. h 2 o 2 is detoxified by glutathione peroxidase through the conversion of reduced glutathione (gsh) to glutathione disulfide (gssg). increased activity of nadph-dependent glutathione reductase is needed to recycle gssg to gsh leading to an increased cellular nadp + /-nadph ratio (less nadph). in a parallel pathway yielding the same result, hydrogen peroxide is detoxified by peroxiredoxins. the oxidized peroxiredoxins are then reduced by thioredoxins, and lastly, the oxidized thioredoxins are reduced by thioredoxin reductase using the reducing power of nadph leading to an increased cellular nadp + /nadph ratio the adp/atp, nad + /nadh, and nadp + /nadph couples control hundreds of cellular reactions. when these levels are altered in cells during a cytokine storm, the cells can no longer effectively perform their primary functions leading to cell dysfunction and death and to pathologies such as ards. blunting the cytokine storm in immune cells, transitioning from an inactive state to an inflammatory and then to a postinflammatory state is accompanied by metabolic reprogramming. this assures that cells have adequate energy and redox potential to perform their new roles, including entering the cell cycle for propagation, performing an oxidative burst, or undergoing regulated apoptosis rather than necrosis. the mitochondrial pdc is well positioned to reprogram metabolism as it is the gatekeeper of carbohydrate flux into mitochondria as well as a major regulator of cellular nad + /nadh. when it is active, it reduces mitochondrial nad + , and when it is inhibited, it redirects pyruvate metabolism to the cytoplasm where lactate dehydrogenase reduces pyruvate by oxidizing nadh. pdc activity, by modulating the nad + /nadh ratio, also affects the flux through glycolysis and the mitochondrial citric acid cycle, fatty acid oxidation, and oxidative phosphorylation. oxidative medicine and cellular longevity likely through type i interferon signaling [76] , which increases proinflammatory cytokine production. in this regard, treatment with tnf-α was shown to downregulate the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (pgc-1α), a master regulator of mitochondrial gene expression [77] , decreasing mitochondrial etc function and oxygen consumption in mouse lung aec [8] . downregulation of pgc-1α would likely increase ros production as pgc-1α also induces antioxidant enzymes such as sod2 and catalase [78] . unexpectedly, peripheral blood mononuclear cells (pbmcs) from sars patients were shown to have increased expression of mitochondrial-encoded subunits of the etc [79] , which could lead to increased ros production due to incompletely formed etc complexes when the nuclear-encoded subunits are downregulated. sars-cov-2 rna has been shown to localize to mitochondria [80, 81] , likely to attempt to hide from cellular immune surveillance systems. this could partly explain how sars-cov-type coronaviruses have been shown to be quite effective in blocking the type i interferon β response during the initial stages of the infection [82, 83] . targeting the viral-induced decrease in pdc activity. in mice infected with iav, atp levels greatly decreased and the level of a negative regulator of pdc, pyruvate dehydrogenase kinase 4 (pdk4), increased substantially. administration of diisopropylamine dichloroacetate (dada), an inhibitor of pdk4, significantly delayed mortality from the infection [17] . however, the infection led to severe anorexia, which also increases pdk4 levels in some tissues such as those in the muscle [84] and liver [85] due to increased foxo1, ppar-α, and glucocorticoid receptor (gr) [86] transcriptional activity. this inactivation of pdc during starvation likely evolved to save glucose and lactate (which is converted back into glucose in the liver as part of the cori cycle) for neurons, which do not efficiently oxidize fatty acids. the full extent to which the virus directly upregulated pdk4 levels is unknown. the levels of the proinflammatory cytokines tnfα, il-6, and il-1β were shown to increase following iav infection [17] . as stated above, a tlr3-and type i interferon-dependent response to viral rna has been shown to reduce the expression of four subunits of mitochondrial etc complex i [87] . this likely contributes to decreased atp production. dada administration significantly increased pyruvate dehydrogenase (pdh) activity and atp levels in the skeletal muscles, heart, lungs, and liver and tended to normalize plasma levels of glucose, lactate, free fatty acids, and r-bhb [17] . dada administration also suppressed the iav-induced increase in il-6, il-2, ifn-α, tnfα, and ifn-γ levels, but not that in ifn-β or il-1β [17] . pdk inhibitors have also been shown to have protective antiinflammatory effects. this may partly result from their effects on t lymphocytes, as proinflammatory th17 cells have high levels of pdk1 and show primarily glycolytic metabolism, while anti-inflammatory th1 and treg cells have low pdk1 levels and show primarily oxidative metabolism. knockdown of pdk1 suppressed th17 cells and increased treg cell numbers to restore immune function in mice with experimental autoimmune encephalomyelitis [88] . oxidative medicine and cellular longevity in another study of iav infection in mice, glucose administration during the period of anorexia following iav infection was found to decrease the mortality rate [89] . glucose administration likely stimulated the insulin receptor-akt signaling pathway to decrease foxo1 activation to blunt the increase in pdk4 levels resulting from anorexia to maintain energy generation [86] . other activators of mitochondrial energy metabolism, such as the peroxisome proliferation-activated receptor-gamma (ppar-γ) agonist pioglitazone or rosiglitazone and the amp kinase activator, aicar, have also been shown to protect mouse mortality from iav infection [90] . these compounds are all known to decrease pdk4 levels in the muscle and liver [91] . therefore, foxo1 hyperactivation may be a pathological event in mouse iav infection as it is induced by both increased ros levels [92] and the anorexia that occurs following iav infection. a mechanism through which antioxidant administration protects against viral infection may be through preventing foxo1 induction of pdk4. the use of ppar-γ agonists to treat the cytokine storm in covid-19 has been reviewed [93] . figure 3 shows the central role of pdc at the gateway between glycolytic and citric acid cycle metabolism. figure 6 shows the multiple transcription factors that control the expression of the kinases and phosphatases that regulate pdc activity as well as controlling the expression of the three enzymes that comprise the pdc. figure 6 also shows that r-bhb is catabolized into two acetyl-coa molecules that enter the citric acid cycle and bypass pdc inhibition. an nadh, which fuels complex i of the etc, is also generated during r-bhb oxidation to acetoacetate. r-bhb has also been shown to increase pgc-1α levels [94] and mitochondrial fusion [95, 96] , which are known to increase mitochondrial energy generation. therefore, ketone body catabolism is a substantial source of atp when the cytokine storm leads to the block of the mitochondrial oxidation of carbohydrate catabolites. , and hpo 4 -, which regulate plasma membrane potential and osmotic balance [99] . atp drives the ion pumps that provide the chemiosmotic potential to (r)-3-hydroxybutyrate can restore atp levels, decrease the production of lactate, and decrease dependence on glycolysis. oxidative medicine and cellular longevity maintain the distribution of these ions (figure 7) , preventing edema. the major sars-cov-2 spike (s) protein binds to extracellular ca 2+ to facilitate viral fusion with host cells such as aec ii [100] and signals for the opening of plasma membrane ca 2+ channels through a protein kinase c-α signaling pathway, which may be triggered by er stress [101] . the sars-cov-2 envelope (e) protein is a lipidated viroporin, which forms a cation-selective channel in the endoplasmic reticulum that releases ca 2+ [102] . blocking the viralinduced increase in cellular ca 2+ levels with a chelator decreased infectivity 60-fold [100] . the increased cytoplasmic ca 2+ leads to the activation of the plasma membrane and er ca 2+ pumps, depleting atp levels. increased cytoplasmic ca 2+ stimulates the uptake of ca 2+ into the mitochondrial matrix in a mitochondrial membrane potentialdependent manner. in the presence of high levels of ros caused by viral infection, the rapid uptake of ca 2+ into the mitochondrial matrix may stimulate permeability transition pores to open in the inner membrane [103] , which uncouple mitochondria leading to further energy depletion and cell death. however, mammals have evolved mechanisms to use the viral-induced increase in cytoplasmic ca 2+ as a signal to upregulate host defenses. an increase in the cytosolic ca 2+ concentration by these mechanisms contributes to the activation of the nlpr3 inflammasome and elevation of il-1β and il-18 [102] . increased cellular ca 2+ levels in aec ii lead to increased mitochondrial etc-derived ros and increased ros from nadph oxidases duox1 and duox2, the most abundant isoforms in aec, through ca 2+ binding to their ef-hand motifs [9] . duox2 is also upregulated at the gene expression level by the increased interferon-β and tnf-α produced in response to the respiratory viral infection [104, 105] . hydrogen peroxide can diffuse from aec into adjacent cells leading to oxidative damage, energy depletion, and osmotic imbalance. increased cytoplasmic ca 2+ levels in airway myocytes induce constriction of the airways. several protein kinase c (pkc) isoforms are activated by ca 2+ , and their activation is an important contributor to bronchoconstriction [106, 107] . pkc directly targets and inhibits kv7 k + channels, which are important for the relaxation of airway smooth muscle [108] . inhibition of kv7 channels induces bronchial constriction [109] , which can contribute to ards. figure 7 : maintaining atp levels during respiratory viral infection is the key to maintain proper ion distribution to avoid edema. the active and passive cotransporters and channels maintain the ion gradients between aec and extracellular fluids. the apical ligand-gated channels function to maintain appropriate osmotic pressure to provide sufficient fluid in the airway without causing edema. aec i express superoxideactivated enac na + channels and nox2 that regulates them. abbreviations: 3na + /2k + -atpase: 3 sodium 2 potassium atpase; ae: anion exchange chloride bicarbonate exchanger; bcftr: basolateral cystic fibrosis transmembrane conductance regulator-like channel; bhbdh: βhydroxybutyrate dehydrogenase; birc: basolateral inward rectifying channel; bk ca : large-conductance ca 2+ -and voltage-gated big k + channel; borc: basolateral outward rectifying channel; cacc/tmem: calcium-activated chloride channels/transmembrane protein; cftr: cystic fibrosis transmembrane conductance regulator; cng: cyclic nucleotide-gated ion channel; enac: epithelial sodium channel; kca3.1: calcium-activated potassium channel; kv7.1: voltage-dependent potassium channel; ncx: sodium calcium exchanger; nhe: sodium-hydrogen exchanger; nkcc: sodium potassium chloride cotransporter; nox2: nadph oxidase 2; pit1/2: sodium-dependent phosphate transporters 1 and 2; ryr: ryanodine receptor calcium-induced ca 2+ channel; serca: sarcoplasmic/endoplasmic reticulum ca 2+ -atpase; sk4: sk4 calcium-activated potassium channel. 10 oxidative medicine and cellular longevity the use of r-bhb as an energy source may blunt the cellular energy deficit, the increase in cytoplasmic ca 2+ levels, and the osmotic imbalance to improve lung function. high-fat, low-carbohydrate enteral feeding of patients with type ii respiratory failure (the inability to expel co 2 at a normal rate) reduced the mean length of time on a ventilator by 40%, from 158 hours down to 96 hours, compared to patients on high-carbohydrate, low-fat enteral feeding [110] . arterial blood co 2 levels, an indicator of patient respiratory distress, decreased to 18% for patients in the high-fat group at the time of weaning off the ventilator. the high-carbohydrate group had an even higher partial pressure of co 2 at weaning than at the onset of ventilation. a likely contributor to the difference observed between the groups was the amount of co 2 synthesized from metabolizing the different diets. the amount of co 2 synthesized for every molecule of oxygen consumed is defined as the respiratory exchange ratio (rer). the rers for catabolism of fat, glucose, and r-bhb are 0.7, 1.0, and 0.88, respectively. a high-fat diet was also protective in a mouse model of ventilator-induced lung injury [111] , a model of ards. a high-fat, lowcarbohydrate diet supplemented with fish oil, gammalinolenic acid, and antioxidants was also shown to decrease the time on a ventilator for patients with ards due to sepsis/pneumonia [112] , trauma, or aspiration injury; the findings may be relevant for ards mediated by viral infection as well. due to the lack of published data on the effects of increased r-bhb levels on the human immune system during viral infection, results obtained from several studies of metabolic therapy on iav-infected mice are described below. a shortterm ketogenic diet was shown to protect mice from iav infection, while racemic r-and s-1,3-butanediol (bd), an exogenous ketone precursor, supplemented to a normal chow diet did not [113] . a long-term ketogenic diet that was obesogenic was shown to adversely affect glucose tolerance and immune system function [114] . as described above, glucose gavage during iav-induced anorexia decreased mouse mortality [89] , while a more recent study showed that glucose metabolism through the hexosamine biosynthetic pathway stimulated a cytokine storm [89] . in the sections below, the results from these studies will be described in more detail and analyzed in an attempt to reconcile these findings. the rationale will also be discussed for the consumption of a moderately high-fat, moderate-carbohydrate, ketone estercontaining diet at the onset of viral infection,and transitioning to a moderately high-fat, low-carbohydrate, ketone ester-containing diet if the infection becomes severe to blunt the cytokine storm. activating a γδ t cell response. a recent study showed that mice placed on a ketogenic diet for seven days before infection had decreased mortality from iav [113] . the ketogenic diet increased the number of protective il-17-secreting γδ t cells in the lungs. administration of the ketone precursor bd to mice on a chow diet did not protect the survival or lead to the recruitment of γδ t cells to the lungs in the mice infected with iav, even though the r-bhb blood level was equivalent to that of the ketogenic diet. one point raised regarding the design of this study is that while dietary protein amount (% w/w) was uniform between control and kd mice [115] , the micronutrient and fiber profiles were not [116] . while this did not likely impact the conclusions of the study, it should be a point of emphasis for future experiments. bd administration may not have shown protection due to a lack of improvement of the cellular redox environment in the lungs that likely occurred during the ketogenic diet. during times of high r-bhb oxidation in the brain, the cytoplasmic nadp + /nadph becomes more reduced and the cytoplasmic nad + ]/[nadh becomes more oxidized [16] . the ketogenic diet, which is very high in fat content, may have improved the cytoplasmic redox environment, in part, through inhibition of fatty acid synthesis, an nadphconsuming pathway, by increasing levels of palmitoyl-coa, an inhibitor of fatty acid synthase activity. so, the combination of increased r-bhb metabolism and decreased fatty acid synthesis may lead to a decrease in the cytoplasmic nadp + /-nadph ratio that may lead to the recruitment and enhanced function of γδ t cells in the lungs, which does not occur when r-bhb is catabolized when mice are fed a normal chow diet. a high-fat but nonketogenic diet was shown to be ineffective in decreasing iav-induced weight loss and mortality, even though it increased the recruitment of il-17-secreting γδ t cells to the lungs. il-17 binds to receptors on lung epithelial cells and possibly other lung cell types to increase the expression of il-33. il-33 secretion leads to the recruitment of type 2 innate lymphoid cells (ilc2s) to the lungs, where they play a role in regulating inflammation and barrier function by secreting the cytokines il-5, il-9, il-13, and amphiregulin. in lung tissue, the ketogenic diet upregulated mitochondrial etc gene expression and the expression of the oxct1 gene encoding scot (succinyl-coa:3-ketoacid coa transferase), the rate-limiting enzyme for ketolysis, suggesting that r-bhb catabolism in the lungs plays an important role in the protective effects of the ketogenic diet against viral infection [113] . it is hypothesized that supplementation of ketone ester to mice on a moderately high-fat diet will lead both to the recruitment of γδ t cells to the lungs and to decreased mortality of iav-infected mice. while one week of ketogenic diet prior to iav infection was shown to be anti-inflammatory and decrease mouse mortality, three months of the ketogenic diet in the absence of iav infection increased white adipose tissue (wat) inflammation, decreased γδ t cell recruitment to the wat, and led to obesity and glucose intolerance [114] . oxidative medicine and cellular longevity therefore, current evidence from mouse studies where the animals were fed obesogenic ketogenic diets suggests that only short-term ketogenic diets will activate γδ t cells to boost immune function [114] . however, another research group identified a ketogenic diet of a different composition that was shown to induce weight loss in mice [117] . the dietary components responsible for the different effects on weight are currently unknown, although the leptogenic diet contained only half as much protein and used lard, butter, and vegetable oil as fat sources, while the obesogenic diet used hydrogenated soybean oil as the fat source [114, 117] . future studies are needed to determine if a ketogenic diet that induces weight loss can provide long-term preservation of the protective γδ t cell response to provide long-term antiviral immunity. it is also hypothesized that increased r-bhb levels improve cellular energy metabolism and redox status to enhance fatty acid beta-oxidation to overcome the metabolic inflexibility mediated by pdc inhibition. r-bhb is known to inhibit adipose tissue lipolysis [118] , so a high-fat diet may be needed, along with exogenous ketones, to provide sufficient fatty acid beta-oxidation for increased metabolic flexibility to overcome a cytokine storm. the initiation of this diet in humans for covid-19 poses challenges because it may take many days to adapt to a ketogenic diet to fully upregulate the expression of genes for ketogenesis, ketolysis, and fatty acid oxidation. proinflammatory cytokines also inhibit ketogenesis [119] . in addition, starting a ketogenic diet, also called ketoinduction, may be accompanied by flu-like symptoms [120] [121] [122] that may limit its application to covid-19 patients. however, studies could be performed to determine the ability of covid-19 patients to tolerate a ketogenic diet supplemented with exogenous ketones, to attempt to decrease the early adverse effects of the diet that likely result from decreased energy production when glucose levels initially decline [122] . if well tolerated, further studies determining the ability of this diet to activate a protective immune response could follow. further experiments are needed to delineate the molecular mechanisms involved if r-bhb precursors such as ketone esters are to be used for the treatment of iav and sars-cov-2 infections. to test the effectiveness of exogenous ketone administration on iav infection, mice could be supplemented with or without a ketone ester and fed a moderately high-fat, moderate-carbohydrate diet or a moderately high-fat, low-carbohydrate diet or a control chow diet, and weight loss and mortality could be monitored following iav infection. as explained in more detail below, glucose (from carbohydrate metabolism) stimulates important proinflammatory antiviral functions early in infection [123] , but these proinflammatory actions also increase the cytokine storm late in infection [124] . so, it is unknown which of these effects will predominate to affect mortality in the presence of high r-bhb levels. it is hypothesized that the moderately high-fat, moderate-carbohydrate, ketone ester-containing diet will provide the most metabolic flexibility to stimulate host cell defense mechanisms. this flexibility should be able to preserve energy metabolism and redox status to boost immune function to decrease iav titer in the lungs. how-ever, the moderately high-fat, low-carbohydrate diet with ketone ester will likely show a stronger ability to blunt the cytokine storm, as increased glucose and insulin levels have recently been shown to block an important antiinflammatory action of r-bhb [125] . mortality that is greatly blunted by gavage of glucose, but not fat or protein. as alluded to earlier, mice became anorexic following iav infection and the anorexia contributed to their mortality, as glucose gavage was able to decrease the mortality. in that study, gavage of olive oil (fat) or casein (protein) did not decrease mortality [89] . the proinflammatory cytokines il-1, il-2, il-6, il-8, tnf-α, and ifn-γ, several of which increase following iav infection, have been shown to suppress appetite [126] . so, it is likely that administering a ketogenic diet simply decreased cytokine levels, allowing for an increase in the amount of food consumed to decrease the mortality of the mice. consistent with this interpretation, mice on the ketogenic diet lost less weight following infection than the chow-fed mice [113] . these results may be applicable to sars-cov-2 infection, as 40% of covid-19 patients reported lack of appetite as a symptom [127] . a major research question that arises from these studies is whether a twice-daily isocaloric gavage of ketone ester starting on the day of infection, to mimic the protective effect of the twice-daily gavage of glucose [89] , can protect mice fed a chow diet from iav infection. this hypothesis is reasonable given that the protective ketogenic diet was composed of roughly 90% fat, 10% protein, and only 0.1% carbohydrate [113] . so, the carbohydrate content of the diet was likely too low to provide adequate glucose for protection, and gavage of fats or protein was unable to provide protection [89] . the ineffectiveness of the ketone precursor bd against iav infection [113] suggests that ketone ester alone may be ineffective and that gavage of fat and ketone ester together as a cotherapy, to better mimic a ketogenic diet, may be needed for protection. experiments probing possible additive or synergistic effects among glucose, ketone ester, and fats on increased survival during iav infection in mice would provide valuable insights relevant to the protection against a cytokine storm in humans. so, how may glucose gavage of mice during viral-induced anorexia decrease mortality from iav infection? glucose has been shown to stimulate a proinflammatory antiviral response through increased flux through the hexosamine biosynthesis pathway. the increased flux increases levels of the pathway end product udp n-acetylglucosamine (udp glcnac), which increases the o-glcnacylation of the antiviral protein mavs to increase its function (figure 8(c) ) [123] . the sars virus synthesizes the nps15 protein, which partially inhibits mavs function to block host antiviral signaling [128] . in addition, viral nucleic acids and type i interferon signaling in macrophages lead to the increased oxidative medicine and cellular longevity expression of the glycolytic activator 6-phosphofructose-2kinase and fructose-2,6-bisphosphatase (pfkfb3), which are required for the increased engulfment of viral-infected cells [129] . surprisingly, mortality from iav infection in mice has been linked with viral induction of an er stressinduced apoptotic pathway in the brain [89] . both glucose and r-bhb are important protective fuels for neurons, potentially reconciling findings of how either glucose or a ketogenic diet is protective. in addition, high glucose levels may compensate for the energy and redox crisis occurring as a result of viral-induced pdc inhibition by increasing flux through glycolysis for the synthesis of atp and by increasing flux through the pentose phosphate pathway (ppp) for the synthesis of nadph. glucose flux through the hexosamine biosynthesis pathway has been shown to stimulate the cytokine storm during iav infection in mice by increasing the o-glcnacylation of the transcriptional regulator irf5, which increases its activity to stimulate proinflammatory cytokine production (figure 8 (c)) [124] . this may explain in part why people with diabetes who are infected with sars-cov-2 have a higher mortality rate [130] . therefore, inhibitors of irf5 or inhibitors of o-glcnacylation, such as osmi-1, are potential treatments for the sars-cov-2 cytokine storm. ketone ester treatment has been shown to decrease blood glucose levels [131, 132] , which would likely decrease flux through the hexosamine biosynthesis pathway in immune cells to decrease cytokine production. monocarboxylate transporters are expressed in aec ii [133] , allowing the entry of r-bhb into the cytoplasm. however, these cells have low expression of ketolytic enzymes, so they may be unable to substantially catabolize the r-bhb produced from the consumption of exogenous ketones [134] . however, a ketogenic diet was shown to increase the expression of ketolytic genes in the lungs [113] , so it is possible that these enzymes can be induced in aec ii and are responsible, in part, for the protective effects of the ketogenic diet. even if r-bhb is not catabolized by aec ii, the presence of r-bhb in aec ii may still greatly protect these cells through signaling pathway activation, through enzyme inhibition, and through gene expression pattern alteration as described in detail below. depend upon the metabolic state of the cell. r-bhb inhibits the nlrp3 inflammasome [135] . s-bhb, the enantiomer of r-bhb, was also effective, but not butyrate. the molecular target through which r-bhb and s-bhb inhibit the inflammasome is still unknown, but treatment decreased cellular k + efflux and reduced inflammasome activator asc (apoptosis-associated speck-like protein containing a card) oligomerization (figure 8(b) ). r-bhb-mediated inhibition of the inflammasome did not require ketone body catabolism, since sirna of the ketolytic enzyme scot did not block inhibition. inflammasome inhibition was also shown to be independent of the effects of r-bhb on gpr109a gprotein-coupled receptor (gpcr) signaling and histone acetylation. in addition to immune cells, several types of epithelial cells express the genes for a functional nlrp3 inflammasome; r-bhb likely protects aec ii from a cytokine storm in part through this mechanism [136] . recently, high insulin or high glucose levels were shown to decrease r-bhb-mediated inhibition of the nlrp3 inflammasome in macrophages in vitro, and 2-deoxyglucose, a glycolysis inhibitor, was shown to potentiate nlrp3 inflammasome inhibition by r-bhb [125] . therefore, the metabolic state of the cell appears to influence the effect of r-bhb on the nlrp3 inflammasome. somewhat surprisingly, a single dose of exogenous ketones was shown to increase inflammasome activation in lps-stimulated blood cells and increase plasma il-1β and il-6 levels of healthy young people after a 10-hour overnight fast [137] . the mechanisms remain unknown, but increased levels of these proinflammatory markers may have been due to an r-bhb-mediated increase in nadph levels stimulating nadph oxidase activity to increase ros levels and possibly also due to increased mitochondrial ros production that occurs when r-bhb and glucose are oxidized simultaneously, as increased ros stimulates nlrp3 inflammasome activity [138] . however, a follow-up study by the same group administering exogenous ketones to obese subjects found no difference in inflammasome activity and the levels of many proinflammatory markers but a slight decrease in il-1β and tnf-α levels in the exogenous ketone-treated group [139] . in a study of well-trained cyclists, acute bd administration was shown to slightly increase interferon-gamma expression in pbmcs, while anti-inflammatory cytokine expression was unaltered [140] . overall, the lack of nlrp3 inflammasome inhibition and the lack of strong antiinflammatory effects of exogenous ketones in the above human studies are likely due to the metabolic state of the subjects when the exogenous ketones were administered. it is possible that the glucose levels and insulin levels were too high to allow r-bhb to inhibit the inflammasome [125] . sars-cov-2 infection can cause ketosis and ketoacidosis, and these patients with high blood r-bhb levels had longer hospitalization and an increased mortality rate [141] . also, covid-19 patients with type i or ii diabetes mellitus (dm) have an increased risk of developing diabetic ketoacidosis (dka), which contributes to mortality [142] . the molecular basis for these findings is not entirely clear as ketone bodies do not directly cause dka. recent findings, however, indicate that the increased glucose levels in mouse models of diabetes can decrease the expression of the ketolytic genes r-bhb dehydrogenase (bdh1) and oxct1 in the heart [143] . if this also occurs in other tissues such as skeletal muscle, it would likely lead to increased blood ketone levels. expressing an exogenous transgene to increase o-glcnacylation in the mice further decreased bdh1 levels demonstrating a role for the hexosamine biosynthetic pathway in this downregulation of ketolytic gene expression. the scot enzyme (oxct1 gene product) was shown to be directly modified by o-glcnacylation. therefore, administration of an o-glcnacylation inhibitor together with exogenous ketones to diabetic patients with covid-19 may be beneficial to prevent decreased ketolysis and dka. 14 oxidative medicine and cellular longevity multiple factors may be contributing to acidosis in covid-19 patients. respiratory acidosis occurs due to a buildup of carbon dioxide in the body, while lactic acidosis occurs due to mitochondrial etc dysfunction or pdc inhibition. r-bhb metabolism, unlike glucose metabolism, does not raise the levels of lactic acid and may even decrease acidosis by lowering the rate of glycolysis and lactic acid synthesis. differential diagnosis and treatment of acidosis have been reviewed [144] . consumption of a ketone ester has been shown to lower glycemic response in both healthy and obese people [131] . fatty acid lipolysis in white adipose tissue is inhibited by ketones [118] , so in most cases, exogenous ketones will inhibit the synthesis of endogenous ketones. before insulin was available, a ketogenic diet that limited carbohydrates to ≤10 g/day was a commonly used effective therapy for type i diabetes [145] . during dka, there are imbalances in the levels of glucagon and insulin and elevation of the stress hormones epinephrine, cortisol, and growth hormone. these changes can be triggered by a stressful event such as covid-19. therefore, care would need to be taken in administering exogenous ketones in a clinical trial for covid-19. coadministration of sodium bicarbonate may also be beneficial for diabetic covid-19 patients to buffer changes in blood ph. in this regard, a recent study showed that cyclists administered ketone ester had a 20% decrease in blood bicarbonate levels and a slight decrease in blood ph, while blood r-bhb levels rose to 2-3 mm. administering bicarbonate together with ketone ester prevented these alterations in the blood and increased blood r-bhb levels another 0.5-0.8 mm, and this resulted in a 5% increase in power output [146] . if diabetics are to be included in a covid-19 trial testing the effects of exogenous ketones, the ph of arterial blood gas (abg) and the blood levels of ketones would need to be monitored by experts in managing dka to identify early-stage ketoacidosis so that interventions according to best practices [147] could be implemented. to avoid risks, patients with naturally high ketone levels should avoid exogenous ketones, so dka may be an exclusion factor in trials. but ultimately, a clinical trial will likely be necessary to determine the effects of exogenous ketone consumption or a ketogenic diet on covid-19 in both diabetic and nondiabetic patients. the lack of protective effects of high r-bhb levels under certain metabolic conditions is likely due to the same underlying molecular mechanism that prevented supplementation with the ketone precursor bd from preventing mortality in iav-infected mice [113] . a protective anti-inflammatory γδ t cell response was likely not initiated in these studies. in the studies with exogenous ketones, this was likely due to the acute nature of the exogenous ketone treatment and to the lack of the high-fat, low-carbohydrate diet that may be necessary to initiate this protective anti-inflammatory response. however, there are several other potential mechanisms that may have also prevented the acute ketone ester treatment from influencing the activation state of the inflammasome. for example, a 24-hour fast in human subjects has been shown to lead to nlrp3 inflammasome inactivation, due to increased mitochondrial nad + /nadh activating the nad + -dependent sirt3 protein deacetylase to decrease ros production [148] . therefore, the 10-hour overnight fast may have led to a partial inhibition of nlrp3 inflammasome activity so that ketone ester treatment was unable to decrease the activity any further. it is also possible that at least five days of a moderately high-fat, low-carbohydrate diet with exogenous ketone treatment may be needed to show large anti-inflammatory effects, as it was shown to take five days to fully upregulate the activity of the fatty acid betaoxidation system after initiating a ketogenic diet [149, 150] . decrease inflammation. r-bhb was shown to be a class i and iia histone deacetylase (hdac) inhibitor (k i = 2 − 5 mm) that induced expression of several antioxidant genes and the transcriptional regulator foxo3a (figures 9(a) and 9(b) ) [151] . administration of the other hdac inhibitors, butyrate or trichostatin a, showed antiinflammatory effects on lung ilc2s, while adding both compounds together showed no additive benefit [152] . this suggested that hdac inhibition is a protective mechanism through which r-bhb and the ketogenic diet prevent lung inflammation. there is a nuclear pool of pdc that contributes to acetyl-coa synthesis for histone acetylation [153] . pdk1 also shows a partial nuclear localization [154] , so the upregulation of pdk1 expression during viral infection could disrupt nuclear histone acetylation, which could be restored by hdac inhibitors such as r-bhb. in studies with macrophages, butyrate was shown to function as an hdac inhibitor to decrease il-6, il-12, and nitric oxide levels, but not tnf-α or mcp-1 levels [155] . in a co-culture model of raw264.7 macrophages and 3t3-l1 preadipocytes, addition of butyrate decreased the production of tnf-α, mcp-1, and il-6 and decreased nf-κb expression in the macrophages [156] . another study found that hdac inhibition decreases nf-κb transcription, which may be responsible for the anti-inflammatory effects [157] . therefore, increasing r-bhb levels will likely lead to similar antiinflammatory effects on lung macrophages to dampen a cytokine storm, although butyrate has been reported to be a superior hdac inhibitor compared to r-bhb in some cell types such as myotubes and endothelial cells [158] . this decreased efficacy of r-bhb as an hdac inhibitor in some cell types may result from different rates of transport into the cell or into the mitochondrial matrix, different rates of r-bhb oxidation, or different endogenous nuclear histone acetyltransferase or hdac activities. dietary therapies that increase both butyrate and r-bhb levels may have additive anti-inflammatory effects [159] . the antibacterial effect of butyrate on intestinal macrophages was shown to be due to hdac3 inhibition, not gpr109a signaling. hdac3 inhibition led to a decreased rate of glycolysis and increased flux through the ppp increasing amp levels and amp kinase activity and decreasing mtor activity to stimulate autophagy [160] . in the lung, butyrate inhibition of the class iia hdac, hdac7, decreased bacterial-induced inflammation [161] . during infections, mitochondrial damage leads to the oxidation and release of the inner membrane phospholipid cardiolipin, leading to ppar-gamma sumoylation and recruitment of hdac3 to the promoter of il-10, an antiperoxynitrite s-nitrosylates hif-1α to inhibit its von hippel-landau factor-induced ubiquitination and proteasomal degradation. this stabilizes hif-1α leading to increased pdk1 expression. this stabilization is likely inhibited by r-bhb, which increases nadph to decrease ros/rns levels. (e) nrf2 is activated by oxidized dj-1, and this is regulated by the redox potential of nadp + /nadph that controls the cellular antioxidant potential. the dj-1 chaperone protein, which is activated by moderate levels of hydrogen peroxide, stimulates the release of nrf2 from keap1 allowing nrf2 to enter the nucleus and induce antioxidant response element gene expression. (f) pgc-1α is the master regulator of mitochondrial biogenesis. it is a coactivator of err-α, foxo1, foxo3a, ppars, and nuclear respiratory factor 1 (nrf1). abbreviations: ac: acetate; are: antioxidant response element; ca9: carbonic anhydrase 9; ccr7: c-c chemokine receptor type 7; err-α: estrogen-related receptor alpha; etc: electron transport chain; k: lysine; hdac: histone deacetylase; fih-1: factor inhibiting hif-1; foxo1: forkhead box o1; foxo3: forkhead box o3; foxp3: forkhead box p3; g6pc: glucose-6phosphatase; g6pdh: glucose 6-phosphate dehydrogenase; γgclm: glutamate cysteine ligase modifier subunit; glut1: glucose transporter 1; gsrx: glutathione reductase; hif-1α and hif-1β: hypoxia-inducible factor 1 alpha and beta; hre: hypoxia response element; ifng: interferon gamma gene; il7r: interleukin-7 receptor; inos: inducible nitric oxide synthase; irf7: interferon regulatory factor 7; keap1: kelch-like ech-associated protein 1; maf: musculoaponeurotic fibrosarcoma; nrf1: nuclear respiratory factor 1; nrf2: nuclear factor erythroid 2-related factor 2 (nfe2l2); rag1 and rag2: recombination activating genes 1 and 2; onoo -: peroxynitrite; onooh: peroxynitrous acid; p300/cbp: binding protein 300/creb-binding protein; pdk1 and pdk4: pyruvate dehydrogenase kinases 1 and 4; pgc1-α: pparg coactivator 1 alpha; phd2: prolyl-hydroxylase domain protein 2; pvhl: von hippel-landau tumor suppressor protein; scot/oxct1: succinyl-coa-3-oxaloacid coa transferase also known as 3-oxoacid coa-transferase 1; sell: selectin l; sod2: superoxide dismutase 2; ub: ubiquitin; vegf: vascular endothelial growth factor. 16 oxidative medicine and cellular longevity inflammatory cytokine, to decrease gene expression. gene expression of tnf-α was unaffected, so increased inflammation was observed. butyrate administration increased il-10 gene expression to normalize the level of inflammation [162] . coronaviruses have been shown to increase the oxidation of phospholipids, which stimulate toll-like receptor 4 (tlr4) signaling on macrophages, leading to cytokine production and acute lung injury [163] , so hdac inhibition with r-bhb appears to be a viable treatment to decrease cytokine levels and inflammation. 7.3. r-bhb binds to the gpr109a gpcr to stimulate anti-inflammatory signaling. the gpr109a (hydroxycarboxylic acid receptor 2 (hca2), expressed from the hcar2 gene) gpcr is bound and activated by r-bhb (ec 50 of 0.7 mm [118] ), s-bhb, or butyrate and is expressed in the lung and many types of epithelial cells, macrophages, neutrophils, and dendritic cells, but not in b or naïve t lymphocytes [164] . however, gpr109a was shown to play a role in the expansion of cd4 + and cd8 + t cells [165] . the expression pattern of gpr109a suggests that it could play a role in the protective effects of the ketogenic diet against iav infection [113] . gpr109a has been shown to be activated by zika virus infection and protect cells by inhibiting viral replication [166] . a major mechanism through which gpr109a signaling exerts its anti-inflammatory effects is through suppressing the activation of the transcriptional regulator nuclear factor-kappa b (nf-κb) [167] , required for the transcription and secretion of several proinflammatory cytokines [168] . studies with gpr109a-knockout mice have identified gpr109a signaling as essential for the increase in thermogenesis induced by its ligands [169] . consistent with this, gpr109a-knockout mice were obese, showing hepatic steatosis due to upregulation of enzymes of fatty acid synthesis (acc1 and fatty acid synthase (fas)) and downregulation of enzymes of fatty acid oxidation (cpt-1α). ppar-α, the master regulator of ketogenesis, was decreased in the liver, while ppar-γ, the master regulator of adipogenesis, was increased in wat. so, it is likely that stimulation of gpr109a plays an important role in the induction of fatty acid beta-oxidation and weight loss induced by the ketogenic diet. macrophages and dendritic cells from gpr109a-deficient mice were defective in inducing naïve t cells to differentiate into treg cells and il-10-producing t cells [170] . lack of gpr109a also decreased the expression of il-18 [170] . gpr109a signaling has been shown to be protective by activating the nrf2 transcriptional regulator through an amp kinase signaling pathway to decrease oxidative stress [171] . gpr109a was also shown to play an important role in maintaining epithelial barrier function during bacterial sepsis [172] , so it may play a similar role during viral infection. antiviral peptide and protect it from inactivation. cathelicidins are a class of antimicrobial host defense peptides. ll-37 is one of two human cathelicidins and released by bronchial epithelial cells, macrophages, and neutrophils as part of the innate immune response against respiratory viral infections [173] . ll-37 has many functions, including binding to nucleic acids, strengthening the viral rna-induced tlr3 signaling response to increase type i interferon production [174] , stimulating inflammasome activation [175] , and reducing viral load and virion release [176, 177] . the peptide has both proinflammatory and anti-inflammatory properties [178] , but the anti-inflammatory properties may predominate in the lungs as ll-37 administration decreased the expression of the proinflammatory cytokines il-8 and il-6 and the chemokine ccl5 in response to respiratory viral infection [179] . respiratory viral infection increases the expression of peptidyl arginine deiminase 2 (pad2) in the lungs. this class of enzymes catalyzes the removal of a positively charged amino group from protein arginine to form citrulline, through a process called citrullination. ll-37 has five arginine residues essential for its antiviral function, which are targets of pad2 function following the viralinduced increase in pad2 expression in the lungs [179] . increased levels of nadph decrease the catalytic activity of peptidyl arginine deiminases to limit ll-37 citrullination [180, 181] . hdac inhibitors such as butyrate have been shown to increase the expression of ll-37 [182] to decrease pathogen infection [183] . therefore, r-bhb may mitigate respiratory virus infection both by increasing ll-37 levels and by increasing nadph levels [16] that protect ll-37 from inactivation. diet and its level in tissues is regulated by redox-sensitive coenzyme ratios. cortisol, an adrenal gland-secreted hormone, has anti-inflammatory properties through activation of the glucocorticoid receptors. subjects on either a ketogenic diet [184, 185] or a severe calorie restriction diet [186] show transient increases in cortisol levels. in mice, seven days of ketogenic diet led to the transcriptional activation of targets of the glucocorticoid receptor [113] . if this transient increase in cortisol levels that occurs in humans also occurs in mice on the ketogenic diet, the increased cortisol levels may contribute to the blunting of the cytokine storm in animals that were fed the ketogenic diet for a week before iav infection [113] . mice that were fed the ketogenic diet for three months showed increased inflammation, which could have been in part due to the return of cortisol to baseline levels [114] . as may be expected due to the function of cortisol as a stress hormone, when healthy subjects were administered ketone ester in nonketogenic states, no change in cortisol levels was observed [22, 187] . in peripheral tissues, such as the lungs, the level of cortisol is regulated by the 11βhydroxysteroid dehydrogenase (11β-hsd) system consisting of the two enzymes, 11β-hsd1 and 11β-hsd2. the conversion of cortisone to the active steroid hormone cortisol is catalyzed by nadph-dependent 11β-hsd1 [188] , while the reverse reaction that regenerates the precursor cortisone is catalyzed by the nad + -dependent 11β-hsd2 enzyme (figures 1 and 2) . therefore, the level of active cortisol in tissues is under tight control by the nad + /nadh and nadp + /nadph redox ratios. ketone ester treatment has been shown to normalize these coenzyme ratios in diseased mouse tissue [16] . corticosteroid hormone administration has been used in an attempt to blunt the cytokine storm in several human respiratory viral infections [28] . to be successful, this therapy must be administered at the appropriate time late in the infection cycle to allow the immune system to first mount a proper antiviral response. since identifying the appropriate timeframe for treatment for different patients is challenging, glucocorticoid therapy has been largely unsuccessful and may have even contributed to detrimental patient effects when used to treat influenza infection [189] . however, emerging data suggest that short-term dexamethasone treatment may be beneficial for sars-cov-2 infection [190] , and dexamethasone treatment was shown to decrease the mortality of patients with severe sars-cov-2 infection who were placed on a ventilator [191] . increasing r-bhb levels, together with a moderately high-fat diet, may be able to stabilize nucleotide coenzyme ratios to allow virally infected tissue to increase endogenous cortisol levels at the appropriate time in the infection cycle to decrease inflammation and blunt the cytokine storm. signaling through hdac inhibition. sars-cov-2 virions bind to angiotensin-converting enzyme 2 (ace2) receptors [192, 193] on the surface of host cells, such as aec ii, as a first step in viral entry [194] . the level of ace2 receptors decreases during aging and may also decrease due to endocytosis during sars-cov-2 infection [195, 196] . in this reninangiotensin signaling system, renin catalyzes the conversion of angiotensinogen into angiotensin 1 (ang i). angiotensin-converting enzyme 1 (ace1) then catalyzes the conversion of ang i into angiotensin ii (ang ii), which binds to the at1r receptor leading to vasoconstriction and proinflammatory, prooxidative, and profibrotic effects leading to tissue injury. ace2 is normally able to blunt these effects by cleaving ang i and ang ii into peptides that bind to the at2r and masr receptors that signal for vasodilation and anti-inflammatory, antioxidative, and antifibrotic effects leading to tissue protection (figure 8(a) ) [197] [198] [199] [200] [201] . increased levels of ang ii stimulate the synthesis of the proinflammatory cytokines il-6, ifn-γ, tnf-α, and il-1β [202] , but also the anti-inflammatory cytokines tgf-β1 and il-10, which may induce m2 macrophage polarization [203] and prevent the γδ t lymphocyte activation needed to initiate the antiviral immune response [204] . butyrate and other hdac inhibitors have been shown to decrease the expression of angiotensinogen, renin, and at1r to block this proinflammatory signaling [205, 206] . therefore, the use of exogenous ketones could lead to a balancing of signaling through the different arms of the renin-angiotensin system when proinflammatory signaling through ang ii predominates such as in aged individuals and subjects infected with sars-cov-2. cytokine production in different cell types. the effects of r-bhb on proinflammatory cytokine production in different cell types can vary greatly. for example, r-bhb, when given to isolated macrophages challenged with streptococcus uberis, was shown to increase the expression of il-1β and il-10 and the chemokines cxcl2 and ccl5 but had no effect on the expression of tnf-α and tgf-β [207] . in another report using isolated m1 peritoneal macrophages, r-bhb was shown to decrease the expression of il-15, but not il-1β, tnf-α, or il-6 [208] . in calf hepatocytes, r-bhb was shown to increase nf-κb activity and the expression of il-1β, tnf-α, and il-6 [209] , while ketosis had similar effects in the liver of cows [210] . the absence of ketolytic enzymes and presence of ketogenic enzymes in the liver may contribute in part to the proinflammatory response. in lps-stimulated bv-6 microglial cells, r-bhb was shown to decrease nf-κb activation and the expression of tnf-α, il-1β, and il-6 [211] . when infused into the rat brain prefrontal cortex for 21 days in a model of depression (chronic unpredictable stress paradigm), r-bhb was shown to prevent the increase in tnf-α and the decrease in corticosterone brought about by the depression-inducing stress [212] . peripheral injection of r-bhb was also able to decrease il-1β and tnf-α levels in the hippocampus of rats in this depression model [213] . in bovine aorta endothelial cells stimulated with lps, r-bhb was shown to decrease the expression of tnf-α and interferon [214] . the different results in different cell types and conditions clearly indicate that more research needs to be done to understand the complex regulation of cytokine production by r-bhb. sirtuin deacetylases. a major mechanism through which r-bhb restores metabolism and redox balance is through epigenetic regulation of gene expression by increasing histone beta-hydroxybutyrylation (figure 9 (c)) and inhibiting class i and iia histone deacetylases (hdacs) to increase histone acetylation. transcription factors and coactivators such as foxo1 [215, 216] , foxo3a [151] , and pgc-1α [217] are induced. complexly, hdac inhibitors can also lead to increased acetylation of foxo1, which reduces its activity at the promoters of genes for gluconeogenic enzymes in the liver to decrease blood glucose levels [218] . this is likely beneficial for inhibiting the cytokine storm as discussed above. these transcriptional regulators increase mitochondrial etc gene expression to help restore the nad + /nadh. they initiate an antioxidant gene expression program together with the induction of ppp enzymes [219, 220] to restore the nadp + /nadph. in tissues such as liver, increased r-bhb levels lead to hdac inhibition at the foxo1 promoter and increased gene expression decreasing proinflammatory cytokine expression [216] . amp kinase [221] and nad + -dependent sirt1 deacetylase [222] enzymes also act on foxo1 to increase its activity. insulin signaling through the akt pathway inhibits foxo1 activity [223] . the anti-inflammatory action of foxo1 may be due in part to its activity in lung macrophages where it binds to irf4 and stimulates the m2 state [224] . however, in tumor-localized macrophages, foxo1 has been shown to stimulate the proinflammatory 18 oxidative medicine and cellular longevity m1 state and il-1β production [225] . so, the effects of foxo1 on macrophage function appear to be dependent upon the environmental conditions. increasing nad + levels in the cytoplasm activates sirt2 to deacetylate glucose-6phosphate dehydrogenase (g6pd) to increase ppp flux and nadph production [226] , linking the ratios of the pyridine nucleotide coenzyme couples. sirt2 also deacetylates the inflammasome, inhibiting its function [227] , while sirt3 function also inhibits inflammasome activity by decreasing mitochondrial ros levels [148] . increased nucleocytoplasmic nad + level also increases the activity of sirt1, which deacetylates pgc-1α to stimulate mitochondrial etc function [228] leading to increased mitochondrial nad + /nadh. the increased mitochondrial nad + /nadh activates mitochondrial sirt3 to deacetylate and activate mitochondrial sod2 [229] , isocitrate dehydrogenase 2 (idh2) [230] , and the 39 kd subunit of etc complex i [231] to decrease ros levels and decrease the mitochondrial nadp + /nadph. the decreased mitochondrial nadp + /nadph maintains reduced mitochondrial gssg/gsh to prevent the glutathionylation of etc complex i and the oxidation of cardiolipin that decrease complex i activity and decrease the matrix space nad + /nadh [232] . other important genes induced by the foxo1 transcriptional regulator to restore metabolism and decrease inflammation and ros production include gpr109a, lactate dehydrogenase b (ldhb), thioredoxin 2 (txn2), pepck1, and the nad + synthesis genes nampt and nmnat2 [233] . expression of pgc-1α and err-α. pgc-1α and err-α (estrogen-related receptor-α), a binding partner of pgc-1α involved in the induction of mitochondrial etc gene expression [234] , are the transcriptional regulators that are known to induce oxct1 gene expression in myotubes to increase the levels of its gene product scot (figure 9 (f)) [235] . err-α, which is widely expressed, is also required for adipose tissue thermogenesis [236] and is induced by fasting, calorie restriction, cold exposure, and exercise [237, 238] . the ketogenic diet has been shown to increase pgc-1α levels in muscle [239] , neurons [95] , and brown adipose tissue [240] . these data suggest that err-α and pgc-1α are likely the transcriptional regulators that induce oxct1 and etc gene expression in the lungs during the ketogenic diet to increase ketolysis and mitochondrial biogenesis [113] . scot activity has been shown to be decreased by tyrosine nitration [241] and increased by tryptophan nitration [242] . scot activity was also inhibited by acetylation and activated by sirt3-mediated deacetylation [243] . high-fat diets can decrease pgc-1α levels in the liver, decreasing its suppression of nf-κb and leading to increased cytokine production [244] . contrary to this result, a relatively highfat diet in the presence of ketone ester was shown to increase pgc-1α levels and mitochondrial function in brown adipose tissue to stimulate thermogenesis [94, 245] . therefore, the consumption of ketone ester may reverse the effects of a high-fat diet on pgc-1α expression in some tissues to stimulate fatty acid oxidation and prevent the accumulation of fat in tissues that is associated with negative health outcomes. uncoupling can restore the nad + /nadh and nadp + /nadph ratios. once the viral-and cytokine storm-induced changes in the mitochondrial nad + /nadh have been partially restored through r-bhb-mediated signaling, enzyme inhibition, and upregulation of gene expression, the nad + -dependent bdh1 enzyme can more effectively catalyze the conversion of r-bhb to acetoacetate. acetoacetate is then metabolized to acetoacetyl-coa, which is metabolized into two molecules of acetyl-coa. as mentioned above, this pathway of acetyl-coa synthesis becomes especially important under conditions of viral infection because pdk4 expression is upregulated leading to pdc inhibition. 8.3.1. nicotinamide nucleotide transhydrogenase. nicotinamide nucleotide transhydrogenase (nnt) is an enzyme that uses energy from the mitochondrial inner membrane proton gradient to synthesize nadph and nad + from nadp + and nadh. nnt gene expression is likely induced by foxo3a, since there are binding sites for foxo3a in the nnt promoter [233] and since the c. elegans nnt homolog nnt-1 is induced by the c. elegans foxo homolog daf-16 [246, 247] . nnt activity is likely high during times of mitochondrial etc dysfunction, such as during a cytokine storm, as decreased mitochondrial nad + /nadh and increased mitochondrial nadp + /nadph stimulate nnt function in the normal nadph-synthesizing and nadhhydrolyzing direction. status. recent evidence suggests that when glucose levels and ppp activity are low, serine and glycine are catabolized in mitochondria, which stimulates one-carbon metabolism, to generate nadph [248] . there are mechanisms in place to use the mitochondrial matrix space-synthesized nadph to prevent product inhibition of enzyme function, the most important being the fueling of glutathione reductase and thioredoxin reductase to combat ros. alternatively, the nadph equivalents can be shuttled to the cytoplasm using the citrate-pyruvate shuttle. this involves the catabolism of glutamine and glutamate to alpha-ketoglutarate, which can lead to idh2 functioning in the opposite direction of its normal citric acid cycle activity to oxidize nadph and form isocitrate in the process called reductive carboxylation [249, 250] . isocitrate can then be further metabolized to citrate. this citrate, together with other citrate molecules, such as those derived from r-bhb metabolism, gets shuttled into the cytoplasm though the mitochondrial citrate carrier protein (cic) as part of the citrate-pyruvate shuttle (see figure 3 ). in the cytoplasm, the citrate can be converted to acetyl-coa and oxaloacetate by atp-citrate lyase (acly). the acetyl-coa can function in histone acetylation or fatty acid synthesis, while the oxaloacetate can be converted to malate by malate dehydrogenase 1 (mdh1) to restore the cytoplasmic nad + /nadh. malate can then be converted to pyruvate by nadp + -dependent malic enzyme (me1), which concurrently synthesizes nadph [248] . in the final step, the pyruvate is shuttled back into the mitochondrial matrix space, where it is metabolized by pyruvate carboxylase to form oxaloacetate. the net result is 2atp + nadh + nadp + − >2adp + nad + + nadph, which contributes to the restoration of the redox state. the result on the redox state is very similar to that which occurs due to the nnt reaction, except the nad + and nadph are formed in the cytoplasm instead of the mitochondrial matrix. increased levels of citrate and acetyl-coa in the cytoplasm, which would likely occur as a result of increased shuttle function, inhibit glycolysis at phosphofructokinase [251] and pyruvate kinase [252] , respectively, which would further aid in the restoration of nad + /nadh. in m1-polarized macrophages, citrate-pyruvate shuttle function can provide nadph that fuels nadph oxidasemediated ros production and contributes to inflammation. these m1 macrophages upregulate the expression of cis-aconitate decarboxylase (irg1/acod1) to convert the citric acid cycle metabolite cis-aconitate to itaconate, which exerts anti-inflammatory actions to restrain the m1 response, by inhibiting etc complex ii activity to decrease ros production and by activating the nrf2 (nfe2l2) transcriptional regulator. therefore, inhibitors of cic and acly have been shown to be anti-inflammatory compounds [253] , but these inhibitors may have deleterious effects on the cytoplasmic and mitochondrial redox states in other cell types. in a related redox shuttle, the citrate-malate shuttle, the cytoplasmic malate is imported into the mitochondrial matrix and therefore no cytoplasmic nadph is synthesized. however, this shuttle is slightly more energy efficient, using the hydrolysis of only one molecule of atp. a third shuttle system that exports citrate from the mitochondrial matrix is the citratealpha-ketoglutarate shuttle. in this shuttle, cytoplasmic citrate is converted into isocitrate and then further into alphaketoglutarate by cytoplasmic aconitase (aco1) and isocitrate dehydrogenase 1 (idh1), respectively, with the latter reaction synthesizing nadph to decrease the cytoplasmic nadp + /-nadph [14] . the alpha-ketoglutarate can then be transported back into the mitochondrial matrix. idh1 expression is downregulated in m1-polarized macrophages when measured 24 hours after stimulation with lps [254] to turn off citrate-alpha-ketoglutarate shuttle flux and stimulate citratepyruvate shuttle function. however, two to four hours after lps stimulation of macrophages, the alpha-ketoglutaratedependent histone demethylase genes kdm6b [255] and phf2 [256] are induced to increase inflammation [257] . therefore, the citrate-alpha-ketoglutarate shuttle proteins cic, aco1, and idh1 may play a role early in the m1 polarization process to provide nucleocytoplasmic alphaketoglutarate for the function of these histone demethylase enzymes before the shuttle is turned off. another important mechanism through which a ketogenic diet restores the mitochondrial nad + /nadh during mitochondrial etc dysfunction is through inducing the expression of mitochondrial uncoupling proteins. the rate of nadh oxidation at complex i is normally limited by matrix space adp levels. the presence of uncoupling proteins removes this limitation by allowing protons to flow back into the matrix space to produce heat. this increases the rate of complex i nadh oxidation to increase the mitochondrial nad + /nadh. the cytoplasmic nad + /nadh can also be altered by mitochondrial redox changes through malate-aspartate shuttle function. another benefit of partial mitochondrial uncoupling is the slight decrease in the mitochondrial membrane potential that greatly decreases the generation of superoxide at etc complexes i and iii [258, 259] . one drawback of the increased expression of uncoupling proteins is the decrease in atp generation. the ketogenic diet increases ucp1 expression in brown adipose tissue [240] and ucp2 [95] , ucp4, and ucp5 [260] in the brain. increases in uncoupling protein levels have been shown to parallel increases in pgc-1α levels. administration of ketone ester to mice has been shown to increase the cytoplasmic nad + /nadh and decrease the cytoplasmic nadp + /nadph [16] and likely utilizes mitochondrial uncoupling and the other mechanisms listed above to restore redox balance. conditions to restore the redox state. the mildly elevated ros production from r-bhb metabolism from a ketogenic diet leads to the activation of the nrf2 transcriptional regulator [261] , which stimulates antioxidant response element (are) gene expression. paraquat, a redox cycling agent, which greatly increases superoxide production from etc complex i, was shown to decrease nrf2 levels, by a mechanism described in detail below, which was restored by the administration of r-bhb [262] . activation of nrf2 blunts the cytokine storm through inducing the expression of antioxidant system enzymes including heme oxygenase-1, sod2 [263] , nadp(h) quinone oxidoreductase (nqo1), gamma-glutamylcysteine synthetase (gclc), thioredoxin (txn), thioredoxin reductase 1 (txnrd1) [264] , and multiple enzymes for nadph synthesis including idh1, malic enzyme 1 (me1), and four enzymes of the ppp including g6pd, 6-phosphogluconate dehydrogenase (pgd), transaldolase, and transketolase [265] . nrf2 can be activated by hydrogen peroxide when the ratio of nadp + /nadph is not too high or too low as shown in figure 9 (e). for nrf2 activation to occur, superoxide produced by the etc in the mitochondrial matrix is converted into hydrogen peroxide by sod2. hydrogen peroxide is then transported out of the mitochondrial matrix through aquaporins present in the inner mitochondrial membrane. in the cytoplasm, the redox-sensitive chaperone dj-1 is activated when cysteine 106 sulfhydryl is oxidized to sulfenic acid by hydrogen peroxide. this activated form of dj-1 is able to release keap1 from nrf2 allowing nrf2 to enter the nucleus and induce gene expression. when the nadp + /-nadph is too low, it prevents dj-1 from being oxidized and activated. when the nadp + /nadph is too high, cysteine 106 in dj-1 becomes overoxidized to sulfonic acid and dj-1 is destabilized, ubiquitinated, and degraded by the proteasome, so nrf2 is not activated [266] . therefore, r-bhb metabolism likely preserves the function of nrf2 by providing the proper nadp + /nadph ratio, which maintains low to moderate levels of cytoplasmic hydrogen peroxide. 20 oxidative medicine and cellular longevity 8.5. hif1-α stabilization by rns leads to proinflammatory cytokine production. hypoxia-inducible factor-1α (hif-1α) is the master transcriptional regulator of hypoxic gene expression. during normoxia, hif-1α is hydroxylated on prolines, which stimulates its binding to von hippel-lindau (vhl) protein that targets it for proteasomal degradation. hif-1α is also hydroxylated on asparagine residues by fih-1 (factor inhibiting hif-1-1) to prevent hif-1α from binding to its coactivator cbp/p300. during hypoxia, the oxygendependent prolyl and asparaginyl hydroxylases are inactive stabilizing the transcriptionally active form of hif-1α. hif-1α can also be activated by ros or rns. hif-1α induces the expression of glucose transporters, glycolytic enzymes, and pdk1 to inhibit pdc and shunt glycolysis-derived carbon flux away from mitochondria when oxidative phosphorylation is compromised. figure 9 (d) shows the snitrosylation and activation of hif-1α by peroxynitrite. s-nitrosylation of hif-1α prevents the interaction with vhl for stabilization and inhibits asparagine hydroxylation for activation [267] . hif-1α can also induce expression of the proinflammatory cytokines tnf-α and il-6 by upregulating nf-κb [268] . hif-1α has been shown to be stabilized by increased pyruvate levels [269] , such as those that occur following pdc inhibition, or by increased succinate levels [270] . the circadian transcriptional regulator bmal1, which can be induced by the hormone melatonin [271] , decreases hif-1α stability and levels to increase mitochondrial oxidative metabolism [272] . in lung aecs, stabilization of hif1-α was shown to cause er stress and chop-mediated apoptosis [273] . iav infection was shown to induce the nuclear translocation of hif-1α by activating the c-jun n-terminal kinase (jnk) signaling pathway to increase proinflammatory cytokine expression [274, 275] . however, hif-1α may also play a role in suppressing iav infection as hif-1α deficiency stimulated iav replication in aec ii cells by increasing autophagy [276] . rsv infection was shown to stabilize hif-1α by increasing nitric oxide and peroxynitrite levels [277] . this resulted in increased nf-κb, proinflammatory cytokine levels, and viral replication [278, 279] . therefore, increasing levels of r-bhb to decrease rns levels will likely be able to decrease the activation of hif-1α and its downstream inflammatory mediators to mitigate sars-cov-2 and other respiratory viruses. 9. the effects of r-bhb on cells of the immune system 9.1. increased nadph can increase or decrease ros levels depending upon the cell type. nadph has roles both in the synthesis of superoxide through its role as a cofactor for nox2 and in peroxide detoxification through its roles as cofactors for glutathione reductase and thioredoxin reductase. in most cell types, the lower k m of nadph for the reductase antioxidant enzymes allows the antioxidant effect to predominate [280] [281] [282] . however, in some cell types such as macrophages and neutrophils, the high expression level of nox enzymes allows ros production to predominate. lung aecs also have relatively high levels of nox2, nox4, duox1, and duox2 [9] . future studies should address how altered nadp + /nadph regulates ros production in these cells and if increased ketone body levels increase nadph levels in macrophages, neutrophils, and lung epithelial cells to increase nadph oxidase activity to stimulate host defenses against pathogens. in lung epithelial cells, knockdown of g6pd to decrease nadph synthesis reduced nox2 activity to decrease the antiviral response [283] . in mice, it has been shown that decreasing nadph synthesis by inhibiting g6pd and ppp flux with 6-aminonicotinamide decreased lps-induced inflammation in a model of acute lung injury [284] . this data is consistent with the protective effects of the nox2 inhibitor for iav infection [68] . the expression of sod2 and catalase is induced by r-bhb-mediated hdac inhibition [151] , so when r-bhb increases nadph levels to stimulate nadph oxidase activity, it also increases ros detoxification enzymes to prevent excessive oxidative stress that may lead to a cytokine storm. increased nadph levels also have been shown to stimulate antiviral immunity by decreasing the level of the nadph sensor protein hscarg, which is a negative regulator of nf-κb transcription. through this mechanism, increased nadph levels were shown to increase expression of the mx1 and tnf-α genes to decrease human coronavirus infection [285] . one other potential proinflammatory action of the high nadph levels from r-bhb metabolism is the reduction of dihydrobiopterin (bh2) to tetrahydrobiopterin (bh4). bh4 is an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases [286] . so, increased nadph levels may increase nitric oxide synthase activity leading to rns production and inflammation. however, when bh4 levels are low, nitric oxide synthases synthesize superoxide instead of nitric oxide [287] . in this way, increased nadph decreases ros production as it increases rns production. this function could potentially decrease toxic peroxynitrite levels when superoxide is limiting for its synthesis. function. lung-resident macrophage polarization to either a proinflammatory m1 state or an anti-inflammatory m2 state is largely controlled by the cytokines secreted by the other cells present in the environment [288] . r-bhbmediated hdac inhibition, gpr109a signaling, and inflammasome inhibition in these cells likely increase the amounts of anti-inflammatory cytokines, such as il-10, produced to stimulate more macrophages to the m2 state. these cytokines also influence the catabolic pathways that are activated to fuel cellular energy needs. m1 macrophages have increased glycolytic activity and lactate production due to the presence of a dysfunctional citric acid cycle [289] . increased nitric oxide levels may inactivate citric acid cycle enzyme aconitase (aco2) and the pdc e3 subunit dihydrolipoyl dehydrogenase (dld). nitric oxide also may decrease the activities of etc complexes i, ii, and iv in m1 macrophages [290] . the dysfunctional citric acid cycle resulted in the accumulation of citrate that increased fatty acid synthesis [291] and succinate that increased mitochondrial ros production [270] . this metabolic programming in m1 macrophages likely 21 oxidative medicine and cellular longevity evolved to increase proinflammatory cytokine production. m2 macrophages are programmed to oxidize pyruvate and fatty acids into acetyl-coa for normal citric acid cycle function and oxidative phosphorylation [292] , which has been shown to facilitate anti-inflammatory il-10 secretion [293] . since macrophages lack the enzyme bdh1, they cannot oxidize r-bhb [294] , but a ketogenic diet also increases the systemic levels of the ketone body acetoacetate that can be oxidized by macrophages. the r-bhb/acetoacetate ratio produced by the liver is proportional to the mitochondrial nad + /nadh ratio [295] , which is normally around five [14] , but varies from three to seven. in this regard, acetoacetate but not r-bhb was shown to be metabolized by macrophages to ameliorate liver fibrosis in mice [294] . neutrophils, another type of phagocyte contributing to the cytokine storm, have few mitochondria and very low expression of ketolytic genes and therefore likely cannot catabolize r-bhb or acetoacetate to a significant extent. reducing glucose uptake into ilc2s. a recent study examined the effects of reducing glucose levels, using a ketogenic diet, on allergen-induced lung inflammation in mice. results showed that ilc2s in the lungs must increase their uptake of both fatty acids and glucose from the environment to elicit allergen-dependent inflammation. a ketogenic diet reduced systemic glucose levels to decrease lung ilc2 glucose uptake to prevent airway inflammation in response to the allergen [296] . the γδ t cell-ilc2 response activated by the ketogenic diet in mice [113] was shown to be active in human infants and children on a normal diet, where it protected them from influenza infection [297] . however, this response appears to be suppressed starting in adolescence and replaced by the ilc1 system, until it is likely reawakened by the ketogenic diet. we speculate that the higher activity of the γδ t cell-ilc2 response in children may be one factor responsible for the less severe symptoms when the sars-cov-2 virus infects individuals in this age group. the increased mortality rate of infected older adults may also be in part due to the increase in inflammation and decline in mitochondrial function and cellular nad + and nadph levels with aging that decreases metabolic flexibility and the ability to "weather" the cytokine storm. 9.4. hdac inhibition and gpr109a signaling have anti-inflammatory effects on dendritic cells. dendritic cells play an important function in presenting antigens to t lymphocytes. dendritic cells have a high level of oxidative metabolism until they are activated through their tlrs, at which point they switch to a primarily glycolytic metabolism [298] , which is essential for their activation by providing cytoplasmic atp for phospholipid synthesis and signals for the remodeling and expansion of the secretory system for its enhanced function in the activated state [299] . the increased rate of glycolysis in dendritic cells was also required for their secretion of interferon-α to mitigate iav infection. vaccination against iav was able to increase glycolytic function in the dendritic cells [300] . human monocyte-derived dendritic cells cultured with butyrate showed decreased pro-inflammatory cytokine and chemokine production [301] . this is likely due to the ability of hdac inhibition and gpr109a signaling in dendritic cells to promote treg cells [302] . the mechanism may be at least partially metabolic in nature. dendritic cells were activated by exposure to lps in the presence or absence of butyrate. butyrate decreased the oxygen consumption rate and blocked the increase in extracellular acidification rate (due to lactate export) used as an indicator of the glycolytic rate [302] . butyrate functioning as an hdac inhibitor also inhibited the formation of dendritic cells from bone marrow stem cells [303] . administration of ethyl pyruvate, a cell-permeable pyruvate precursor with known anti-inflammatory properties [304] , was shown to inhibit the activation of dendritic cells by single-strand rna that binds to tlr7. ethyl pyruvate decreased glycolytic and oxidative metabolism and blocked dendritic cell activation through decreasing erk and akt signaling and decreasing the production of nitric oxide [298] . therefore, ethyl pyruvate administration may be a potential therapy to block the cytokine storm during the later stages of sars-cov-2 infection. restore redox and energy levels. increasing r-bhb levels may also stimulate the immune system by enhancing b or t lymphocyte function. recent evidence links the ketogenic diet in mice with increasing levels of δγ t cells in adipose tissue [114] , where the δγ t cells stimulate a thermogenic program [305] , in part responsible for the weight loss effects frequently provided by the ketogenic diet. the ketogenic diet did not alter the level of ketone body metabolism genes in lung γδ t cells, but it did upregulate the expression of mitochondrial etc genes in these cells [113] . cells of the immune system express varied amounts of scot, used specifically for ketolysis, and bdh1, used for both ketogenesis and ketolysis. b and t lymphocytes possess the greatest amounts of bdh1 and scot of the blood cell types [134] , so the ketogenic diet or exogenous ketone treatment may enhance the energy levels and redox status in these cells more effectively. 9.5.1. a ketogenic diet normalizes the increased th17/treg ratio that occurs due to disease. there are two important subtypes of cd4 + t lymphocytes, th17 and treg cells. a ketogenic diet was shown to normalize the proinflammatory th17/treg ratio in the blood from epileptic patients [306] . a high-fat diet may favor the expansion of treg cells over th17 cells as tregs can take up and utilize fatty acids from the environment, while th17 do not have this ability [307] , so th17 cells must synthesize fatty acids from glucose, which is present at slightly lower levels when consuming the ketogenic diet [308] . intermittent fasting that increases r-bhb levels was also able to restore this ratio in mice with experimental autoimmune encephalomyelitis (eae), a model of multiple sclerosis [309] . hdac inhibition in naïve t cells leads to the expression of the foxp3 transcriptional regulator and treg conversion [310] . the longevity-promoting mtor inhibitor and antiaging calorie restriction mimetic rapamycin also decreased the th17/treg balance. this occurred through inhibition of glycolysis in th17 cells and stimulation 22 oxidative medicine and cellular longevity of fatty acid oxidation in tregs. fatty acid oxidation was stimulated by amp kinase activation [311] . a moderate dose of butyrate only induced differentiation of t cells to treg cells when administered together with tgf-β1. as tgf-β1 level is increased by viral infection, this should not pose a problem for the treatment of sars-cov-2 with ketone ester. in addition, hdac inhibition induces the expression of tgf-β1 in epithelial cells. however, administering a high dose of butyrate, even when added together with tgf-β1, was ineffective at inducing differentiation into il-10-producing treg cells. high doses of butyrate resulted in either normal t cells or ifn-γ-producing tregs [312] . therefore, butyrate or r-bhb may need to be present in the cell nucleus within a specific narrow concentration range for the partial inhibition of hdac function for the optimal treatment of sars-cov-2. 9.5.2. r-bhb stimulates the formation of cd8 + memory t cells. cd8 + memory t cells readily oxidize fatty acids and, like hepatocytes, have the rare ability to simultaneously express the genes for gluconeogenesis and ketogenesis [313] . the high level of expression of pepck1, the rate-limiting step in gluconeogenesis, resulted in the depletion of cellular oxaloacetate levels. therefore, the acetyl-coa formed from fatty acid beta-oxidation was not able to enter the krebs cycle and was therefore used for ketogenesis. the increased r-bhb levels led to increased beta-hydroxybutyrylation of histone proteins in the foxo1 and pgc-1α promoters leading to increased gene expression. increased gluconeogenesis led to increased glucose levels that stimulated the ppp synthesis of nadph required for the long-term protection of the cd8 + memory t cells against ros [314] . metabolic therapy with ketone ester should enhance this endogenous epigenetic program to promote the survival of cd8 + memory t cells to facilitate immune function when a patient is reexposed to sars-cov-2. the data presented here suggest two types of clinical studies with covid-19 patients that would provide data on the efficacy of a ketone-based metabolic therapy. these include the following: (1) a determination of forced vital capacity by spirometry of covid-19 patients consuming ketone ester and a moderately high-fat diet (forced vital capacity is a measure of lung function based on taking a full breath and exhaling as much volume of air as possible) (2) a randomized trial of covid-19 patients consuming ketone ester with a moderately high-fat diet with evaluations of length and severity of infection and patient mortality a ketone-based metabolic intervention for covid-19 patients will likely be initiated at one of the three general stages of disease progression. during all three stages, the basic treatment of raising blood ketone levels to 1 to 2 mm with exogenous ketones, increasing consumption of dietary fats, and taking enteric-coated sodium bicarbonate to buffer blood ph will likely be beneficial. the first stage is the onset of disease symptoms. during this stage, a moderate carbohydrate diet will allow normal blood glucose levels to boost immune cell function. the second stage is when the severity of symptoms requires hospitalization and/or ventilation. at this middle stage of infection, a limited carbohydrate diet would be beneficial to lower glucose metabolism and its associated proinflammatory signaling. the third stage is after ventilator use ceases and difficulty in breathing ensues. once again, a low-carbohydrate diet at this late stage is hypothesized to facilitate beneficial anti-inflammatory processes. the expected outcomes no matter when treatment is initiated are a decrease in the incidence of progression to ards, protection of organs from oxidative and inflammatory damage, and increased clearance of the virus to shorten the duration of the infection. patient data analyzed independently for the different stages of therapy initiation may yield important insights into the relative time when ketone-based metabolic intervention is most effective. the studies suggested above, together with long-term patient monitoring, may also yield important insights into the significant post-covid-19 patient complications. there is evidence from sars-cov-1 infections that psychiatric and chronic fatigue issues continued for four years after infection [315] . unfortunately, there are anecdotes from recovered covid-19 patients describing similar lingering morbidities. we further hypothesize that the severity of these morbidities including chronic fatigue, depression, posttraumatic stress disorders, panic disorders, and somatoform pain disorders may be blunted by a ketone-based metabolic therapy due to the mitigation of cell death and tissue damage. the sars-cov-2 virus may become a sustained threat to global health. this review has described many of the molecular mechanisms through which an exogenous ketone-based metabolic therapy together with a moderately high-fat diet may stimulate host cell metabolism and defenses as a possible treatment to blunt the cytokine storm associated with severe sars-cov-2 infection. a clinical trial testing this therapy on patients with sars-cov-2 is warranted. in addition, further mouse iav infection studies will aid in the determination of permissive dietary conditions under which exogenous ketone supplementation enhances immune function to facilitate viral clearance and decrease mortality. the authors declare no competing interests. dr. william seeds has no current role in the operation and no financial interest in drseeds.com, and his legal separation from that entity is pending. a transcriptome analysis identifies potential preventive and therapeutic approaches towards covid-19 how immune t-cell 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cells repression of transcriptional activity of forkhead box o1 by histone deacetylase inhibitors ameliorates hyperglycemia in type 2 diabetic rats peroxisome proliferator-activated receptor γ coactivator 1α (pgc-1α) promotes skeletal muscle lipid refueling in vivo by activating de novo lipogenesis and the pentose phosphate pathway foxo3 coordinates metabolic pathways to maintain redox balance in neural stem cells an ampk-foxo pathway mediates longevity induced by a novel method of dietary restriction in c. elegans deacetylation of foxo by sirt1 plays an essential role in mediating starvation-induced autophagy in cardiac myocytes foxo1 regulates tlr4 inflammatory pathway signalling in macrophages foxo1 regulates allergic asthmatic inflammation through regulating polarization of the macrophage inflammatory phenotype foxo1 is a regulator of mhc-ii expression and anti-tumor effect of tumor-associated macrophages regulation of g6pd acetylation by sirt2 and kat9 modulates nadph homeostasis and cell survival during oxidative stress an acetylation switch of the nlrp3 inflammasome regulates aging-associated chronic inflammation and insulin resistance sirt1 functionally interacts with the metabolic regulator and transcriptional coactivator pgc-1α calorie restriction reduces oxidative stress by sirt3-mediated sod2 activation sirt3 mediates reduction of oxidative damage and prevention of agerelated hearing loss under caloric restriction a role for the mitochondrial deacetylase sirt3 in regulating energy homeostasis interplay between nadh oxidation by complex i, glutathione redox state and sirtuin-3, and its role in the development of insulin resistance the harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins estrogen-related receptor alpha and mitochondria: tale of the titans skeletal muscle pgc-1α modulates systemic ketone body homeostasis and ameliorates diabetic hyperketonemia in mice orphan nuclear receptor 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dj-1, a cancer-and parkinson's diseaseassociated protein, stabilizes the antioxidant transcriptional master regulator nrf2 nitric oxide donor, (±)-s-nitroso-n-acetylpenicillamine, stabilizes transactive hypoxia-inducible factor-1α by inhibiting von hippel-lindau recruitment and asparagine hydroxylation hypoxia-induced neutrophil survival is mediated by hif-1α-dependent nf-κb activity hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the warburg effect in carcinogenesis succinate is an inflammatory signal that induces il-1β through hif-1α melatonin: roles in influenza, covid-19, and other viral infections bmal1 integrates mitochondrial metabolism and macrophage activation hif-1α triggers er stress and chop-mediated apoptosis in alveolar epithelial cells, a key event in pulmonary fibrosis nuclear translocation of hif-1α induced by influenza a (h1n1) infection is critical to the production of proinflammatory cytokines mechanism of nuclear translocation of 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through a transporter (slc5a8)-dependent inhibition of histone deacetylases pyruvate is an endogenous anti-inflammatory and anti-oxidant molecule γδ t cells producing interleukin-17a regulate adipose regulatory t cell homeostasis and thermogenesis the effects of ketogenic diet on the th17/treg cells imbalance in patients with intractable childhood epilepsy de novo fatty acid synthesis controls the fate between regulatory t and t helper 17 cells the biochemistry of ketogenesis and its role in weight management, neurological disease and oxidative stress intermittent fasting confers protection in cns autoimmunity by altering the gut microbiota metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation rapamycin modulate treg/th17 balance via regulating metabolic pathways: a study in mice the microbial metabolite butyrate induces expression of th1-associated factors in cd4(+) t cells ketogenesis-generated βhydroxybutyrate is an epigenetic regulator of cd8 + t-cell memory development a pck1-directed glycogen metabolic program regulates formation and maintenance of memory cd8 + t cells mental morbidities and chronic fatigue in severe acute respiratory syndrome survivors: long-term follow-up we would like to thank dr. brianna stubbs for reading the paper and offering several helpful suggestions, david lamps for drawing the cells and organs for the graphics, todd king 23 oxidative medicine and cellular longevity for proofreading the paper, and frank llosa for providing the idea to look into the potential effects of ketones on covid-19 and offering helpful discussion. key: cord-314825-fzba05wn authors: chauhan, ravendra p.; gordon, michelle l. title: a systematic review analyzing the prevalence and circulation of influenza viruses in swine population worldwide date: 2020-05-08 journal: pathogens doi: 10.3390/pathogens9050355 sha: doc_id: 314825 cord_uid: fzba05wn the global anxiety and a significant threat to public health due to the current covid-19 pandemic reiterate the need for active surveillance for the zoonotic virus diseases of pandemic potential. influenza virus due to its wide host range and zoonotic potential poses such a significant threat to public health. swine serve as a “mixing vessel” for influenza virus reassortment and evolution which as a result may facilitate the emergence of new strains or subtypes of zoonotic potential. in this context, the currently available scientific data hold a high significance to unravel influenza virus epidemiology and evolution. with this objective, the current systematic review summarizes the original research articles and case reports of all the four types of influenza viruses reported in swine populations worldwide. a total of 281 articles were found eligible through screening of pubmed and google scholar databases and hence were included in this systematic review. the highest number of research articles (n = 107) were reported from asia, followed by americas (n = 97), europe (n = 55), africa (n = 18), and australia (n = 4). the h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses were the most common influenza a virus subtypes reported in swine in most countries across the globe, however, few strains of influenza b, c, and d viruses were also reported in certain countries. multiple reports of the avian influenza virus strains documented in the last two decades in swine in china, the united states, canada, south korea, nigeria, and egypt provided the evidence of interspecies transmission of influenza viruses from birds to swine. inter-species transmission of equine influenza virus h3n8 from horse to swine in china expanded the genetic diversity of swine influenza viruses. additionally, numerous reports of the double and triple-reassortant strains which emerged due to reassortments among avian, human, and swine strains within swine further increased the genetic diversity of swine influenza viruses. these findings are alarming hence active surveillance should be in place to prevent future influenza pandemics. influenza viruses are the members of orthomyxoviridae family and have a wide host range [1] [2] [3] [4] [5] [6] . due to unique physiology, swine are considered the "mixing vessel" for influenza viruses [7] . four types of influenza viruses have been reported in swine i.e., influenza a virus (iav), influenza b virus (ibv), influenza c virus (icv), and influenza d virus (idv). the genomes of iav and ibv have eight gene segments of single-stranded negative sense rna while the genomes of icv and idv have seven gene segments [8] . among the eight gene segments of iav and ibv, the hemagglutinin (ha) and neuraminidase (na) are most significant and crucial for the pathogenicity of these viruses which neuraminidase (na) are most significant and crucial for the pathogenicity of these viruses which determine the antigenic properties. the ha gene regulates the attachment of virus particles to the host receptor while na gene regulates the release of progeny virus into the host cell. co-infection of swine with two or more iav strains may trigger the reassortment [9] which in turn, could facilitate the emergence of new influenza virus strains [10] [11] [12] . point mutations which occur due to an errorprone rna polymerase that lacks the ability of proof-reading and corrections during replication may also complement the genetic diversity of the influenza viruses [13] . the mechanisms of reassortment and point mutations may give rise to "antigenic shift" and "antigenic drift" within ha and na genes, respectively, facilitating the emergence of new subtypes and lineages of influenza viruses. as a result, total 18 ha and 11 na subtypes of iav [14] [15] [16] and two lineages (victoria/b and yamagata/b) of ibv have been reported so far in different hosts [17, 18] . the host range of iav and ibv is determined by their specificity to sialic acid receptors. the ha proteins of iav can bind to α-2,3 and α-2,6 sialic acid receptors present in avian and human trachea, respectively [19] [20] [21] . interestingly, swine trachea has both, α-2,3 as well as α-2,6 sialic acid receptors, due to which swine can become infected with avian and human strains of influenza viruses [22] . the genomes of icv and idv have a gene segment termed as "hemagglutinin-esterase-fusion" (hef) which carries out the functions similar to that of ha and na genes of iav and ibv. the hef is responsible for attachment and release of icv and idv virus particles into the host cell [23] [24] [25] . the particles of both virus types icv and idv bind to 9-o-acetylated sialic acid receptors of the host [25] . several studies have shown that human and avian origin influenza viruses can be transmitted to swine in natural settings and thus may evolve into new strains of reassorted influenza viruses [26, 27] . historically, the first flu pandemic (spanish flu) hit the human population in 1918 [28] and killed approximately 50 million people globally [29] . the 1918 influenza pandemic emerged as a result of reassortment in which human h1 virus acquired avian (poultry) n1 neuraminidase along with internal protein genes and evolved into what is now termed as "classical h1n1" virus [30] ( figure 1 ). the second flu pandemic occurred in 1957 (asian flu) and was traced to the h2n2 virus which killed approximately two million people [31] . the third flu pandemic hit the human population in 1968 (hong kong flu) with an h3n2 outbreak and killed approximately two million people [31, 32] . the second flu pandemic occurred in 1957 (asian flu) and was traced to the h2n2 virus which killed approximately two million people [31] . the third flu pandemic hit the human population in 1968 (hong kong flu) with an h3n2 outbreak and killed approximately two million people [31, 32] . the most recent flu pandemic (swine flu) originated in swine in mexico during march-may 2009 [33] databases. this systematic review is part of a research project which has already obtained the relevant ethical approvals from the animal research ethics committee (arec), university of kwazulu-natal, durban, south africa; arec reference: arec/041/019d. additionally, the authors have the required permission to do research in terms of section 20 of the animal diseases act, 1984 (act no. 35 of 1984) from the department of agriculture, forestry and fisheries (daff), government of the republic of south africa; daff reference: 12/11/1/5/4 (1425). the original research articles and case reports on the serological and virological prevalence of all the four genera of influenza viruses i.e., iav, ibv, icv and idv were downloade [51] [52] [53] nigeria h1n1, h3n2, a(h1n1)pdm09, h5n1 none [52, [54] [55] [56] [57] [58] [59] ghana h3n2 none [57] egypt h5n1, h5n2, h9n2, a(h1n1)pdm09 none [60, 61] kenya h1n1, h3n2, a(h1n1)pdm09 none [62] [63] [64] benin, cote d'ivoire none none [65] reunion island a(h1n1)pdm09 none [66] togo a(h1n1)pdm09 none [67] uganda iav none [68] [51] [52] [53] nigeria h1n1, h3n2, a(h1n1)pdm09, h5n1 none [52, [54] [55] [56] [57] [58] [59] ghana h3n2 none [57] egypt h5n1, h5n2, h9n2, a(h1n1)pdm09 none [60, 61] kenya h1n1, h3n2, a(h1n1)pdm09 none [62] [63] [64] benin, cote d'ivoire none none [65] reunion island a(h1n1)pdm09 none [66] togo a(h1n1)pdm09 none [67] uganda iav none [68] asia china h1n1, h1n2, h3n2, a(h1n1)pdm09, h5n1, h9n2, h4n1, h4n6, h5n3, h10n5, h4n8, h6n6, h7n9, h3n8 icv, idv bhutan h1n1, a(h1n1)pdm09 none [107] cambodia h1n1, h3n2, a(h1n1)pdm09 none [108, 109] japan h1n1, h1n2, h3n2, a(h1n1)pdm09 icv [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] south korea h1n1, h1n2, h3n2, a(h1n1)pdm09, h7n2, h5n2, h3n1 none [127] [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] thailand h1n1, h1n2, h3n2, h3n1, a(h1n1)pdm09 none [144] [145] [146] [147] [148] [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] viet nam h1n1, h1n2, h3n2, a(h1n1)pdm09, h5n1 none [160] [161] [162] [163] india h1n1, h2n2, h3n2, a(h1n1)pdm09 none [164, 165] lebanon h9n2 none [166] malaysia h1n1, h3n2 none [167] laos h3n2 none [168] russia h1n1 none [169] taiwan iav ibv [170, 171] indonesia h5n1 none [172] sri lanka h3n2, a(h1n1)pdm09 none [173] kazakhstan h1n1, h3n2 none [174] australia australia h1n1, h1n2, h3n2, a(h1n1)pdm09 none [175] [176] [177] [178] [179] [180] [181] denmark h1n1, h1n2, h3n2 none [182] [183] [184] united kingdom h1n1, h1n2, h3n2, a(h1n1)pdm09, h1n7 ibv, icv [185] [186] [187] [188] [189] [190] [191] [192] finland h1n1, h1n2, h3n2, a(h1n1)pdm09 none [193, 194] france h1n1, h1n2, h3n2, a(h1n1)pdm09 none [195] [196] [197] [198] [199] germany h1n1, h1n2, h3n2, a(h1n1)pdm09 none [200] [201] [202] [203] greece h1n1, h1n2, h3n2, a(h1n1)pdm09 none [204] italy h1n1, h1n2, h3n2, a(h1n1)pdm09 idv [205] [206] [207] [208] [209] [210] [211] [212] [213] [214] [215] [216] [217] spain h1n1, h1n2, h3n2 none [218] [219] [220] [221] netherlands h1n1, h1n2, h3n2 none [222] norway a(h1n1)pdm09 none [223] [224] [225] [226] poland h1n1, h1n2, h3n2, a(h1n1)pdm09 none [227] [228] [229] czechoslovakia h3n2 none [230] hungary h1n1 none [231] czech republic h1n1, h1n2, h3n2 none [232] republic of ireland h1n1, h1n2, h3n2 none [232] luxembourg none idv [46] multiple european nations h1n1, h1n2, h3n2 none [233] north america canada h1n1, h1n2, h3n2, a(h1n1)pdm09, h4n6, h3n3 none usa h1n1, h1n2, h3n1, h3n2, a(h1n1)pdm09, h4n6, h2n3 ibv, idv mexico h1n1, h1n2, h3n2, a(h1n1)pdm09, h5n2 none [304] [305] [306] [307] [308] [309] guatemala h3n2, a(h1n1)pdm09 none [310] cuba h1n1, a(h1n1)pdm09 none [311, 312] trinidad & tobago h3n2, a(h1n1)pdm09 none [313] south america argentina h1n1, h1n2, h3n2, a(h1n1)pdm09 none [314] [315] [316] [317] [318] brazil h1n1, h1n2, h3n2, a(h1n1)pdm09 none [319] [320] [321] [322] [323] [324] [325] [326] [327] [328] [329] colombia a(h1n1)pdm09 none [330] peru a(h1n1)pdm09 none [331] chile iav, h1n2 none [332] [333] [334] pathogens 2020, 9, 355 8 of 54 the first report of iav in cameroonian swine appeared when a(h1n1)pdm09 virus was documented during 2009-2010. the youngest infected swine was four-month old which suggested that a(h1n1)pdm09 virus can infect the young piglets [51] . nine more swine herds in cameroon were found infected with a(h1n1)pdm09 and h1n1 viruses during may-june 2011 [52] . a multiple-site study including free-roaming and penned swine along with domestic poultry and columbiformes birds between december 2009 and august 2012 identified one iav positive swine at each of the two study sites. the inter-species transmission of iav was ruled out as all the birds were negative for the iav [53] . the first evidence of the past iav infection in nigerian swine appeared in 2008 when h1n1 and h3n2 virus antibodies were detected in swine sampled at three different locations [54] . shortly after that, in 2009, the first report of a(h1n1)pdm09 virus appeared in the nigerian swine when one swine herd was found seropositive for a(h1n1)pdm09 virus. interestingly, eight other swine herds were found seropositive for the h1n1 and four herds were found positive for human-like h3n2 viruses. the seroprevalence of iav further increased as 66 swine herds were detected positive for a(h1n1)pdm09 virus and 53 herds were found seropositive for h1n1 virus in 2012 [52] . the active infection (viral rna) of a(h1n1)pdm09 virus was first reported in the nigerian swine between july 2010-june 2012 when 18 a(h1n1)pdm09 virus isolates were retrieved from the swine in lagos. the zoonotic transmission of a(h1n1)pdm09 virus to the exposed human workers was ruled out as all the human samples were negative for the a(h1n1)pdm09 virus [55] . nineteen more a(h1n1)pdm09 and five human-origin h3n2 viruses were identified in the nigerian swine during 2013-2015 [56] . later one more report of the human strain of h3n2 virus appeared in swine during january-february 2014 [57] . a high seroprevalence of iav in a commercial piggery was reported in lagos. total 197 human and 281 swine sera samples were screened which determined that 87% human and 67% swine sera had antibodies for iav depicting the past exposure [58] but the active infection was absent given that all nasal swabs were negative for iav infection [58] . lately, highly pathogenic avian influenza virus (hpaiv) strain h5n1 was detected in 22 swine samples between december 2015 and february 2016 during an ongoing h5n1 disease outbreak in nigerian poultry [59] which indicated the inter-species transmission of h5n1 virus from poultry to swine [59] . a molecular study reported the negative prevalence of avian-like h5n1 and h5n2 viruses in egyptian swine in may 2008 [60] but the serological investigation identified h5n1 virus antibodies in seven and h5n2 virus antibodies in four swine sera samples [60] which suggested a past exposure of these swine to the viruses. the active h5n1 infection in egyptian swine was again ruled out in october 2013 as the viral rna could not be detected in 36 swine samples but interestingly, the antibodies against avian-like h5n1, h9n2, and a(h1n1)pdm09 viruses were detected in swine sera samples which suggested a past exposure [61] . interestingly, 122 of the 157 swine nasal swab samples collected during 2014 and 2015 were found positive for iav active infection using rt-pcr. as a result, ha subtyping identified 46 avian-origin h5n1, seven h9n2 and 69 a(h1n1)pdm09 viruses [61] . the first report of iav in swine in kenya appeared in may 2010 when a(h1n1)pdm09 virus were detected in eight swine samples collected from the asembo and kibera counties and at a nairobi based abattoir. the extended serological study further identified h1n1 and h3n2 viruses in swine during august 2011 to december 2012 [62] . the active iav infection was reported in four household members pathogens 2020, 9, 355 9 of 54 having acute respiratory illness while the backyard swine were negative for the iav and ibv in kiambu county during september 2013-august 2014. on the contrary, the serology identified the iav antibodies in 230 swine sera samples suggesting a past exposure of iav [63] . the a(h1n1)pdm09 virus was again reported in five swine samples collected from a slaughterhouse in kenya during september 2013-september 2014. interestingly, all the 288 human subjects including the slaughterhouse workers or the traders and farmers who had visited the slaughterhouse were negative for iav, hence ruled out the zoonotic transmission [ [66] and in togo during october 2012-january 2014 [67] . the human strain of h3n2 virus was detected in swine in ghana during january-february 2014 [57] . one more report documented iav in swine in two districts of uganda in 2015 [68] . overall, influenza viruses have been reported in swine from eight african countries including cameroon, nigeria, egypt, kenya, reunion island, togo, ghana, and uganda ( figure 4a ). the a(h1n1)pdm09 virus, which originated in mexican swine in 2009, has been reported in all except ghana and uganda. interestingly, the hpaiv strain of h5n1 has been reported in swine in nigeria and egypt while hpaiv strain h5n2 and low pathogenic avian influenza virus (lpaiv) strain h9n2 have also been reported in the egyptian swine (table 1) . china is considered the epicenter of influenza viruses [69] . the first seroprevalence of iav in chinese swine was documented during 1977-1982 when antibodies for 38 h1n1, 22 h3n2, 12 h4n6, 12 h5n3, and seven h9n2 viruses was detected in swine sera obtained from apparently healthy swine [70] . the first ever report of icv in swine was documented from the apparently healthy swine in beijing when 15 icv isolates were retrieved during january-december 1981 [38] . three isolates of reassortant h1n2 virus were identified after an influenza-like illness triggered abortions and mortalities in sows on a swine farm in november 2004 [71] . the same year, lpaiv strain h9n2 was isolated from the sick or dead swine in china which was the first ever isolate of h9n2 virus retrieved from a swine [72] . first human-origin h1n1 and four human-origin h3n2 virus isolates in chinese swine were retrieved during 2005-2006 [73] . further, two isolates of swine h3n2 viruses, four isolates of avian-origin hpaiv strain h5n1 and two isolates of h1n1 viruses were detected in swine nasal swab and lung tissue samples collected from swine in central provinces of china during 2004-2006 [74] . surprisingly, two isolates of equine influenza virus h3n8 were also detected in swine during december 2005 and january 2006 [74] . another report of avian-origin h9n2 virus in chinese swine was documented during 2006-2007 when four h9n2 virus isolates with closely related nucleotide sequences were retrieved from swine [75] . each of the two different investigations reported 19 h1n1, one h1n2 and nine h3n2 virus isolates from chinese swine during 2006-2009 [76, 77] ; the h1n2 virus and all nine isolates of h3n2 viruses were either double or triple-reassortant viruses [76] . the first report of hpaiv strain h5n1 in swine was documented during october 2008-may 2009 when two h5n1 virus isolates were retrieved from apparently healthy swine [78] . the third report of avian-origin h9n2 virus in chinese swine appeared when 144 apparently healthy swine across four provinces viz., yunnan, guangdong, fujian and zhejiang were found h9n2 positive over a four-year period during march 2008-march 2012. the frequent interactions of birds to the swine at the study sites was reported which was suspected to be the most likely source of infection [79] . further, a novel strain of avian-origin h4n1 virus was isolated from a chinese swine in 2009 [80] . several classical and avian-like h1n1, eurasian avian-like h1n1, triple-reassortant h1n1, h1n2, h3n2 and a(h1n1)pdm09 viruses were reported in chinese swine between 2009 and 2016 [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] . a triple-reassortant h1n1 virus having the internal genes of avian, human, and swine lineages of influenza viruses was reported from a two-month old piglet on a guangdong based swine farm in january 2010 [92] . three reassortant h3n2 virus isolates having internal genes of a(h1n1)pdm09 virus were reported in swine between november 2010 and june 2011 [93] . a three-year old boy was diagnosed with european origin avian-like h1n1 virus on a family swine farm in a rural area of the jiangsu province in december 2010 which speculated a zoonotic transmission from swine to the boy [94] . the first report of h10n5 avian-origin influenza virus in a domestic swine in hubei province further extended the diversity of swine influenza viruses and provided another evidence of interspecies transmission of avian influenza virus to the swine under natural conditions [95] . several other avian-origin h3n2, h4n8, h6n6, h7n9, h5n1, and h9n2 virus antibodies were detected in swine in china during april 2010-june 2014 [86, [96] [97] [98] . another interspecies transmission of avian-like h1n1 virus in southern china was observed when 219 swine and 61 swine farm workers were identified to be infected with avian-like h1n1 swine influenza virus between march 2011 and march 2013 [99] . further a zoonotic transmission of h9n2 virus was identified at a shandong based swine farm during may 2013-april 2014 when h9n2 virus antibodies were detected in 84 swine and four farm workers. the wild birds visiting swine feeding sites at the swine farm were speculated to serve as the carrier for h9n2 virus [100] . zoonotic transmission of h1n1 virus was reported on a swine farm in shandong province between march 2015 and february 2016 among the swine exposed human workers having influenza-like illness. as a result, five of the 32 (15.6%) nasal swab samples were found iav positive; a married couple exposed to swine were found infected with h1n1 virus [88] . the iav infection was also documented in 44 wild boars in jilin province of china between april 2015 and february 2016 [101] . the first report of the idv prevalence in chinese swine documented 21 idv positive swine in the guangdong province in 2016 [44] . the swine idv sequences shared high similarity (99-100%) with idv sequences reported earlier from the bovine species in china [102] which indicated the transmission of idv from bovine to swine in china. hong kong is a special administrative region while tibet is an autonomous administrative region under the control of people's republic of china. the h1n1 and h3n2 virus isolates were successfully retrieved from apparently healthy swine in hong kong during july 1993-june 1994 [103] . further, classical swine h1n1, h3n2 and avian-like h9n2 viruses were identified in hong kong based swine between march 1998-june 2000; two independent introductions of the avian-like h9n2 viruses were ascertained from avian species to the swine [27, 104, 105] . the first information of iav seroprevalence in tibetan swine appeared during april-december 2010 when antibodies against h1n1 and h3n2 viruses were detected in swine sera collected from tibet [106] . the first report of h1n1 seroprevalence in swine in bhutan appeared when h1n1 virus was detected in backyard as well as breeding swine during october 2011 and february 2012 [107] . the h1n1 virus was reported in swine over a five year-period between 2006-2010 while the a(h1n1)pdm09 and h3n2 viruses were identified only in 2010 [108] . later three triple-assortant h3n2 viruses were isolated and sequenced from the backyard swine between may 2011 and july 2012 [109] . the antibodies against a/hong kong(h3n2) virus termed as "a/swine/wadayama/5/69" were first detected in japanese swine in 1969 [110, 111] . the h3n2 virus seroprevalence in japanese swine was further documented in sendai city during 1977 to 1980 [112] ; the transmission between human and swine was also suggested [112] . the first active iav infection was reported when two reassortant h1n2 virus isolates were retrieved from the japanese swine having influenza-like disease in 1978. the isolated h1n2 virus was believed to be a recombinant of h1n1 and h3n2 viruses [113] . further 340 swine were diagnosed with h1n1 antibodies in toyama prefecture between 1978-1982. a lower seroprevalence was observed during the summer months while the seroprevalence was relatively higher during the winter season [114] . again, one more h1n2 virus was isolated and characterized from the sows in ehime prefecture in september 1980 [115] . intriguingly, 18 h1n1, h1n2 and h3n2 viruses were detected in swine imported from the united states, however, all the imported swine from the europe were negative for the iav infection. this was the first report of the iav infection in the imported swine [116] . the icv seroprevalence (19%) in japanese swine was first reported in the hyogo prefecture during july 1981-june 1982 [117] but swine in yamagata prefecture were found seronegative for the icv between august 1979 and march 1986 which suggested a localized transmission of icv in swine within hyogo prefecture [118] . several other reassortant h1n2 virus isolates were reported in japanese swine after 1991 [119] . one novel reassortant h1n2 virus appeared to have emerged from the a(h1n1)pdm09 virus was reported in swine in gunma prefecture while two other h1n2 viruses appeared to have emerged from the japanese h1n2 viruses with internal genes from a(h1n1)pdm09 virus. one more h1n2 virus was detected in swine which was closely related to the japanese h1n2 virus [120] . the immunohistochemistry identified lesions in the lungs of the sick swine infected with reassortant h1n2 virus [121] . additionally, several h1n1 and h3n2 viruses have also been reported in japanese swine between 1990 and 2017 [122, 123] . interestingly, six h1n1 virus isolates were identified with reassorted genes from a(h1n1)pdm09 virus while one h1n1 isolate appeared to have h1 gene from japanese swine influenza virus with internal genes of a(h1n1)pdm09 virus. further, one h3n2 virus isolate was determined to have genes of japanese swine influenza and a(h1n1)pdm09 viruses [124] . these results reflected the occurrence of the reassortment events between japanese swine influenza and a(h1n1)pdm09 viruses. iav seroprevalence has lately been reported in wild boars (sus scrofa leucomystax) in japan. three wild boars in the yamaguchi prefecture were found seropositive for a(h1n1)pdm09 virus while nine wild boars in tochigi prefecture were seropositive for the swine h1n1 virus. but, the active iav infection could not be identified in these wild boars as all the nasal swab samples were negative for iav and ibv [125] . in a more recent investigation, fifteen wild boars were found seropositive for a(h1n1)pdm09 virus in kagoshima prefecture between november 2014-december 2017 while two of these fifteen wild boars had antibodies against h1n2 and h3n2 viruses as well [126] . this reflected a past exposure of the japanese wild boars to the iav strains. the first active iav infection in the korean swine was identified in december 1998 when three h3n2 virus isolates were recovered from the swine experiencing an acute influenza-like respiratory disease. the close relatedness of these korean swine h3n2 isolates with human-origin h3n2 viruses reported from korea between 1987-1999 suggested the events of reverse zoonosis [127] . one unique h7n2 virus isolate was detected in swine which had seven gene segments originated from hong kong avian-origin h7n2 virus isolated in 1978 and the ns gene originated from hong kong h5n3 virus isolated in 1977. additionally, four typical swine influenza h1n1 viruses were identified in swine [128] . several h1n1, h1n2, and h3n2 viruses were detected in symptomatic south korean swine after 2000 [129] [130] [131] [132] [133] [134] . the iav localization in the swine lung tissues was confirmed by immunohistochemistry [130] . total 35 avian-origin h5n2 viruses of eurasian lineage were identified in swine in different south korean provinces during 2004-2008 which suggested cross-species transmission of h5n2 virus [135] . three h1n1 virus isolates closely related to us isolates of h1n1 were obtained from 45-day-old piglets in korea in january 2005. the other swine farms in the proximity of this index farm were negative for the h1n1 virus [136] . further, one h1n1, two h1n2, and one h3n2 subtypes of iav identical to the american strains based on their ha and na gene sequences were obtained from swine nasal swab, lung, and thoracic fluid samples during 2005-2006 which suggested that there was no probability of arising of these iav strains in korea through recombination [137] . two novel isolates of swine h3n1 virus with high genomic similarity to each other were retrieved from two different swine farms in korea during march-april 2006 which would be due to a common origin of these isolates. these viruses had human-like h3 gene while other gene segments originated from swine influenza viruses within korea. high reactivity of the 52 swine sera samples to h3n1 virus antibodies suggested a previous exposure and probability of the swine to swine transmission of h3n1 virus [138] . the human to swine transmission of a(h1n1)pdm09 virus was reported in chungbuk province where 42 a(h1n1)pdm09 virus isolates were recovered from swine lung tissues [139] . the reassortment between a(h1n1)pdm09 and swine h1n2 viruses emerged into a novel reassortant h1n2 virus in swine [140] . a triple-reassortant h3n2 virus was identified in swine during december 2011-may 2012 which indicated the iav reassortment was taking place in korean swine [141] . a swine fever eradication campaign identified nine a(h1n1)pdm09, two classical h1n1 and one h1n2 viruses in wild boars which were hunted and killed in south korea during 2012 [142] . more recently, a complete genome sequence of h1n1 virus was reported from a domestic swine in korea in 2016 [143] . the occurrence of iav in thai swine was first reported during november-december 1978. active h3n2 infection was detected in one swine while several other swine had h3n2 antibodies [144] . two h1n1 virus isolates from thai swine were first recovered in january 1988 [145] . several studies reported h1n1, a(h1n1)pdm09, h1n2, and h3n2 viruses in swine exhibiting respiratory disease symptoms between 2000 to 2014. intriguingly, one swine sample was found co-infected with four iav subtypes including h1n1, h1n2, h3n1, and h3n2 viruses [146] [147] [148] [149] [150] [151] [152] . the first evidence of h5n1 seroprevalence in thai swine was documented in 2004 when eight h5n1 positive swine sera samples were identified [153] . later ten h1n1 and two h3n2 virus isolates were retrieved from piglets aged between 4 to 12 weeks during 2008-2009 [154] . interestingly, most of the virus isolates retrieved in this study were obtained from 4 to 8 week-old piglets which was in agreement of a previous report stating that swine influenza viruses can be successfully retrieved from piglets less than ten weeks of age [155] . a zoonotic transmission of iav was reported at a thai swine farm where all the swine were found positive for either h1n1 or h1n2 virus. interestingly, two farm owners, 46 swine handlers, four veterinarians, five farm cleaners and two farm office workers also reported iav seroprevalence. this study claimed that there was transmission of swine influenza viruses from swine to human however the possibility of human to swine transmission was ruled out [156] . after a respiratory disease outbreak in nursery piglets, 15 nasal swabs were found positive for a(h1n1)pdm09 virus between december 2009 and march 2010. fifteen sera samples of the farm workers along with three sera from dogs and one serum obtained from a cat were negative for iav, hence the interspecies transmission of iav was ruled out [157] . the first report of active infection with reassortant h1n1 virus in thai swine appeared in february 2010 but the follow up screenings conducted after two and three months, respectively confirmed the cessation of the active infection as the viral rna was not detected anymore [158] . the reshuffling and reassortment of iav internal genes were reported in thai swine in february 2012. the ha and na genes of h1n1 virus isolates clustered with the eurasian swine-like iav lineage while the h3n2 viruses diverged and formed a separate group. all the internal genes of h1n1 and h3n2 virus isolates appeared to be derived from a(h1n1)pdm09 viruses which confirmed the events of reassortments [159] . the events of reverse zoonoses were suggested after the detection of a(h1n1)pdm09 virus seroprevalence in vietnamese swine during october 2009-march 2010 [160] . one more evidence of reverse zoonosis was identified during february-march 2010 after six triple-reassortant h3n2 viruses having a novel cluster of the triple reassortant internal gene (trig) cassette were isolated. the ha and na genes of these reassortant h3n2 isolates originated from human h3n2 viruses reported between 2004-2006 while the other six internal genes had a high similarity with the korean and american isolates [161] . two more studies reported the h1n1, a(h1n1)pdm09, hin2, and h3n2 virus isolates during february 2010-december 2013 from clinically healthy swine with no influenza disease symptoms [162, 163] . additionally, the antibodies for a(h1n1)pdm09 and h3n2 viruses were detected in swine which suggested a past exposure of swine to these viruses [163] . a high seroprevalence of h1n1, h2n2 and h3n2 viruses was detected in human and swine sera in calcutta, india during 1982-1990 [164] . the first active infection of iav in indian swine appeared in 2009 when a(h1n1)pdm09 virus isolates were reported from a swine farm located in uttar pradesh. interestingly, the retrieved a(h1n1)pdm09 virus sequences were similar to the north american and korean viruses which might be either because of trade or long-distance transmission [165] . after an influenza outbreak on lebanese poultry farms in 2005 the farmers fed the carcasses of the dead flocks to the swine. intriguingly, a following investigation found that three swine were seropositive for the h9n2 virus while approximately one-third of the poultry farm workers were seropositive either for h1 or h9 viruses [166] . these results revealed the interspecies transmission of iav among poultry, farm workers and swine. the seroprevalence of h1n1 and h3n2 viruses in four to six-month-old malaysian swine at 41 swine farms was reported during may-august 2005. co-infections of h1n1 and h3n2 were detected in 29 swine samples [167] . the seroprevalence of h3n2 virus in swine samples obtained from the slaughterhouses in laos was reported between may 2008 to january 2009 [168] . a full-length genome sequence of a reassortant h1n1 virus was reported from a russian swine in 2016. the ha and na genes of this virus isolate shared 90% identity with the h1n1 viruses that were reported from humans in the usa in the 1980s [169] . the human to swine transmission of iav was speculated after iav antibodies were detected in 147 taiwanese swine during june 1969-may 1970. the results were further confirmed with virus isolation which retrieved 13 iav isolates [170] . more recently, ibv of victoria/b lineage was detected in swine nasal swab samples collected from apparently healthy swine at three swine farms in 2014 [171] . an active iav infection in 52 swine within four provinces in indonesia was identified during 2005-2009. interestingly, 39 h5n1 virus isolates were successfully retrieved and sequenced [172] . the first report of influenza in sri lankan swine was documented during 2004-2005 after one human-like h3n2 virus was identified. later, a(h1n1)pdm09 virus isolates were identified in swine during 2009-2012. a spillover of these viruses from human to swine was speculated [173] . one recent investigation in kazakhstan during 2017-2018 identified nine h1n1 and eight h3n2 viruses in human while seven h1n1 and four h3n2 viruses were identified in swine. interestingly, 10 of the human samples were also positive for ibv infection while the swine samples were negative for ibv [174] . in summary, the influenza viruses have been reported in swine in 16 asian countries including china, japan, thailand, south korea, viet nam, cambodia, taiwan, india, bhutan, russia, laos, malaysia, lebanon, indonesia, kazakhstan, and sri lanka ( figure 4b ). apart from the most common iav strains of h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses, several avian-origin h5n1, h5n3, h4n1, h4n6, h4n8, h6n6, h7n9, h9n2, and h10n5 influenza viruses were also reported in chinese swine. horse to swine transmission of equine influenza virus h3n8 was reported in china. additionally, avian-origin h7n2, h5n2 viruses were identified in south korean swine while h5n1 was reported in indonesian swine. interestingly, after the swine were fed upon dead poultry carcasses in lebanon the h9n2 virus was detected in lebanese swine. the ibv was reported in asian swine only in taiwan while strains of icv were reported in swine in china and japan while idv was recently reported in chinese swine (table 1) . swine influenza was first reported in australian swine only in 2009 after a swine farm owner reported coughing symptoms among swine. simultaneously, some of the human workers on the farm also developed influenza like symptoms and hence stayed out of the farm until recovery. later, the farm owner also developed similar symptoms following which he was tested for a(h1n1)pdm09 virus which resulted positive. as a result, a representative number of swine showing coughing symptoms and loss in appetite were sampled for molecular diagnostics and serology which confirmed that 12 swine were positive for h1n1 virus [175] . second report of iav in australian swine appeared on a queensland farm in august 2009 when a veterinarian observed elevated temperature, coughing and loss of appetite in swine. simultaneously, two of the staff members on the farm exhibited influenza-like symptoms and hence were sampled for diagnostic testing using nasal swabs. interestingly, both the staff members and four of the swine were found positive for the a(h1n1)pdm09 virus. sequencing identified that the ha gene of a(h1n1)pdm09 virus retrieved from a staff member was identical to the virus retrieved from the swine which suggested transmission of a(h1n1)pdm09 virus between swine and human [176] . third report of iav in australian swine appeared when a respiratory disease outbreak in swine and the farm workers occurred in perth, western australia during 2012 which identified 43 iav positive swine. sanger sequencing of ha and na genes identified six novel hin2, three novel h3n2, one a(h1n1)pdm09 and two seasonal h3n2 viruses in swine. on the contrary, only one out of eight human workers were found positive for seasonal h3n2 virus. this study could not conclude the event of zoonotic transmission of iav between swine and human workers at the farm [177] . the fourth report of iav was documented when 14 iav positive swine were identified at a commercial swine farm in western australia during july-september 2012 and later during september-november 2016. additionally, 17 swine were determined to be iav positive in southern queensland. the complete genomes of 10 iav isolates retrieved in western australia and queensland were successfully sequenced which identified seven h1n2, two human-like h3n2 and one h1n1 virus [178] . overall, four reports of iav outbreaks in swine in new south wales, queensland and western australia were available ( figure 4c ). the h1n1, h1n2, h3n2 and a(h1n1)pdm09 subtypes have been reported from australian swine with relatively low prevalence. the h1n1 virus was identified in swine lung tissues or trachea of two of the deceased sows after an influenza-like disease erupted at two swine farms in january 1979. interestingly, it was also reported that the identical virus was detected in wild ducks in germany [179] . since it was already established that h1n1 from wild ducks can successfully infect swine if inoculated via intranasal route [179] hence this observation suggested the transmission of h1n1 from wild ducks to the swine [180] . a second investigation isolated three avian-like h1n1, two h1n2 and twelve human-like h3n2 viruses from eight commercial swine farms in march 1999 [181] . denmark has been running a passive surveillance program for iav detection in swine since 2011. the h1n2 virus having the h1 gene which evolved from h1n1 avian-like viruses and n2 gene which evolved from human h3n2 viruses was reported in swine during 2011-2013 [182] . this was an example of how iav can evolve through reassortment and may emerge into a new iav strain. the other investigation included swine sampling at different time intervals to assess the persistence of iav shedding in danish swine which detected one avian-like h1n1 and 107 reassortant h1n2 viruses. this study observed that most of the swine were shedding iav right before achieving six weeks of age. surprisingly, a piglet as young as just three days was found infected with iav [183] . two h3n2 isolates having h3 genes from seasonal human influenza along with internal genes that originated from a(h1n1)pdm09 virus and na genes from contemporary n2 swine influenza viruses that have been in circulation in denmark were retrieved from young piglets at two locations during 2011-2014 [184] . h3n2 virus was also detected from piglets having respiratory illness and from sows with reproductive problems in commercial piggeries in 2014 [184] . the h3n2 virus antibodies were first detected in english swine in 1973 which revealed the past exposure of swine to h3n2 virus [185] . later, the antibodies for h1n1 and h3n2 viruses were detected in swine at a slaughterhouse in england during 1991-1992 [186] . interestingly, this serological investigation also reported the antibodies for ibv in eight and for icv in 198 swine [186] . a molecular investigation identified a novel h1n7 virus in swine in england which had six of its rna segments closely related to those of human viruses while two rna segments were identical to those of equine viruses which concluded that the h1n7 strain may have evolved due to reassortment between human h1 and equine h7n7 viruses [187, 188] . the first report of a(h1n1)pdm09 virus in english swine appeared in september 2009 when histology and immunofluorescence assays followed by molecular diagnostics and sequencing confirmed four a(h1n1)pdm09 virus infected swine in the northern ireland [189] . after this, 17 more a(h1n1)pdm09 virus isolates were reported in swine in england during september 2009-october 2010 which revealed that a(h1n1)pdm09 virus was in circulation in english swine during the 2009 flu pandemic [190] . the same year, four h1n2 virus isolates were reported in english swine which had six internal genes of a(h1n1)pdm09 virus along with ha and na genes of h1n2 virus hence were identified as the novel reassortant h1n2 strains [191] . in a more recent study, two more iav positive swine were identified in the united kingdom in 2016 [192] . however the first report of seroprevalence of h1n1 virus in finnish swine appeared in 2008 during an investigation which detected h1n1 virus antibodies in swine at seven swine farms which further increased to 24 swine farms in 2009 [193] but the first isolate of avian-like swine h1n1 virus (indicative of active infection) was detected from the lung tissues of a swine in february 2009. later, the first a(h1n1)pdm09 virus in finnish swine was detected in november 2009 [193] . three more swine were identified with iav antibodies during may 2011-january 2014 which was due to a past exposure to iav [194] . the h1n1 viruses in turkey and swine were identified after the swine influenza outbreak hit the turkey population in brittany, france in february 1983 which suggested that iav transmission happened from swine to turkey [195] . later two strains of h1n2 virus were isolated from six swine exhibiting influenza-like illness in brittany during 1987-1988 [196] . another investigation reported h1n1, h1n2, and h3n2 viruses in swine herds experiencing respiratory disease in brittany region [197] . a negative prevalence of iav was reported in wild boars in camargue during september 2009-november 2010 given that all the 315 nasal swabs obtained from either hunted or trapped wild boars along with all the sera samples were negative for iav [198] . a more recent investigation reported the zoonotic transmission of a(h1n1)pdm09 virus from swine to a farmer in january 2018. this farmer along with a veterinarian collected nasal swab samples from three pregnant sows exhibiting influenza-like illness on the swine farm and submitted to a local diagnostic laboratory for analysis which, as a result, were found iav positive. few days later, the farmer and the veterinarian both developed the influenza-like symptoms. the farmer was later diagnosed with a(h1n1)pdm09 virus [199] . sixty-five iav positive wild boars were identified across five german states during 1997-2006. cloning and sequencing identified h1n1 and h3n2 viruses in these wild boars [200] . later thirteen h1n1, three reassorted a(h1n1)pdm09 and four h1n2 viruses were detected in swine during 2009-2010. interestingly, the a(h1n1)pdm09 virus isolates had high similarity with the a(h1n1)pdm09 viruses reported earlier in humans within germany which suggested a reverse zoonotic transmission of the a(h1n1)pdm09 virus [201] . a nationwide sero-surveillance identified 12,585 swine with h1n1, 9,566 swine with human-like h1n2, 12,220 swine with human-like h3n2 and 11,086 swine with a(h1n1)pdm09 virus antibodies during june 2009-december 2012 which reflected a high seroprevalence of influenza viruses in german swine population [202] . later 273 iav positive swine exhibiting influenza-like illness were detected between january 2010-december 2013. subtyping successfully distinguished 198 of 273 samples into h1n1, h1n2, h3n2 and a(h1n1)pdm09 viruses. the h1n1 virus was the most widely occurring in german swine while a(h1n1)pdm09 virus had the lowest prevalence [203] . the h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses were detected in swine sera samples collected from apparently healthy swine at 42 swine farms during 2002-2004 and from 46 swine farms during 2010-2012 [204] . the seropositivity of italian swine to h3n2 virus was first reported during december 1976-november 1977 when 24 swine were detected with h3n2 antibodies [205] . the first report of h1n1 active infection in italian swine appeared during an influenza disease outbreak between 1977 to 1986 which identified 63 h1n1 viruses [206] . further, four h3n2 viruses were detected in swine nasal swabs originated from three swine farms and an abattoir during 1981-1982 [207] . later 47 h1n1 and 37 h3n2 viruses were detected in swine during 1992-1995. interestingly, four human sera samples were also positive for h1n1 and 77 samples were positive for h3n2 viruses which might be due to the transmission between human and swine [208] . further iav seroprevalence was detected in the age group of three-month to four-year old swine during 2002-2004 [209] . the first report of a(h1n1)pdm09 virus in italian swine appeared after a respiratory disease outbreak in piggeries in lombardia region of northern italy in november 2009. piglets experienced diarrhea and weight loss while the sows experienced reduction in reproduction rate [210] . two more a(h1n1)pdm09 virus isolates were reported in female swine in sicily in december 2009 [211] while five isolates of a(h1n1)pdm09 virus were identified in swine at three different locations during 2011-2012 [212] . a novel strain of reassorted h1n2 virus having 99-100% identity through six gene segments with a(h1n1)pdm09 virus along with ha and na genes similar to h1n2 virus was reported in swine in mantua province [213] . reassorted h1n2 viruses were again detected in 34 piglets during 2013-2014 [214] . seroprevalence of italian wild boars with one h1n1, ten h1n2, and one h3n2 viruses at two different locations was reported during 2012. on the contrary, active infection was found only in three wild boars whose nasal swabs were positive for the iav [215] . one more investigation reported active infection of iav in 12 wild boars while 78 wild boars had iav antibodies during july-december 2012 [216] . further molecular and serological investigations detected avian-like h1n1 viruses in italian wild boars [216] . the first complete genome sequence of idv in italian swine was retrieved from a symptomatic sow in 2015 which was identified to be closely related to the idv sequence reported in oklahoma swine in 2011 [217] . further idv prevalence in italian swine was reported when 14, three and four swine were found positive for idv antibodies in veneto, emilia romagna and lombardia regions, respectively during june 2015-may 2016. as a result, swine clinical samples collected during 2013-2014 were investigated retrospectively for idv prevalence but were reported negative. an extended serological investigation detected idv antibodies in 364 swine sera samples collected during 2015. these findings suggested that idv was in circulation in italian swine population only after 2014 [45] . isolation and characterization of 12 h3n2, nine h1n1 and one h1n2 viruses reported the prevalence of influenza viruses for the first time in spanish swine herds experiencing the respiratory illness and pneumonia during november 2001-april 2004 [218] . more strains of h1n1, h1n2 and h3n2 viruses were isolated, sequenced and characterized in spanish swine during 2006-2011 [219] [220] [221] . interestingly, five h1n1, three h1n2, and four h3n2 virus isolates retrieved between january 2010 and august 2011 had significant similarities with other european isolates which was an evidence of continent-wide transmission of these swine influenza viruses [220] . a molecular investigation reported a negative prevalence of idv in swine in luxembourg during 2009 but later successfully detected three idv positive swine during 2014-2015. further, the serological investigation confirmed that swine in luxembourg were free from idv during 2012 but interestingly, idv antibodies were detected in 17 swine samples collected during 2014-2015. these observations suggested that idv was not in circulation in swine in luxembourg during 2009-2012 but became prevalent at a low frequency later during 2014-2015 [46] which was almost the same time idv was reported in italian swine populations [45] . a serological investigation of swine in the netherlands identified 601 h1n1, 584 h1n2, and 229 h3n2 virus antibodies in 29 swine herds during january-may 1999 [222] with no further evidence of iav in swine in the country after that. after the 18 swine which were experiencing influenza-like illness were found infected with a(h1n1)pdm09 virus on a norwegian swine farm in october 2009 the surveillance was expanded to the 39 nearby swine farms which determined that 23 of these farms were positive for the a(h1n1)pdm09 virus. intriguingly, one human subject at the index farm who had influenza-like symptoms was also found positive for a(h1n1)pdm09 virus. this study suggested that the symptoms first appeared in the human subject at the index farm and later the disease got transmitted to the swine. hence the findings of this study suggested the reverse zoonosis of the influenza virus from human to pig [223] . further molecular and serological investigations identified 48 more swine herds that were positive for iav during september 2009-october 2010 [224] . a more comprehensive nation-wide surveillance in norwegian swine identified 16 a(h1n1)pdm09 virus positive swine herds during 2009 which later increased to 190 swine herds in 2010 [225] . later 194 more swine were found infected with a(h1n1)pdm09 virus in norway between april and july 2011 and reported that the iav infected swine took longer to weigh 100 kg body mass [226] . the first active iav infection in swine in poland was reported in 2010 when 21 oral fluid samples collected from three swine farms detected iav [227] . soon after, five avian-like h1n1 viruses were reported from the swine lung tissues during 2011-2013 [228] . later a serological surveillance identified 1212 h1n1, 851 h1n2, 1012 h3n2, and 572 a(h1n1)pdm09 virus antibodies in swine during march 2011-february 2015 [229] . surprisingly, 34 of these swine had antibodies against all four iav subtypes i.e., h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses [229] suggesting the past co-infections. the human-like h3n2 virus was isolated from a swine in czechoslovakia during 1969-1972 [230] ; however, no other reports ever appeared from the country in later years. complete genome of an h1n1 virus was reported from a hungarian swine having fever and conjunctivitis in may 2011 [231] . this was the only report of h1n1 virus in the swine in hungary. a large-scale investigation across seven european countries reported a high seroprevalence (>62%) of iav antibodies in swine populations of belgium, germany, spain, italy while a relatively lower (<21.25%) seroprevalence was observed in swine populations of czech republic, poland and ireland during 2002-2003. antibodies against h1n1, h1n2, and h3n2 viruses were reported in swine from the european countries under surveillance except poland where swine had antibodies against only h1n1 virus [232] . a virological surveillance across five european countries including belgium, united kingdom, italy, france and spain reported 169 iav positive swine during 2006-2008. the h1n1, h1n2, and h3n2 viruses were detected in swine from belgium, italy, and spain while the samples from united kingdom and france were found infected with h1n1 and h1n2 viruses [233] . briefly, the virological and/or serological prevalence of influenza viruses in european countries ( figure 4d ) identified the strains of h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses in swine populations of the united kingdom, ireland, italy, germany, france, norway, finland, denmark, belgium, spain, poland, greece, hungary, netherlands, czech republic, and czechoslovakia while the swine in luxembourg and italy were found infected with idv. shortly after a respiratory disease outbreak in swine in manitoba, an autopsy was done on a dead swine on march 1, 1967. the histopathology confirmed the bronchitis in the deceased swine and a strain of iav designated as "s/manitoba/647/67" was characterized using iav antisera [234] . the first report of h1n1 virus in canadian swine appeared in quebec during 1980s-1990s when five genotypes of h1n1 virus were identified [235] . since then several studies have reported h1n1, h1n2 and h3n2 viruses in canadian swine [236] [237] [238] [239] [240] [241] [242] [243] . another study reported nine isolates of swine influenza viruses with an antigenic variant from the sick swine having proliferative pneumonia in quebec, canada during 1990-1991 [244] . in a retrospective diagnosis, only one formalin-fixed paraffin embedded swine lung tissue collected during 1991 was found iav positive with immunohistochemistry. this investigation suggested that immunohistochemistry can be useful in retrospective diagnosis of the swine influenza virus [245] . the broncho-intestinal pneumonia in lung tissues of dead swine was reported on a swine farm which exhibited disease symptoms including coughing, weight loss, and labored breathing. interestingly, before the onset of the disease symptoms, this farm conducted a routine serological surveillance of influenza virus which identified h1n1 virus in only one of the twelve swine samples [246] . following this surveillance, a three-month old swine from the same farm was found positive for avian influenza virus h4n6. the complete genome of this h4n6 virus was reported in 1999. this was the first ever report of an avian-origin h4n6 virus in swine. the proximity of the swine farm to a natural lake where several wild bird species including waterfowls which were reported to visit frequently might be the reason behind the introduction of this avian influenza virus strain to the swine [246] . later three avian-origin h3n3 influenza virus isolates were recovered from swine in eastern ontario exhibiting weight loss and coughing during october 2001. on a nearby farm located approximately 30 kms away, another h3n3 virus isolate was recovered from the swine. there was no recorded movement of the swine between these two farms. since these were avian-origin h3n3 viruses hence the role of birds in transmission cannot be ruled out. later, on a third farm, where an influenza like disease had been affecting mainly the nursery piglets, an h1n1 virus was recovered in may 2002 [247] . reassortant h1n1 and h1n2 viruses were detected in swine nasal swab or lung tissue samples obtained from three-week old piglets and sows exhibiting typical influenza-like symptoms in ontario during 2003-2004 [248] . first triple-reassortant (avian/classical swine/human triple-reassortant) h3n2 viruses from four swine and one human nasal samples were identified in ontario during 2005. the phylogenetic analysis determined that all the virus sequences were 100% identical to each other which apparently emerged from triple-reassortant h3n2 viruses reported in us based swine in 1988 [249] . one more report of triple-reassortant h3n2 (trh3n2) viruses appeared on the swine farms located in saint-hyacinthe, assomption and saint-foy during early 2009. the trh3n2 viruses identified in this study were determined to be closely related to north american/canadian trh3n2 viruses reported earlier [250] . later a(h1n1)pdm09 and h1n1 viruses having internal genes of triple reassortant h3n2 virus were reported in swine in four provinces including manitoba, alberta, saskatchewan and quebec during 2009 [251] . the first evidence of a(h1n1)pdm09 virus in canadian swine appeared in 2009 after the human workers at a swine farm developed influenza-like illness. the investigation identified that two farm workers along with 56 swine were positive for the a(h1n1)pdm09 virus. transmission of a(h1n1)pdm09 virus from human to swine was suggested [252] . the same year 17 more swine were detected with a(h1n1)pdm09 virus after a respiratory disease outbreak hit the alberta swine farms [253] . a reverse zoonotic transmission of a(h1n1)pdm09 virus to swine from a human subject who visited mexico and returned to the swine farm was reported in april 2009. as a result, ten swine having severe disease were sacrificed for necropsy which identified lesions in the bronchioles corresponding to the influenza virus disease. virus isolation and sequencing identified the a(h1n1)pdm09 virus. additionally, a(h1n1)pdm09 virus was identified in two more human subjects who were exposed to the swine hence indicated the occurrence of zoonoses on the swine farm [254] . later during summer 2009, ten more a(h1n1)pdm09 viruses from five swine herds in manitoba were reported. virus shedding was observed up to 20 days post-infection after the appearance of the clinical symptoms in swine [255] . this observation was in agreement of a previous report which documented the experimental infection of swine in the laboratory and determined that virus shedding occurs until 11th day after appearance of the clinical symptoms [256] . another investigation reported nine a(h1n1)pdm09 and four h3n2 viruses after an influenza-like disease outbreak on a quebec based swine farm in december 2010 [257] . the effect of microclimatic conditions on the transmission dynamics of swine iav in the barns was studied which observed that high relative humidity in the environment during summer months suppresses the aerosol transmission of the droplets which in turn decreases the transmission of iav [240] . the high relative humidity in the environment would facilitate the generation of larger droplets which do not tend to shrink easily and hence are less likely to be aerosol transmitted to a longer distance as they fall on the ground quickly after their formation [240, 258] . as a result, a lower transmission of iav is observed usually during the summer months. on the contrary, the iav transmission increases during winter months when relative humidity is relatively lower [258] . the iav was first isolated from the nasal discharge of a swine in the united states in 1931 [34] and from the human in 1933 [35] . the first report of human-origin iav in swine appeared in the united states on 24 may 1937 after an unexpected result was observed when the serum sample of a sick swine obtained from a state prison farm located in new jersey neutralized the antibodies of human influenza virus. a series of investigations made a strikingly new observation that swine had suffered from a human strain of influenza virus [259] . serological investigations conducted during 1950s suggested that the weight loss and mortalities among swine were due to swine influenza viruses [260, 261] . swine influenza viruses were isolated from febrile swine at nine occasions during 1965-1968 in wisconsin and nebraska [262] . additionally, swine influenza antibodies were also detected in swine sera samples collected from six farms [262] . a virological surveillance conducted in memphis, tennessee and madison, wisconsin during may 1976 to june 1977 successfully isolated 478 influenza viruses from swine nasal swabs collected at abattoirs; approximately 300 of which were characterized to be swine h1n1 viruses. additionally, the serological surveillance identified that 21% of the 9400 swine sera samples had swine h1n1 virus antibodies [263] . a small percentage (1.4%) of swine sera samples were found positive for the swine h3n2 viruses which was further confirmed by virus isolation [263] . interestingly, this study identified inter-species transmission of swine influenza viruses between human and swine [263] . a novel swine-origin h1n1 virus termed as "a/new jersey/76 (hsw1n1)" was detected at fort dix army training camp in new jersey in january 1976. the outbreak was localized and was limited to fort dix only. as a result, 230 soldiers were found infected with this novel virus; 13 of which had severe respiratory disease with one death due to viral pneumonia [264] [265] [266] . since this novel swine-origin h1n1 virus quickly disappeared from fort dix hence the epidemiology and the origin of the disease could not be ascertained [264] . the h1n1 and h3n2 virus antibodies were detected in swine sera collected from an abattoir in north-west united states. interestingly, a higher iav seroprevalence was observed during the fall and early winter months. virus isolation and sequencing identified that the h1n1 viruses were closely related to the classical h1 swine influenza virus [267] . classical swine-like h1n1 and triple-reassortant h3n2 viruses were identified in swine samples collected across 23 states in the usa during 1998-1999 [268] . the minnesota veterinary diagnostic laboratory (mvdl) detected large number of h1n1, h1n2 and h3n2 subtypes of iav in swine samples during 1998-2001 and again during 2007-2009. interestingly, some of the samples were co-infected with h1n1 and h3n2 viruses [269] [270] [271] . a second-generation reassortant h1n2 virus having genes from a reassortant h3n2 and classical h1 swine influenza viruses was obtained from the lung tissue samples of a dead sow at an indiana swine farm in november 1999 [272] . a novel subtype of h3n1 virus termed as "a/swine/minnesota/00395/2004 (h3n1)" was identified during a severe respiratory disease outbreak on a swine farm in minnesota in october 2004. sequencing observed that the ha gene of this strain was closely related to swine influenza h3n2 virus while the na gene was related to classical h1n1 virus which suggested that the novel h3n1 virus emerged due to reassortment between h1n1 and h3n2 viruses in the midwest united states [273] . further an h2n3 subtype of iav which may have emerged as a result of a reassortment between avian and swine influenza viruses was identified on a commercial swine farm in minnesota in april 2006 and again in september 2006 [274] . the first evidence of a(h1n1)pdm09 virus infection in us swine appeared when four a(h1n1)pdm09 and one triple-reassortant h1n2 viruses were identified and characterized in the exhibition swine in the states of minnesota and south dakota in 2009 [275] . during last ten years, a large number of h1n1, h1n2, h3n2, a(h1n1)pdm09 along with reassortant iav subtypes have been reported in the us swine populations [243, [276] [277] [278] [279] [280] [281] [282] [283] [284] [285] [286] [287] [288] [289] . the united states has a large feral swine population which is considered a reservoir of h1n1 and h3n2 viruses [290] . the swine-like h1n1, avian-like h1n1, swine-like h1n2, swine-like h3n2, human-like h3n2, a(h1n1)pdm09 along with avian-like h6n2 and h7n2 viruses were identified in feral swine samples collected across 35 states in the usa between october 2009-september 2013 [291] . histological examination of the lung tissues obtained from two backyard piglets suffering from pneumonia and weight loss in colorado in november 2010 suggested that the piglets were infected with swine influenza virus which were later confirmed to be infected with iav subtype a(h1n1)pdm09 virus. since the piglets were raised at the house of a pharmacist hence a possible human to swine transmission was speculated given the possibility of an occupational exposure of the pharmacist to the a(h1n1)pdm09 virus at the pharmacy [292] . the first report of ibv infection in swine appeared when swine in the midwest united states were found infected with ibv lineages of yagamata/b and victoria/b [37] . this was a new finding because initially ibv was thought to have a host range limited to human, pheasants, horses and seal [1] [2] [3] [4] . a novel strain of swine influenza virus was detected in oklahoma swine exhibiting influenza-like symptoms in april 2011. the nasal swab samples taken from the swine were negative for the iav infection. hence the virus isolation was attempted in swine testicle cells; the cells in culture showed influenza-like cytopathic effects by third day. electron microscopic observations revealed particles typical of a virus of orthomyxoviridae family, but the rt-pcr was negative for the ibv and icv. after ultracentrifugation was used for virus isolation, the genome of the virus was sequenced using ion torrent sequencing. the genome sequence analysis along with genetic and biochemical investigations revealed that the isolated virus was a novel orthomyxovirus having 50% overall identity at amino acid level with human influenza c virus [43] . since this novel virus was genetically and antigenically distinct from icv therefore, later was proposed to be categorized as a new genus of orthomyxoviridae family which was later accepted as influenza d virus (idv) [5] . later, two feral swine which were shot dead in a cotton field in texas in june 2011 were found infected with a(h1n1)pdm09 virus. the significant identity of a(h1n1)pdm09 virus isolated from these two feral swine with human a(h1n1)pdm09 virus suggested a possible transmission between human and the feral swine [290] . another study reported seroprevalence of h3n2 virus in one feral swine from mississippi and in five feral swine from the state of california in 2005 but a negative seroprevalence was reported in the feral swine samples obtained from the states of florida, oklahoma and missouri. additionally, the seroprevalence of iav was reported in feral swine from texas where a total of 68 out of 472 feral swine sera were found positive for h3n2 and h1n1 viruses [293] . another investigation detected h3n2 virus rna in only one feral swine from a pool of samples collected across 31 states in the usa during 2011-2012 which indicated a negligible active influenza infection in us feral swine population. on the contrary, elisa identified iav antibodies in 182 feral swine samples while the serological subtyping identified h3n2 virus antibodies in 76 feral swine samples collected from 19 states which indicated a significant past exposure of us feral swine to the h3n2 virus [294] . further, seroprevalence of idv was reported in 49 feral swine samples collected from oklahoma, texas, hawaii and north carolina during october 2012-september 2013 which provided the first evidence of past idv infections in us feral swine [295] . a study investigating virus shedding in nursery piglets found that all 81 piglets under investigation were shedding h3n2 virus starting seventh day of arrival into the barns until 29th day. shedding was still observed in some piglets until 39th day [296] . interestingly, 48 of these nursery piglets were also identified shedding h1n1 virus starting at the third day of arrival into the barns until 41st day over a 53-day observation period [296] . this was the new information which identified that young nursery piglets could get infected with iav. the oral fluid samples collected from 25 neonatal piglets at four oklahoma based swine farms during may-august 2014 [297] were found infected with different iav subtypes including h1, n1, h3, and n2. this study supported the use of swine oral fluid samples in iav diagnostics [285] . the swine oral fluid samples were also collected in north and south carolina during june to august 2014 using the cotton rope hanging method [298] . in this method of sampling, swine are encouraged to chew the rope, as a result, saliva accumulates on the rope which is later squeezed to collect the sample aseptically. one of the benefits of this method of sampling is that each sample does not represent an individual swine but rather represents multiple swine that chewed the rope while hanging inside the pen [298] . another benefit of this sampling method is that swine oral samples may contain contaminants like feed and feces but this method minimizes the chances of such contaminations in the sample [299] . another investigation carried out metagenomic sequencing of swine nasal and rectal swabs obtained from apparently healthy swine which identified 11 iav positive swine at three abattoirs and a buying station in usa in august 2015 [300] . in a striking observation, an avian-lineage h4n6 virus was isolated and sequenced from 7-8-month-old gilts on a missouri based swine farm in december 2015 [301] . the investigators collected more samples at different time points for next few months at the same farm to assess the transmission of h4n6 virus among swine. no other samples were found positive for the h4n6 virus which suggested that the h4n6 virus did not transmit from swine-to-swine and therefore disappeared from the index farm. interestingly, this extended study identified three h1n1 viruses infecting swine [301] . one large-scale study identified that 23 percent (2 947/12,814) of the swine samples were positive for the iav in mid-west united states between july 2011-march 2017, however, sequencing could identify only 173 h1 and h3 subtypes among positive samples [302] . a human to swine transmission of iav was suggested when two human-like h3n2 virus isolates were identified from an oklahoma based swine farm in 2017 which had high similarity with the human-like h3n2 viruses reported earlier from baltimore [303] . maya people represent ethnolinguistic groups in south and central america. the practice of household swine keeping put the maya people at high risk of contracting the swine influenza viruses. thirty-one sera samples collected from the maya people in mexico were identified having antibodies against h1n1 and h3n2 viruses while 93 other sera had antibodies against the h3 subtype of iav, representing a past exposure to these viruses [304] . however, this study did not include swine samples for investigation but since swine were household animals in their backyard hence the iav seroprevalence of the maya people could be because of a past transmission of these viruses from the backyard swine [304] . a retrospective study identified antibodies against swine-like h1n1, a(h1n1)pdm09, h3n2, and human-like h1n1 viruses in backyard swine in mexico between 2000 to 2009. this investigation retrospectively determined that the classical-swine h1n1 virus was most widely present in mexican swine before the 2009 influenza pandemic [305] . further, a significant number of swine experiencing respiratory illness had h1n1 or h3n2 virus antibodies in commercial piggeries in sonora province of mexico during october 2008-march 2009. the molecular diagnostics and subtyping determined four h1 and two h3 viruses while 19 other iav positive samples could not be subtyped given the low viral load [306] . during the influenza virus pandemic in mexico in 2009, a(h1n1)pdm09 virus was first identified in a single swine nasal swab. additionally, h3n2, a(h1n1)pdm09 and ibv viruses were detected in four symptomatic humans [307] . the a(h1n1)pdm09 virus isolate retrieved from the swine was believed to be the first from the sister lineage of the pandemic influenza virus isolates reported in mexico [307] . further 59 iav isolates were retrieved from mexican swine having respiratory illness during 2010-2014. intriguingly, this study identified 13 reassorted genotypes of iav in mexican swine [308] . this investigation also reported that iav introduction into mexican swine may have occurred through three different routes; human to swine transmission; reassortment between human-like h3n2 and a(h1n1)pdm09 virus; and through the long-distance movement of the swine from usa and europe. a periodic introduction of iav in mexican swine occurred with the import of american and european swine to mexico over two decades in 1980s and 1990s before the 2009 influenza pandemic [33] . fifty-eight iav whole genome sequences were retrieved from mexican swine during 2010-2014. genome sequence analysis identified classical h1n1, h3n2, and a(h1n1)pdm09 viruses. interestingly, the data obtained in this study suggested independent evolution of iav in the mexican swine population in different regions of the country. phylogeny determined that mexico city was the source of the 2009 influenza pandemic which erupted during march-may 2009 [33] . later a reassortant h1n2 virus which had the genes from human and swine influenza viruses was isolated and sequenced from a swine in november 2014 [309] . the molecular diagnostics identified a total of 104 iav positive commercial and backyard swine in guatemala during 2010-2011 which resulted into three a(h1n1)pdm09 and one h3n2 virus isolates [310] . the first report of a(h1n1)pdm09 virus in commercial piggeries in cuba appeared in november 2010 when 24 swine were found positive for a(h1n1)pdm09 virus across five swine farms [311] . further, five more iav positive swine were detected in pinar del rio province of western cuba having respiratory illness and interstitial pneumonia. however only one iav positive sample could be successfully subtyped as a(h1n1)pdm09 virus having reassorted internal genes, all except the na gene [312] . in a more recent investigation, a high seroprevalence of iav (114/309) was detected in swine in trinidad and tobago which later identified h3n2 and a(h1n1)pdm09 viruses in swine [313] . in summary, the h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses were reported in north american swine population. interestingly, the avian influenza virus strain h4n6 was detected in us based swine while h3n3 and h4n6 were identified in the canadian swine and h5n2 was reported in the mexican swine in 2018 ( figure 4e ). mexico city was identified to be the origin of 2009 influenza pandemic. it was also ascertained that a(h1n1)pdm09 virus was present in mexican swine well before 2009 pandemic erupted. after influenza virus outbreak hit a swine farm in buenos aires in november 2008, one of the five dead swine were diagnosed with viral pneumonia through immunohistochemistry. a full genome of h3n2 virus sharing 96-98% nucleotide sequence identity with h3n2 viruses reported in north america during 2000-2003 was recovered from the swine [314] . an h1n1 virus was reported from a swine after a swine farm manager along with his spouse experienced influenza-like symptoms few days before the outbreak erupted in the swine at a buenos aires based farm in june 2009. the influenza disease symptoms lasted for about a week in nursery piglets. immunohistochemistry identified necrotizing bronchiolitis in four of the swine post-mortem samples while one sample had severe inflammation in the bronchiolar epithelia. the serological investigation detected iav antibodies in most of the sera samples collected after 15 days of onset of clinical symptoms however the active infection was reduced to only six swine [315] . the third investigation carried out histopathology which identified lung lesions compatible to the influenza virus infection in nine swine necropsy samples at a buenos aires based swine farm in october 2009 and later in eight swine necropsy samples originated from a santa fe based farm in may 2010. the swine at buenos aires farm were found infected with h1n1 virus while the swine at the santa fe farm retrieved one h1n2 and three human-like reassortant a(h1n1)pdm09 virus isolates which had triple reassortant internal genes. this was the first report of human-like reassortant a(h1n1)pdm09 virus in swine in argentina [316] . later two more investigations using histopathology, immunohistochemistry, serology, and molecular analyses reported h1n2, h3n2, and reassortant h3n2 viruses with a(h1n1)pdm09 internal genes in swine in argentina during 2011-2012 [317, 318] . several h1n1, h1n2, h3n2, human-like h1n1, and a(h1n1)pdm09 viruses have been identified in brazilian swine from the minas gerais, parana, rio grande do sul and sao paulo provinces in brazil during and after 2009 [319] [320] [321] [322] [323] [324] [325] . a technician who visited a minas gerais swine farm experiencing influenza outbreak developed similar respiratory disease symptoms. the nasal swab sample was obtained from the technician, as a result, one a(h1n1)pdm09 virus was isolated which was closely related to the a(h1n1)pdm09 viruses reported from the swine herd in the minas gerais which was recently visited by the technician. hence it was concluded that a zoonotic transmission from swine to the technician occurred at the minas gerais swine farm [326] . an immunohistochemical investigation demonstrated microscopic lesions suggesting broncho-interstitial pneumonia in the lung tissues of four severely sick piglets at a swine farm located in parana province in february 2011. the a(h1n1)pdm09 viruses were isolated from two piglets. additionally, a novel reassortant h1n2 virus was also recovered [327] . one more investigation identified that a(h1n1)pdm09 virus was the most prevalent iav subtype in sows. the co-infections of sows with a(h1n1)pdm09, h1n2, or h3n2 subtypes were also documented in rio grande do sul province. these findings were noteworthy because the coinfections may trigger reassortments and thus may facilitate emergence of novel strains of iav [328] . later two more h1n2 viruses were isolated and characterized from swine in rio grande do sul province during 2013. the sequences of both the isolates had high nucleotide similarity to each other in different genome segments in the range of 98.9% to 100% which suggested a common source of origin of both isolates [329] . the backyard productive systems (bps) for raising swine, cattle, and poultry are popular in chile. a molecular investigation reported a negative active iav infection across 113 bps units within ten counties in chile during 2012-2014 but the serological investigation detected iav antibodies in swine at two bps units which suggested a past exposure of swine to the iav [332] . interestingly, the ha gene sequence of an h12 virus was obtained from a domestic muscovy duck at one of the bps which appeared to have originated from a wild bird. this suggested a spillover of the iav from wild reservoir to the domestic poultry [332] . another study reported the prevalence of h1n2 virus in swine reared at 40 different bps having poultry and swine in el yali wetland during 2013-2014 [333] . one more study identified four swine sera samples (4/64; 6.3%) that were found positive for iav antibodies collected from different bps in central chile. one pool of swine nasal swab samples (1/39; 2.6%) was also detected iav positive with real-time rt-pcr. interestingly, 7.9% chicken, 4.3% ducks and 11.1% geese samples collected from 329 bps in central chile also had active iav infections. the breeding practice of poultry and swine in the bps was determined to be a major risk factor for iav transmission [334] . briefly, the iav strains of h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses have been reported from the swine in argentina and brazil while a(h1n1)pdm09 virus was reported in swine in colombia and peru. swine in chile were found infected with h1n2 virus ( figure 4f ). in summary, total 281 research articles were identified which reported several influenza viruses in swine populations globally. the highest number of studies were reported from asia (n = 107), followed by north america (n = 76), europe (n = 55), south america (n = 21), africa (n = 18) and australia (n = 4). the highest number of reports per country were documented in united states (n = 40) followed by china (n = 39) and canada (n = 24). until february 2020, influenza viruses have been reported from 53 countries worldwide. four subtypes of iav including h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses were most frequently detected in swine populations ( table 1) . most of the large-scale studies used serological investigations including elisa, hemagglutinin inhibition (hi), neuraminidase inhibition (ni), virus neutralization (vn), or microneutralization (mn) assays for the determination of the seroprevalence and subtyping of the influenza viruses in swine. several investigations used virus isolation for the confirmation and subtyping of iav. most of the virological investigations used one-step real-time rt-pcr and/or reverse-transcription pcr for influenza virus detection and subtyping. sanger sequencing or next-generation sequencing using miseq or ion torrent sequencing successfully generated the influenza virus sequences from the swine samples for epidemiological interpretations. histological examinations including immunohistochemistry or immunofluorescence were used to examine the swine lung or other internal organ tissue samples for the influenza virus diagnostics (table 2) . as of february 2020, influenza viruses have been identified and reported in swine from 53 countries worldwide (table 1 ; figure 3 ). the influenza viruses have been detected in different sample types including swine sera, nasal, tracheal, oropharyngeal, nasopharyngeal swabs as well as oral fluids collected from the live swine. nasal and snout wipes, lung homogenates and fecal slurry samples were also used. additionally, the lung as well as other internal organ tissues ( table 2 ) obtained from either dead or sacrificed swine have also been used for the detection of iav symptoms i.e., lesions in lungs, pneumonia, bronchitis, bronchiolitis etc. various methods have been used for the detection of influenza viruses in swine samples depending on the sample type, sample numbers and objective of the study. virus isolation methods, using either mdck, caco-2, hrt18, or swine testicle cells or the pathogen free embryonated chicken eggs, although considered the gold standard [335] [336] [337] have largely been taken over lately by the sequencing approaches which tend to provide a considerably faster identification of the iav subtypes. the additional benefit of sequencing over virus isolation is that the sequences would be useful for analyzing the influenza virus outbreak clusters [338] , virus evolution or reassortment [339] using phylogenetic analyses in different gene segments. a recent study reported that next-generation sequencing can be useful in the influenza virus diagnosis and for the identification of the novel virulence markers and drug resistance [340] . most of the studies have used real-time rt-pcr with matrix-gene specific oligonucleotide primers and taqman probe for iav detection [308, 341, 342] . the conserved sequences of the matrix-gene specific primers can detect any iav subtype in the swine samples [343] . most of the studies used subtype-specific real-time rt-pcr for the iav subtyping, however, few studies opted for the conventional approach of reverse-transcription pcr followed by sanger sequencing for amplification of the ha and/or na genes for retrieving the sequences for phylogenetic analyses to identify the subtypes. although the real-time rt-pcr is a powerful and rapid tool for the subtyping of iav strains, it is more expensive than reverse-transcription pcr. a few studies have reported reverse-transcription pcr based amplification of all the eight gene segments of iav to generate the whole genome sequences [33, 91, 128, 231] but in most cases, the whole genome sequences were generated using miseq next-generation sequencing approach [150, 214, 325, 329] . a great advantage of this sequencing approach is that it can identify novel influenza viruses in the swine samples [80, 90, 301] . most of the serological investigations used one or more methods for influenza virus detection and subtyping in the swine samples e.g., elisa, hi, ni, mn, or vn assays. the serological methods are useful in large-scale surveillances for screening large number of samples in a limited time. however, the molecular detection assays are more reliable than the serological methods given the higher sensitivity, but the serological assays are rapid and affordable hence are preferred for large-scale surveillance studies. the molecular and serological investigations report either active infections (viral rna) or past exposures (antibodies) in swine samples, respectively. the molecular detection approaches followed by sequencing are largely used for the research focusing on the influenza virus epidemiology [73, 78, 82, 87, 94, 128, 146, 150] . [5, 37, 43, 45, 46, 61, 74, 88, 137, 157, 171, 176, 188, 191, 199, 207, 215, [219] [220] [221] 241, 243, 248, 250, 263, 289, 290, 301, 306, 307, 326, 334] 2. [37, 38, 46, 52, 59, 70, 79, 96, 97, 135, 138, 153, 186, 195, 200, 215, 291, 306] 9. lung/liver/internal organ tissues rna extraction, real-time rt-pcr, reverse transcription-pcr, ligation, hi assay, virus isolation (mdck cells/spf chicken eggs), sanger and next-generation sequencing, hematoxylin-eosin staining, immunohistochemistry, immunofluorescence h1n1, h1n2, reassortant h1n1, h3n2, h2n3, a(h1n1)pdm09, h7n2, idv [45, 121, 127, 128, 130, 201, 203, 243, 245, 248, 274, 314, 320, 325, 327] 10. lung homogenate rna extraction, real-time rt-pcr, multiplex rt-pcr, single step rt-pcr, virus isolation (mdck cells/caco-2 cells/spf chicken eggs), sanger sequencing, membrane enzyme immunoassay, hi assay h1n1, h1n2, h3n2, reassortant h1n2, a(h1n1)pdm09 [130, 213, 218, 292] 11. fecal slurry rna extraction, qrt-pcr iav [88] 12. rectal swab nucleic acid extraction, reverse transcription, metagenomic sequencing iav [300] several studies used histological examinations e.g., immunohistochemistry or immunofluorescence to identify the iav symptoms in lung or other internal organs of either dead or severely sick swine sacrificed for the investigations [130, 189, 315, 318] . immunohistochemistry provides a rapid and affordable diagnosis of the influenza virus disease using swine tissue samples [344] . one major benefit of immunohistochemistry is that it can be used in the retrospective analysis of the archived tissue samples [245] . a large number of investigations have reported sub-clinical influenza virus infections in asymptomatic (apparently healthy) swine [78, 103, 150, 161, 171, 278, 300] , indicating that influenza infections can go undetected while the swine may be shedding virus and hence may infect other swine and farm workers in contact [183] . intriguingly, most of the swine samples processed in australia, europe, and north america were obtained from the symptomatic swine while most of the chinese swine samples were collected from asymptomatic swine ( figure 5 ). symptomatic swine may exhibit mild or severe influenza like symptoms [59, 202, 252] , including fever, coughing, sneezing, pneumonia, bronchitis, reduced appetite, diarrhea, nasal and/or ocular discharge, conjunctivitis, weakness, anorexia, prostration, weight loss, abortion in sows, and mortality in some cases [89, 224, 231, 239, 248, 282, 318] . most studies where the swine were severely infected reported reduced appetite and weight loss [129, 246, 247, 292] . due to iav infection, the swine takes longer to weigh 100 kg body mass [226] , hence the iav disease burden affects the swine farmers economically. varying rates of mortality of swine due to iav infections were reported from around the world ranging from 0.5% to 30% [138, 206, 213, 218, 221, 273, 296, 327] . this wide difference in mortality rate could be due to novel virus strains emerged through reassortments within the swine [273, 315, 327] or inter-species transmission, e.g., avian to swine transmission, resulting into severe disease outbreaks [27, 80, 119, 133] . one example of the emergence of a novel influenza virus strain is the emergence of a(h1n1)pdm09 strain due to reassortment between avian and swine iav strains in swine which resulted into 2009 influenza pandemic [33] . additionally, the emergence of iav subtype h1n2 is another classic example of influenza virus reassortment which resulted into severe disease outbreaks in japanese and korean swine populations during 1990s and 2000s [27, 121, 140] . strains of iav can infect the swine of any age group; piglets as young as one week may become infected with iav naturally. interestingly, a study in denmark observed a piglet as young as just three days was infected with iav despite having maternally derived iav antibodies [183] , suggesting that the infection might have occurred from the infected sow which was shedding the virus [183] . however, the symptoms of the influenza-like illness in swine may last only for one week but the virus shedding may still persist until 41 days after appearance of the influenza-like symptoms [183, 296] . this phenomenon may have serious implications in influenza virus spill over to the non-infected swine as well as to the exposed farm workers due to prolonged virus shedding. three other studies observed virus shedding in swine and reported that the virus shedding may persist until the 11 day [256] , 20 day [255] , or 29 day [296] after onset of the clinical symptoms in swine. this variation in the duration of the virus shedding might be strain dependent, which needs to be further investigated. a higher rate of virus shedding and iav prevalence was reported during the fall and early winter months than summer season because the high relative humidity present in the environment during summer decreases the transmission of influenza virus [267] . the high relative humidity in summer facilitates the generation of larger droplets which are less likely to be aerosol transmitted to a longer distance as they tend to fall on the ground quickly after their formation [240, 258] . several cases of inter-species transmission were identified which documented transmission of iav between human and swine or between birds and swine. the occurrence of the avian influenza virus strains in swine in china (h5n1, h9n2, h4n1, h4n6, h5n3, h10n5, h4n8, h6n6 , h7n9), united states (h4n6, h6n2, h7n2), canada (h4n6, h3n3), south korea (h7n2, h5n2), nigeria (h5n1), and egypt (h5n1, h5n2, h9n2 ) serve as the evidence of interspecies transmission of avian influenza viruses to swine. the first evidence of avian influenza virus active infection in swine appeared in 1999 in canada when h4n6 virus was isolated from a swine. later several other avian-origin iav strains were detected and sequenced in swine in china, canada, and south korea ( figure 6) . a large number of investigations have reported sub-clinical influenza virus infections in asymptomatic (apparently healthy) swine [78, 103, 150, 161, 171, 278, 300] , indicating that influenza infections can go undetected while the swine may be shedding virus and hence may infect other swine and farm workers in contact [183] . intriguingly, most of the swine samples processed in australia, europe, and north america were obtained from the symptomatic swine whi egypt is recognized as a "hot spot" for the influenza virus reassortment due to its geographical location [345] . the role of migratory wild birds in the introduction of avian influenza in egypt has been already established [346, 347] , and the highly pathogenic strains of the iav have previously been detected in migratory birds in egypt [348] . since migratory wild birds were reported to harbor in the vicinity of cairo [60] therefore, the probability of the migratory bird-swine interaction in the regions remain high which very well explains the occurrence of highly and low pathogenic strains of avianorigin iav in swine in cairo, egypt. given the "mixing vessel" nature of the swine, the occurrence of avian-origin iav strains in swine is alarming in terms of iav reassortment and evolution which may trigger the emergence of novel iav strains of pandemic potential in the future. further, the multiple reports of double or triple reassortant iav strains in swine are evidence that iav co-infections may facilitate the antigenic diversity of the influenza viruses; and as a result, new ha and na subtypes of iav may be continually added to the existing 18 ha and 11 na subtypes in the future. intriguingly, the frequency of the occurrence of double or triple-reassortant iav strains in swine has dramatically increased in the recent decades [76, 81, 87, 109, 161, 250, 268, 278] . one unique example of the reassortment and evolution of the pandemic strain of iav in swine was the emergence of a(h1n1)pdm09 virus in swine in mexico which evolved due to the reassortment between avian and swine iav strains [33] . while an overwhelming majority of investigations reported iav in the swine across the world ( figure 7a) , there were only a few reports which documented either active infections or past exposures of the swine to the influenza virus types ibv [37, 171, 186, 277] (figure 7b ), icv [38, 117, 186] ( figure 7c ) or idv [5, [43] [44] [45] [46] 217] (figure 7d) . a low prevalence of ibv was observed in swine given that only one study reported the ibv antibodies in swine samples in england during 1991-1992 [186] with no evidence of further spill over to other european countries. the active infection of ibv was later reported in us swine when two ibv isolates were obtained in 2009. a recent study from taiwan reported three strains of the victoria/b lineage of ibv in naturally infected swine in 2014 [171] , again various studies have spotted wild birds visiting the swine farms or in the vicinity which suggested that wild birds may have served as the carriers for the introduction of the different avian-origin iav subtypes to the swine populations [59, 80, 100, 246, 332] . the highest number of avian-origin iav strains were reported in chinese swine which shows frequent avian-swine interaction in china, a country that has historically been an epicenter for influenza virus disease [69] . egypt is recognized as a "hot spot" for the influenza virus reassortment due to its geographical location [345] . the role of migratory wild birds in the introduction of avian influenza in egypt has been already established [346, 347] , and the highly pathogenic strains of the iav have previously been detected in migratory birds in egypt [348] . since migratory wild birds were reported to harbor in the vicinity of cairo [60] therefore, the probability of the migratory bird-swine interaction in the regions remain high which very well explains the occurrence of highly and low pathogenic strains of avian-origin iav in swine in cairo, egypt. given the "mixing vessel" nature of the swine, the occurrence of avian-origin iav strains in swine is alarming in terms of iav reassortment and evolution which may trigger the emergence of novel iav strains of pandemic potential in the future. further, the multiple reports of double or triple reassortant iav strains in swine are evidence that iav co-infections may facilitate the antigenic diversity of the influenza viruses; and as a result, new ha and na subtypes of iav may be continually added to the existing 18 ha and 11 na subtypes in the future. intriguingly, the frequency of the occurrence of double or triple-reassortant iav strains in swine has dramatically increased in the recent decades [76, 81, 87, 109, 161, 250, 268, 278] . one unique example of the reassortment and evolution of the pandemic strain of iav in swine was the emergence of a(h1n1)pdm09 virus in swine in mexico which evolved due to the reassortment between avian and swine iav strains [33] . while an overwhelming majority of investigations reported iav in the swine across the world ( figure 7a) , there were only a few reports which documented either active infections or past exposures of the swine to the influenza virus types ibv [37, 171, 186, 277] (figure 7b ), icv [38, 117, 186] (figure 7c ) or idv [5, [43] [44] [45] [46] 217] (figure 7d) . a low prevalence of ibv was observed in swine given that only one study reported the ibv antibodies in swine samples in england during 1991-1992 [186] with no evidence of further spill over to other european countries. the active infection of ibv was later reported in us swine when two ibv isolates were obtained in 2009. a recent study from taiwan reported three strains of the victoria/b lineage of ibv in naturally infected swine in 2014 [171] , again there was no further report of dissemination to nearby asian countries. the ibv infected swine were apparently healthy with no signs of influenza disease. pathogens 2020, 9, x 32 of 54 there was no further report of dissemination to nearby asian countries. the ibv infected swine were apparently healthy with no signs of influenza disease. the first report of icv appeared in chinese swine after the virus was isolated from apparently healthy swine in 1981 in a routine diagnostic procedure at an abattoir in beijing [38] . later icv seroprevalence was reported in english and japanese swine during 1980s-1990s [117, 186] with no further evidence of circulation anymore thereafter. the idv was first detected and characterized in 2011 in oklahoma based swine in the united states which appeared to have made a species jump from cattle to swine [5, 43] . interestingly, a complete idv genome was retrieved from a symptomatic sow in italy in 2015 which was found closely related to the idv genome reported in 2011 from oklahoma, usa [217] . this might have happened due to the trade of the cattle or swine between italy and the united states. a recent study from china identified idv sequences which shared a high similarity (99%-100%) with the idv sequences reported earlier from the cattle in china [102] which was another evidence of bovine to swine transmission of idv. the idv has been in circulation in swine in the current decade with reports emerging from swine in italy, luxembourg, china and the united states. in summary, iav was first isolated from a swine in usa in 1930 [34, 349] and later antibodies for the human influenza viruses were reported in swine at the state prison of new jersey in 1937 [259] . more iav outbreaks and cases in swine in north america were reported during 1981-2000; the frequency has now dramatically fallen in the last two decades (figure 8 ). this might be due to the first report of icv appeared in chinese swine after the virus was isolated from apparently healthy swine in 1981 in a routine diagnostic procedure at an abattoir in beijing [38] . later icv seroprevalence was reported in english and japanese swine during 1980s-1990s [117, 186] with no further evidence of circulation anymore thereafter. the idv was first detected and characterized in 2011 in oklahoma based swine in the united states which appeared to have made a species jump from cattle to swine [5, 43] . interestingly, a complete idv genome was retrieved from a symptomatic sow in italy in 2015 which was found closely related to the idv genome reported in 2011 from oklahoma, usa [217] . this might have happened due to the trade of the cattle or swine between italy and the united states. a recent study from china identified idv sequences which shared a high similarity (99%-100%) with the idv sequences reported earlier from the cattle in china [102] which was another evidence of bovine to swine transmission of idv. the idv has been in circulation in swine in the current decade with reports emerging from swine in italy, luxembourg, china and the united states. in summary, iav was first isolated from a swine in usa in 1930 [34, 349] and later antibodies for the human influenza viruses were reported in swine at the state prison of new jersey in 1937 [259] . more iav outbreaks and cases in swine in north america were reported during 1981-2000; the frequency has now dramatically fallen in the last two decades (figure 8 ). this might be due to improved swine influenza surveillance and vaccination in north america in recent decades. the h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses were reported from the commercial, backyard, exhibition, feral swine and wild boars in the united states. as of february 2020, the highest number of reports of influenza virus infections in swine in a country were documented in the united states (n = 40) followed by china (n = 39) and canada (n = 24). the highest number of iav positive swine samples were reported in the united states (36128/200384) followed by china (5031/90760). one of the factors behind the higher number of iav cases in swine in the united states compared to china would be related to the disease symptoms. a majority of the north american swine samples that were screened for the iav infections had either mild or severe symptoms of influenza-like illness which would have made it visually easier to identify iav infected swine in the united states. on the contrary, a smaller number of chinese swine exhibited influenza-like disease symptoms while a large proportion of the chinese swine population appeared to have sub-clinical infections with no symptoms. this would have made it difficult for identifying the influenza virus infected swine during surveillances in china. the first report of idv in 2011 in oklahoma swine reflected the antigenic diversity and evolution of influenza viruses in the us swine. however, the recent influenza virus disease prevalence in north american swine appeared to have declined, nevertheless, given the large swine population of the continent, the surveillance should continue to track the influenza virus diversity and evolution. the first serological evidence of iav in european swine was documented from the czechoslovakia during 1969-1972, but the first h1n1 virus in european swine was isolated in belgium in 1979 which was apparently transmitted from wild ducks in germany to the swine in belgium. since then several h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses have been detected in commercial and backyard swine as well as in wild boars within europe. the incidence of iav in european swine has increased several folds in the past two decades with a relatively high number of iav positive swine samples (19644/49814). most of the iav positive european swine were reported having influenza-like symptoms at the time of sampling. germany reported the highest number of iav positive swine in europe where the pork industry is considered the third largest globally after china and the united states. importantly, the idv was more recently identified in the european swine, first in italy in 2015, and later a retrospective study identified idv infection in swine samples collected in luxembourg during 2014-2015 which indicated that the circulation of idv in european swine took place only after 2014. the evidence has suggested the bovine to swine transmission of idv. this observation is interesting because until recently more emphasis has been given to the avian-swine interaction and the bovine-swine interactions have been neglected from the influenza virus spill over perspective. the first occurrence of iav in asian swine can be traced back to 1969, but the iav prevalence has increased multi-fold in the recent two decades. the iav subtypes h1n1, h1n2, h3n2, and a(h1n1)pdm09 have become endemic in swine in several asian countries. the highly pathogenic avian-origin iav strains of h5n1 and h7n9 have been reported from chinese swine while h5n1 has been documented in swine in viet nam and indonesia. the highly pathogenic strains of h5n2 have been reported in south korean swine. several lpaiv strains including h4n1, h4n6, h4n8, h6n6, h9n2, and h10n5 have also been documented in chinese swine. the studies suggested a frequent interaction between wild birds and swine in china which appeared to have transmitted avian-origin iav strains in the chinese swine. occurrence of equine influenza virus h3n8 in chinese swine further expanded the genetic diversity of swine influenza viruses. h1n1, h1n2, h3n2, and a(h1n1)pdm09 viruses were reported from the commercial, backyard, exhibition, feral swine and wild boars in the united states. as of february 2020, the highest number of reports of influenza virus infections in swine in a country were documented in the united states (n = 40) followed by china (n = 39) and canada (n = 24). the highest number of iav positive swine samples were reported in the united states despite an avian to human transmission of certain avian influenza virus strains including h5n1 virus, only a limited human to human transmission of avian influenza viruses was established in the past [350, 351] . with the passage of these avian-origin iav strains in a mammalian host like swine, a high probability remains of these avian influenza virus strains to adapt and gain the ability of the human to human transmission, if happens, the consequences would be devastating for the public health. australian swine were free from influenza virus until the year 2009 when a new south wales swine farm reported an influenza-like outbreak in the swine. the zoonotic transmission of the a(h1n1)pdm09 virus was reported to the farm workers and the farm owner. until now, there have only been four iav reports in australian swine which reflects a low prevalence of iav in australian swine. new zealand is yet to officially report the influenza virus prevalence in swine and remains free from the disease. a retrospective study identified that the a(h1n1)pdm09 virus was present in mexican swine as early as 2000, well before the influenza pandemic occurred during march-may 2009. a high genetic diversity of iav in mexican swine due to live swine imports from north america and europe during 1980s laid the foundation of the emergence of zoonotic strain of a(h1n1)pdm09 virus in mexican swine [33] . the highest number of iav positive swine in central america were reported from mexico followed by guatemala. interestingly, a report of the highly pathogenic h5n2 virus in mexican swine in 2018 further triggers the alarm in the context of a potential novel iav reassortment. outbreaks of iav in the south american swine populations occurred during the last two decades, with the highest prevalence reported in the brazilian swine. a considerable proportion of cases showed sub-clinical infections with no symptoms which might have made it more difficult to detect the infected swine. the reports of iav active infections or the seroprevalence appeared in the african swine only during last two decades. until february 2020, iav have been detected in swine in cameroon, nigeria, egypt, kenya, reunion island, uganda, togo, and ghana. however, south africa has a considerable swine population [352] , but currently there is no published report on the prevalence of active iav or other influenza virus infections in the south african swine. this might be because of the lack of an active surveillance for the detection of the influenza virus disease in the swine in south africa. the reports of reassortant, double-reassortant and triple-reassortant influenza viruses in asian, north american and european swine strengthens the concept of swine being the "mixing vessel" in terms of influenza virus reassortment and evolution. the multiple reports of avian-origin iav strains including highly pathogenic h5n1, h5n2 and h7n9 in swine are alarming given the fact that the avian-origin strains may adapt in swine to facilitate the emergence of a reassortant pandemic strain. the highest number of influenza virus studies in swine population have been reported from the united states (n = 40) followed by china (n = 39). also, the united states reported the highest numbers of iav cases in swine. due to widespread active surveillance, the united states 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immunohistochemistry: a rapid and specific diagnostic tool for influenza virus infection in pigs predicting hotspots for influenza virus reassortment avian influenza viruses at the wild-domestic bird interface in egypt an overview of the epidemic of highly pathogenic h5n1 avian influenza virus in egypt: epidemiology and control challenges highly pathogenic avian influenza virus (h5n8) clade 2.3.4.4 infection in migratory birds the etiology of swine influenza detecting human-to-human transmission of avian influenza a (h5n1) barrier to bird flu transmission found in humans pig farming in rural south africa: a case study of uthukela district in kwazulu-natal this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-278523-djjtgbh6 authors: zhou, bei-xian; li, jing; liang, xiao-li; pan, xi-ping; hao, yan-bing; xie, pei-fang; jiang, hai-ming; yang, zi-feng; zhong, nan-shan title: β-sitosterol ameliorates influenza a virus-induced proinflammatory response and acute lung injury in mice by disrupting the cross-talk between rig-i and ifn/stat signaling date: 2020-06-05 journal: acta pharmacol sin doi: 10.1038/s41401-020-0403-9 sha: doc_id: 278523 cord_uid: djjtgbh6 β-sitosterol (24-ethyl-5-cholestene-3-ol) is a common phytosterol chinese medical plants that has been shown to possess antioxidant and anti-inflammatory activity. in this study we investigated the effects of β-sitosterol on influenza virus-induced inflammation and acute lung injury and the molecular mechanisms. we demonstrate that β-sitosterol (150–450 μg/ml) dose-dependently suppresses inflammatory response through nf-κb and p38 mitogen-activated protein kinase (mapk) signaling in influenza a virus (iav)-infected cells, which was accompanied by decreased induction of interferons (ifns) (including type i and iii ifn). furthermore, we revealed that the anti-inflammatory effect of β-sitosterol resulted from its inhibitory effect on retinoic acid-inducible gene i (rig-i) signaling, led to decreased stat1 signaling, thus affecting the transcriptional activity of isgf3 (interferon-stimulated gene factor 3) complexes and resulting in abrogation of the iav-induced proinflammatory amplification effect in ifn-sensitized cells. moreover, β-sitosterol treatment attenuated rig-i-mediated apoptotic injury of alveolar epithelial cells (aec) via downregulation of pro-apoptotic factors. in a mouse model of influenza, pre-administration of β-sitosterol (50, 200 mg·kg(−1)·d(−1), i.g., for 2 days) dose-dependently ameliorated iav-mediated recruitment of pathogenic cytotoxic t cells and immune dysregulation. in addition, pre-administration of β-sitosterol protected mice from lethal iav infection. our data suggest that β-sitosterol blocks the immune response mediated by rig-i signaling and deleterious ifn production, providing a potential benefit for the treatment of influenza. the annual spread of seasonal influenza a virus (iav), particularly the sporadic transmission of highly pathogenic avian influenza (hpai) viruses (e.g., the h5n1 and h7n9 subtypes) continues to constitute a major threat to public health. as of december 2018, avian influenza a h7n9 viruses have caused an estimated 1567 individual infections, with an overall mortality rate of 39.2% (http://www.fao.org/ag/againfo/programmes/en/empres/h7n9/ situation_update.html). recently, the na r292k and ns g540a substitutions in the h7n9 virus have been reported to be associated with increased resistance to oseltamivir and to confer enhanced viral replication in mammalian cells [1, 2] . this raises concerns about a potential human pandemic. iav initiates infection in humans via entry through the upper respiratory tract, eliciting symptoms that can be mild and self-limited, but in some patients, it can lead to acute respiratory distress syndrome (ards), which is characterized by the impairment of gas exchange resulting in a fatal outcome [3, 4] . a series of reports have indicated that a robust and dysregulated innate immune response is primarily responsible for the high mortality rate associated with influenza [5, 6] . however, there are few alternative medicine strategies available for the treatment of influenza virus-infected patients who have an intense inflammatory response. the innate immune system is armed with an array of pattern recognition receptors (prrs) that provide the principal barrier defense against infectious agents [7] . during the process of viral replication, influenza virus synthesizes viral rna containing a 5′triphosphate end and is then transported to the cytoplasm [8] . this pathogen-associated molecule (pam) is detected by the cytosolic sensor retinoic acid-induced gene i (rig-i) and subsequently activates multiple cellular signal cascades [9] . ultimately, these signaling pathways lead to the activation of transcription factors including nf-κb, ap-1 (atf2:c-jun), and irf3, which together bind to specific sites of the ifn-β promoter and initiate ifn-β synthesis for the antiviral response [10] . targeting p38 map kinase with a specific inhibitor affects the induction of ifn-β [11] , which inhibits the downstream phosphorylation of atf2 by p38 map kinase [12] . studies have demonstrated the crucial role of ifn production mediated by rig-i signaling in protecting against lethal iav challenge [13, 14] . in vivo studies have shown that the increased susceptibility of both rig-i-and ifn-deficient mice to several rna viruses [15] [16] [17] , including vesicular stomatitis virus (vsv), newcastle disease virus (ndv), and iav, is associated with the failure of ifns levels to increase or a lack of ifn signaling. there is evidence that infection with hpai h5n1 viruses triggers the hyperinduction of proinflammation via rig-i signaling cascades despite the crucial role of rig-i signaling in defense against iav [18] . the induction of downstream genes of rig-i signaling, such as il-6, tnf-α, il-8, and il-1β, is significantly upregulated in the bronchoalveolar lavage fluid (balf) of patients with sustained ards [3, 19, 20] . type i and type iii ifns secreted by infected cells bind to distinct receptors but trigger similar signal transduction through the jak/ stat cascades [21, 22] . upon ifn stimulation, the phosphorylation of the stat1:stat2 heterodimer leads to its interaction with irf9 and the formation of the interferon-stimulated gene factor 3 (isgf3) complex [23] . then, the complex moves from the cytoplasm to the nucleus where it binds to the interferonstimulated response element (isre) and drives the expression of interferon-stimulated genes (isgs). these genes possess direct antiviral and immunomodulatory activity [24] . for example, the mx protein, which was the first isg identified to restrict influenza virus infection to be identified, has been demonstrated to impair vrnp nuclear import [25] . however, despite the abundant production of ifns and hundreds of isgs in response to viral infection, the ifnmediated antiviral response is blocked by various viral products. the non-structural (ns1) protein of iav is a typical multifunctional protein involved in the blockade of the antiviral effect of isgs, such as 2′,5′-oligoadenylate synthetase (oas) and dsrnadependent protein kinase r (pkr) [26, 27] . in addition, iav infection has also been shown to suppress the antiviral response mediated by type i and iii ifns by upregulating the negative regulators socs3 and socs1 [28, 29] . given that viruses can overcome the ifn-mediated antiviral response through various mechanisms, it is possible that excessive ifn production during the antiviral response may contribute to the harmful effects. surprisingly, studies have found that increased host susceptibility to secondary bacterial co-infections correlates with ifn induction during iav infection [30] . moreover, a deficiency in ifn-α/β signaling increases survival through a reduction in excessive inflammation and apoptotic injury to lung epithelial cells [31] . similarly, high levels of trail in balf collected from patients with influenza-associated ards may be a result of increased trail expression induced by macrophage-derived ifn-β [32] , which contributes to lung alveolar epithelial cell injury. in addition, recent evidence has also revealed that ifn-mediated signaling drives immunopathologic injury in response to various viral infections, including respiratory syncytial virus infection (rsv) [33] and severe acute respiratory syndrome cov (sars-cov) [34] . given these findings, it is clear that the disease-promoting effects of ifn contribute to deleterious outcomes, and should be limited by pharmacological intervention. the identification of agents derived from medicinal plants that can prevent influenza is valuable. chinese medicinal plants, including lonicera japonica [35] , chrysanthemum morifolium [36] , taraxacum mongolicum [37] , forsythia suspense [38] , and isatis indigotica [39] have been prescribed for the common cold, heatclearing, and detoxication for thousands of years, but the bioactive ingredients of these plants that mediate these pharmacological effects is unknown. phytosterols contain structural features that resemble those of cholesterol and are abundant in vegetables, fruits, and medicinal plants [40, 41] . among phytosterols, β-sitosterol (24-ethyl-5-cholestene-3-ol) is the most common sterol and has been shown to possess antioxidant, antiinflammatory, antitumor, and antiasthmatic effects [42] [43] [44] [45] . in the present study, we hypothesized that β-sitosterol is the bioactive component of five types of medicinal plants. to test this hypothesis, we investigated the effects of β-sitosterol and the underlying mechanisms by which it may exert a therapeutic effect against influenza-mediated injury and dysregulated inflammation. preparation of extracts and quantitative analysis of β-sitosterol samples of four kinds of different heat-clearing and detoxifying traditional chinese medicines samples (l. japonica, c. morifolium, t. mongolicum, and f. suspense) were purchased from local markets in bozhou, china. i. indigotica was supplied by hutchison whampoa guangzhou baiyunshan chinese medicine co., ltd (guangzhou, china). a β-sitosterol standard was purchased from sigma (san francisco, usa), and hplc-grade methanol was purchased from fisher scientific (fisher, usa). a sample of each of the five medicinal materials was crushed into a coarse powder, and 2.0 g was placed in a 100-ml flask. extraction was performed using ultrasonic waves for 15 min and the addition of 50 ml of chloroform and was repeated three times. the samples were then centrifuged at 2500 × g for 10 min. the supernatants were combined and condensed to a proper volume under reduced pressure, and then the concentrates were dissolved with chloroform. the samples were transferred to 5-ml volumetric flasks, diluted with chloroform to 5 ml, and mixed. a total of 2.0 mg of the β-sitosterol standard was accurately weighed and dissolved in 5 ml of chloroform to produce individual stock solutions. hplc analysis of β-sitosterol was performed at 28°c on an hplc instrument (shimadzu 20a, japan) with a dad detector at 205 nm. chromatographic separation was performed on a shimadzu ods column (4.6 × 150 mm, 5 μm, tokyo, japan). the mobile phase was methanol, and the injection volume was 10 μl. the samples were subjected to quantitative analysis, which was performed using the external standard method. the results are expressed as mg/g, and all analyses were performed in triplicate. influenza a/puerto rico/8/34 (h1n1) and a/fm/1/47(h1n1) mouse-adapted viruses were stored in our laboratory and propagated in the allantoic cavities of 9-day-old specific pathogen-free embryonated chicken eggs at 37°c. freshly collected allantoic fluids were clarified by low-speed centrifugation at 72 h postinoculation and then stored in small aliquots at −80°c. the virus titers were determined using a plaque forming assay in monolayers of madin-darby canine kidney (mdck) cells as previously described. mouse experiments and viral challenge four-to six-week-old female balb/c mice (weighing 16-18 g) were purchased from guangdong medical laboratory animal center. all mice were housed and cared for under specific pathogen-free conditions at the state key laboratory of respiratory disease or guangdong laboratory animal monitoring institute. all animal experimental procedures in this study were approved by the ethics committee of the first affiliated hospital of guangzhou medical university and conducted in strict accordance with the approved guidelines. the 50% lethal dose (ld 50 ) of the mouse-adapted h1n1 virus was estimated in mice after the stock virus was serially diluted. the mice were treated intragastrically with β-sitosterol (50 mg·kg −1 ·d −1 , 200 mg·kg −1 ·d −1 ) or pbs (vehicle group) 2 days prior to viral challenge. the mice were anesthetized (5% isoflurane inhalation) and challenged intranasally with 5 ld 50 of mouse-adapted h1n1 virus. cell culture and viral infection human alveolar epithelial a549 cells and 293t human embryonic kidney cells were grown in dulbecco's modified eagleʼs medium (dmem/f12, 1:1 mixture) (gibco) supplemented with 10% fetal bovine serum (fbs) (gibco) in a humidified incubator at 37°c and 5% co 2 . for the viral infection, a549 cells (5 × 10 6 cells/ml) grown in 6well tissue culture plates (guangzhou jet bio-filtration co., ltd, tcp-010-006) were inoculated with a/pr/8/34 (h1n1) in serumfree dmem/f12 medium at an indicated multiplicity of infection (moi). after 2 h absorption, the inoculum was discarded and replaced with fresh serum-free dmem/f12 medium containing diluted compounds. antibodies and recombinant protein the following primary antibodies were used in the current study: anti-rig-i, anti-nf-κb p65, anti-phospho-nf-κb p65 (ser 536 ), anti-p38 mapk, anti-phospho-p38 mapk ( protein preparation and immunoblot analysis excised lungs and cells were lysed using ice-cold ripa buffer (25 mm tris·hcl ph 7.6, 150 mm nacl, 1% np-40, 1% sodium deoxycholate, and 0.1% sds) supplemented with protease inhibitors (sigma). the crude lysates were centrifuged at 14,000 rpm (13,000 × g) for 15 min at 4°c, and the supernatant was collected. equal amounts of cell or tissue extracts were loaded onto 10% sds-polyacrylamide gels for separation. then, proteins were transferred to 0.2 μm pvdf membranes (bio-rad), which allowed subsequent probing with primary antibodies. after overnight incubation at 4°c, the membranes were washed with 0.1% tbst (tbs/0.1% tween 20) and incubated with corresponding horseradish peroxidase (hrp)conjugated secondary antibodies (multisciences). the signals were visualized using enhanced chemiluminescence (ecl) reagents (perkin-elmer). quantification of the relative band intensities was performed using imagej software version 1.43. ppprna generation, rna isolation, and real-time rt-pcr viral rna (5′-triphosphate rna, 5′ppp-rna) and cellular rna were generated as previously described [46] . briefly, total rna was isolated from a549 lung epithelial cells infected with a/pr/8/34(h1n1) for 24 h (viral rna) and uninfected a549 lung epithelial cells (cellular rna) using trizol reagent (takara, usa). then, the 5'-phosphate group was dephosphorylated by treatment with calf-intestinal alkaline phosphatase (ciap) (takara). a549 lung epithelial cells were transfected with the indicated rna using lp2000 (thermo, usa). for qpcr, total rna (1 μg) was reverse transcribed into cdna and subjected to real-time quantitative pcr with gene-specific primers and probes on an abi7500 real-time pcr system. relative gene expression was calculated using the 2 −δδct method [47] . the specific primer and probe sets are shown in supplementary table s1 . small interfering rna (sirna), plasmid transfection, and reporter gene assays transient transfection of plasmids into cells, including an isre luciferase reporter plasmid (beyotime) and a flag-rig-i overexpression plasmid (geneppl), was performed by using lipofectamine 2000 (invitrogen). after isre luciferase reporter plasmid (0.5 μg) and flag-rig-i (0.5 μg) overexpression plasmid were transfected into a549 cells for 6 h, the transfected cells were stimulated with ifns or iav (moi = 0.1). in the other experiment, hek293 cells stably co-transfected with pnf-κb-tata-f-luci and pqcxip-egfp plasmid were stimulated with tnf-α (20 ng/ml) or iav for the indicated times. firefly luciferase activity was assayed using the luciferase reporter system (promega, usa) and normalized to renilla luciferase activity or the levels of gfp expression. rig-i-specific sirnas (rig-i #1 sirna and, rig-i #2 sirna) were purchased from guangzhou ribobio co., ltd. and transfected into cells with lipofectamine 2000 according to the manufacturer's instructions. histology and immunohistology lung tissue was excised, fixed in 10% formalin and embedded in paraffin using routine procedures. tissue sections (4 μm) were stained with hematoxylin-eosin for histological examination. for immunohistochemical staining, the sections were deparaffinized with xylene and rehydrated in a graded alcohol series. endogenous peroxidase was blocked with 3% hydrogen peroxide (h 2 o 2 ) in methanol for 20 min at room temperature and antigen retrieval was performed in 0.01 mm citrate buffer (ph 6.0). afterward, the sections were blocked with 5% normal serum and incubated with primary antibody at 4°c overnight. the sections were then incubated for 1 h in hrp-labeled secondary antibody solution and then visualized using a dab reagent kit (maixin, china). the sections were counterstained with mayer's hematoxylin for 2 min before mounting. apoptosis detection by annexin v and flow cytometry cells collected from both suspension and adherent were washed twice with cold pbs, and subsequently resuspended in 1× annexin binding buffer (bioscience, usa) at a concentration of 2 × 10 6 cells/ml. one hundred microliters of cell suspension was stained with 5 μl of annexin v-fitc and 5 μl of propidium iodide (pi) for 15 min at room temperature in the dark. the cells were analyzed using a novocyte flow cytometer within 4 h. bronchoalveolar lavage and flow cytometry mice were euthanized intraperitoneally (i.p.) with an overdose of sodium pentobarbital at the indicated time point. the trachea was cannulated via ventral middle incision. the lungs were lavaged three times with 600 μl of sterile saline. bronchoalveolar lavage fluid (balf) was centrifuged at 300 × g for 10 min at 4°c and the supernatants were collected and stored at −80°c for biochemical analysis. the balf pellets were resuspended in pbs with 0.5% bsa and stained with fluorochrome-conjugated monoclonal antibodies (mabs) (bioscience, usa) that bound to surface molecules for 20 min at 4°c. all samples were analyzed on a novocyte flow cytometer. statistical analysis all data are presented as the mean ± sem. statistical analysis was performed using spss software version 18.0. one-way anova followed by the student-newman-keuls (snk) test was carried out to assess differences between the study groups, and p values less than 0.05 were considered significant. quantitative analysis of β-sitosterol in five medicinal materials medicinal plants including l. japonica, c. morifolium, t. mongolicum, f. suspense, and i. indigotica are traditionally used for heatclearing and detoxification. we hypothesized that β-sitosterol is the common substance in these medicinal plants that mediates their pharmacological actions against respiratory diseases, such as β-sitosterol ameliorates iav-induced inflammation and ali bx zhou influenza. first, we quantified the content of β-sitosterol in these plants. as shown in table 1 , β-sitosterol was detected in all samples at concentrations ranging from 0.51 to 2.23 mg/g. β-sitosterol treatment inhibits the activation of the cellular signaling pathway in iav-infected cells to clarify whether β-sitosterol possesses antiviral activity for the treatment of iav infection, cytopathic effect (cpe) inhibition assays and plaque reduction assays were performed to investigate the antiviral effects of β-sitosterol. as shown in supplementary table s2 , β-sitosterol showed no activity against a/gz/gird07/ 09(h1n1), a/pr/8/34(h1n1), a/hk/8/68(h3n2), a/hk/y280/97 (h9n2), or b/lee/1940 (flub). these findings were further confirmed by plaque reduction assays ( supplementary fig. s1 ). the activation of multiple cellular signaling events has been implicated in the molecular pathogenesis of iav infection [48] . to identify the pharmacological effects exerted by β-sitosterol during iav infection, we assessed the impact of β-sitosterol on the activation of cellular signaling pathway in cells infected with iav. hek293 cells in which an nf-κb-luc reporter was stably expressed were stimulated with either tnf-α (20 ng/ml) (fig. 1a) or a/pr/8/ 34 (h1n1) (fig. 1b) , and then incubated with β-sitosterol for 24 h. we observed that hek293 cells stimulated with tnf-α or a/pr/8/ 34 (h1n1) exhibited robust nf-κb activation, on which β-sitosterol treatment had a dose-dependent suppressive effect ( fig. 1a, b) . furthermore, to test whether β-sitosterol possesses other pharmacological properties, we assessed the activation of signaling pathways in iav-infected a549 cells in the presence or absence of β-sitosterol by immunoblotting. nf-κb and mapk (p38, erk1/2, and jnk/spak) signaling was activated by iav infection (fig. 1c) . as expected, β-sitosterol inhibited the iav-induced p38 mapk activation and p65 phosphorylation, which is consistent with the data gathered using the nf-κb-luc reporter cell line. however, βsitosterol had no inhibitory effect on the erk1/2 or jnk mapk pathway. together, these data demonstrate that β-sitosterol has the potential to inhibit the activation of nf-κb and p38 mapk signaling in response to iav infection. β-sitosterol treatment decreases the expression of iav-induced proinflammatory mediators both the nf-κb and p38 mapk signaling cascades have been implicated as major contributors to hypercytokinemia during human hpaiv h5n1 infection [49, 50] . therefore, we next investigated the effect of β-sitosterol on the expression of proinflammatory mediators in iav-infected cells. a549 cells were infected with the pr8/h1n1 virus (moi = 0.1) in the presence of increasing concentrations of β-sitosterol (150-450 μg/ml) for 24 h. subsequently, the transcript levels of proinflammatory mediators were measured by qpcr. our data showed that the gene expression of an array of cytokines and chemokines, including il-6, tnf-α, ip-10, il-8, mcp-1, mip-1β, and rantes, was elevated dramatically following iav infection, and that this elevation was attenuated by β-sitosterol treatment (fig. 2a) . the kinetics of iavinduced proinflammatory mediator release showed that the protein levels of these proinflammatory mediators (including il-6, tnf-α, il-8, ip-10, rantes, and mcp-1) reached their expression peak at 24 h after viral infection (fig. 2b) , and that the increases in the levels of these mediators were reversed by β-sitosterol treatment (fig. 2c) . the production of cyclooxygenase-2 (cox-2) and its derivative prostaglandin e2 (pge2), has been shown to occur via nf-κb and p38 mapk signaling and play a pathogenic role during iav infection [51, 52] . indeed, increasing the expression of iav-induced cox-2 at both the mrna and protein levels at 24 h p.i., while treatment with β-sitosterol profoundly reduced cox-2 production (fig. 2d, e) . furthermore, we measured the effect of β-sitosterol on the iav-induced expression of iav-induced cox-2 and carried out elisa to quantify cox-2-derived pge2 in the culture supernatants. as expected, β-sitosterol treatment of iav-infected cells led to a significant dose-dependent reduction in pge2 levels (fig. 2f) . these results indicate that β-sitosterol treatment decreases the expression of iav-induced proinflammatory mediators through the inactivation of the nf-κb and p38 mapk signaling pathways. β-sitosterol treatment suppresses the iav-mediated induction of interferon expression and signal transduction by targeting rig-i in response to iav infection, transcription factors including nf-κb, atf2/c-jun, and irf3/7 bind cooperatively to the promoter regions of ifn genes, which are secreted to establish an antiviral state, to initiate their expression [10] . the activation of p38, which mediates the activation of its downstream target atf2, is a prerequisite for optimal ifn-β induction [12] . since β-sitosterol inhibits the iav-induced activation of p38, we hypothesized that β-sitosterol treatment might reduce the expression of ifns. to test this hypothesis, culture supernatants from iav-infected a549 cells treated with or without β-sitosterol were collected at 24 h p.i. and transferred to uninfected a549 cells. after 15 min of incubation, we assessed ifn levels in the culture supernatant by monitoring the phosphorylation and downstream signaling of ifns by immunoblot analysis. as shown in fig. 3a , supernatants from infected a549 cells stimulated the phosphorylation of stat1 tyr701 and stat2 tyr690 , while supernatants from donor cells treated with β-sitosterol attenuated the phosphorylation of stat1 tyr701 . our results indicated that the suppression of stat1 tyr701 phosphorylation by β-sitosterol was associated with reduced ifns production. to further confirm that β-sitosterol inhibits the iav-induced expression of ifns, ifn concentrations in supernatants collected at 24 h p.i. were measured using luminex. as demonstrated in fig. 3b , the iav-induced elevation of ifns, including type i ifn (ifnβ), type ii ifn (ifn-γ), and type iii ifn (ifn-λ1), was significantly decreased in the supernatants of β-sitosterol-treated cells. these observations led us to ask whether β-sitosterol directly affects the ifn signaling transduction pathway. to address this question, we tested whether signal transduction induced by exogenous ifn-β is altered by β-sitosterol treatment. then, we pretreated iav-infected a549 cells with β-sitosterol for 4 h before stimulating them with ifn-β for 15 min. the phosphorylation of stat1 tyr701 , but not stat2, was markedly inhibited by β-sitosterol treatment (fig. 3c) . however, at later time points (8 h p.i.), the ifnβ-mediated phosphorylation of stat1 tyr701 was reduced in iavinfected a549 cells regardless of whether they were pretreated with β-sitosterol or not (fig. 3d) . our data demonstrated that β-sitosterol reduced ifn production through the inhibition of nf-κb and p38 map kinase and that it blocks ifn-mediated signal transduction. we next sought to elucidate the mechanism by which β-sitosterol disrupts ifn production and signaling. the intracellular prr rig-i senses iav 5′ppp-rna, resulting in the activation of nf-κb and p38 map kinase which preferentially promote the initiation of ifn transcription [53] . it is worth mentioning that rig-i, an interferon-stimulated gene (isg), has been reported to augment stat1 activation and inhibit leukemia cell proliferation [54] . thus, it is reasonable to speculate that cross-talk between the rig-i and ifn signaling pathways is affected by β-sitosterol. to confirm this assumption, we investigated the effect of β-sitosterol on the iavinduced expression of rig-i. our results show that β-sitosterol decreased the induction of rig-i in infected a549 cells (fig. 3e, f) . furthermore, cells transfected with the flag-rig-i overexpression plasmid transfection were found to have elevated p-stat1 levels, which were diminished by β-sitosterol treatment (fig. 3g) . the tyrosine kinases jaks are well recognized to be involved in the activation of stat1. therefore, it is possible that β-sitosterol affected stat1 activation by inhibiting jaks. interestingly, we found that a549 cells pretreated with β-sitosterol for 12 h prior to ifn-β (20 ng/ml) stimulation did not exhibit decreased ifn-βmediated activation of jak1, stat1, and stat2 levels (fig. 3h) . together, these data indicate that β-sitosterol can exert an inhibitory effect on rig-i, which leads to decreased iav-induced ifn production and ifn-β signal transduction. β-sitosterol treatment attenuates the ifn-mediated amplification of the proinflammatory response during iav infection via the inhibition of rig-i it is widely recognized that ifn signaling plays a vital role in host antiviral immunity [55] . furthermore, ifns possess immunomodulatory activities on the induction of both chemokines and cytokines and on the recruitment of various immune cells [56] . to examine the effect of rig-i inhibition by β-sitosterol on the transcription of ifn signaling-related molecules, we analyzed iav-mediated ifn-stimulated response element (isre) activation with an assay that utilized a transient isre reporter plasmid. the results presented in fig. 4a show that iav-mediated isre-dependent transcription was significantly inhibited by βsitosterol treatment. rig-i knockdown in iav-infected cells by specific sirnas in iav-infected cells was confirmed to significantly inhibit the iav-mediated isre transcriptional activity (fig. 4b, c) . to further determine the effects of β-sitosterol on ifn-induced proinflammatory responses during iav infection, isre reporter plasmid-transfected cells were stimulated with ifn-β (500 ng/ml) 4 h prior to iav infection. prestimulation with ifn-β led to much higher isre activity than iav infection alone (fig. 4d) . similarly, ifn-β stimulation following iav infection also resulted in increased isre activity, but to a lesser extent than what was seen after ifn-β prestimulation (fig. 4d) . this indicates that ifn-β signaling contributes to the amplification of isre activity during iav infection. nevertheless, this increase in isre activity was diminished in a dose-dependent manner by β-sitosterol treatment representative results from at least three independent experiments are shown. d the band intensities of p-p65, p-p38, p-erk1/2, and p-jnk were semiquantified using imagej (normalized to the loading control gapdh). the data are presented as the mean ± sem (n = 3-5). **p < 0.01, ***p < 0.001 versus the group treated with tnf-α or iav. β-sitosterol ameliorates iav-induced inflammation and ali bx zhou immunoblot analysis was performed to evaluate the protein expression of cox-2. gapdh was used as the internal control (e). f quantification of pge2 in the culture supernatants using elisa. *p < 0.05, **p < 0.01, ***p < 0.001 versus the iav control group. ( fig. 4d) . we next assessed the effects of β-sitosterol on the expression of proinflammatory genes in iav-infected cells pretreated with or without ifn-β. consistent with the previously observed increase in isre activity, the mrna and protein levels of cytokines and chemokines, including il-6, ip-10, tnf-α, il-8, mcp-1 and gm-csf, were robustly increased in iav-infected cells after stimulation with ifn-β (fig. 4e, f) . a similar cytokine and chemokine expression pattern was observed in response to stimulation with ifn-λ1 (data not shown), which signals through the jak/stat pathway leading to isre activation. as expected, the elevation in cytokine and chemokine levels induced by ifn stimulation was decreased by β-sitosterol treatment (fig. 4e, f) . to understand whether the attenuation of the amplified proinflammatory response in ifn-β pretreated cells was solely due to a reduction in ifn-β levels, cells with ifn-β prestimulated for 4 h were treated with an ifn-β neutralizing antibody (2.5 μg/ml) prior to virus infection. however, we observed that the amplification effects of proinflammatory mediators (il-6 and ip-10) in cells prestimulated with ifn-β were not abrogated by ifn-β neutralizing antibody treatment (fig. 4g) . this indicates that cells prestimulated with ifn-β become sensitized and thereby amplify proinflammatory responses. to understand whether the observed inhibitory effect of βsitosterol was due to stat1 inhibition, we measured the phosphorylation of stat1 tyr701 in a549 cells stimulated with ifn-β (500 ng/ml). stat1 was significantly activated in cells pretreated with ifn-β (lane 4), but not in cells infected with iav prior to ifn-β stimulation (lane 8). treatment with β-sitosterol abrogated stat1 activation in a dose-dependent manner (lanes 5-7) (fig. 4h) , indicating that the inhibitory effect of β-sitosterol on stat1 reduced the ifn-mediated amplification of cytokine and chemokine expression. notably, pretreatment with ifn-β significantly increased the iav-triggered expression of rig-i in a549 cells, which was inhibited by β-sitosterol treatment (fig. 4i, j) . together, these data demonstrate that β-sitosterol blocks the iav-induced amplification of the proinflammatory response in ifn-β-activated a549 cells, which is due to inhibition of rig-i levels by β-sitosterol, leading to the inactivation of stat1, and thereby diminishes the transcriptional activity of interferon-stimulated gene factor 3 (isgf3). in addition to playing a role in ifn induction, rig-i signaling has been demonstrated to be involved in apoptosis [57] , which is implicated in iav-induced lung epithelial cell damage and injury. therefore, we investigated whether β-sitosterol affects rig-imediated apoptosis using intracellular viral rna (vrna, 5′ppp-rna) stimulation. stimulation with cellular rna (crna) or vrna and treatment with calfintestine alkaline phosphatase (ciap) to fig. 3 effect of β-sitosterol treatment on iav-induced ifn production and ifn signal transduction. a after 24 h, iav-infected cells with or without the indicated concentration of β-sitosterol treatment were harvested, and the supernatants were transferred to uninfected cells. after 15 min of incubation, the cells were lysed, and the expression of phosphorylated stat1 and stat2 was analyzed by immunoblotting. equal loading of protein was verified by immunoblotting for gapdh. b ifns (ifn-β, ifn-γ, and ifn-λ1) secretion into the culture media was measured at 24 h p.i. using a multiplex luminex assay. c, d after allowing 2 h for iav absorption, a549 cells were incubated with the indicated concentration of β-sitosterol for 4 h (c) or 8 h (d). then, the cells were stimulated for an additional 15 min with human ifn-β (20 ng/ml). the cells were lysed, and total extracts were processed for immunoblotting. e, f effect of β-sitosterol on the expression of rig-i. rig-i mrna expression levels were assayed by quantitative real-time pcr (e). rig-i protein levels were assessed by western blotting (f). g a549 cells were transfected with a flag-rig-i overexpression plasmid, and then treated with β-sitosterol. after 24 h, the cell lysates were collected for immunoblotting. h a549 cells were pretreated with β-sitosterol for 12 h, stimulated with ifn-β (20 ng/ml) for 15 min, and immunoblotted for the indicated proteins. *p < 0.05, **p < 0.01, ***p < 0.001 versus the iav control group. dephosphorylate 5′-triphosphate did not induce apoptosis, excluding the possibility that crna or the non-phosphate at the 5′ end of rna has a pro-apoptotic effect (fig. 5a) . strikingly, the apoptosis of vrna-transfected cells was reduced by β-sitosterol treatment (fig. 5a) . we confirmed the anti-apoptotic effect of βsitosterol by measuring the active caspase-3 and its substrate parp, observing that these products were found in cells transfected with vrna but not in those treated with β-sitosterol fig. 4 (continued) β-sitosterol ameliorates iav-induced inflammation and ali bx zhou (fig. 5b) . to determine whether the inhibitory effect of β-sitosterol on rig-i-mediated apoptosis is related to alterations in the expression of pro-apoptotic factors, we quantified trail and sfas ligand levels in the supernatants of viral rna-transfected cells. trail and sfas ligand levels were significantly reduced by βsitosterol treatment (fig. 5c ). next, we determined the effect of βsitosterol on the apoptosis of iav-infected cell. β-sitosterol treatment reduced iav-mediated apoptosis and active caspase-3 and parp (fig. 5d, e) . last, we observed that β-sitosterol treatment blocked the increase in the release of trail and sfas ligand in iavinfected cells (fig. 5f) . furthermore, rig-i knockdown by specific sirnas was demonstrated to significantly decrease iav-mediated apoptosis and the release of trail (fig. 5g, h) . however, the combination of rig-i sirnas and β-sitosterol did not have an additive effect on the inhibition of iav-mediated apoptosis and trail release, which indicated that β-sitosterol decreased iavmediated apoptosis via the inhibition of rig-i. given that vrna (5′ ppp-rna) recognition is associated with the rapid induction of ifns, we wondered whether β-sitosterol treatment affects ifn induction in the context of vrna transfection. as shown in fig. 5i , the upregulated expression of both type i ifn (ifn-β) and type iii ifn (ifn-λ1) in response to vrna was reversed by β-sitosterol treatment in a dose-dependent manner. to further explore whether the signaling cascade underlying rig-i-mediated apoptosis and ifn induction is affected by βsitosterol, we performed immunoblot analysis 24 h after the fig. 4 effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory mediators. a the effect of β-sitosterol on isre luciferase reporter activity in iav-infected cells. a549 cells were co-transfected with 0.5 μg of pisre-ta-luc reporter plasmid and 0.05 μg of prl-tk plasmid as described in the materials and methods. at 6 h posttransfection, the cells were infected with iav and treated with βsitosterol. after 24 h, the cells were lysed, and luciferase activity was measured. ## p < 0.01 versus the control group. *p < 0.05, **p < 0.01 versus the iav control group. b rig-i knockdown by specific sirnas in iav-infected cells was confirmed by immunoblotting. c the effect of rig-i knockdown by specific sirnas on isre luciferase reporter activity in iav-infected cells. ### p < 0.001 versus the control group. ***p < 0.001 versus the iav control group. d the effect of β-sitosterol on isre luciferase reporter activity induced by stimulation with a combination of ifn-β and iav. after 6 h of transfection, a549 cells were pretreated with ifn-β (500 ng/ml) (columns 4-7) for 4 h or infected with iav prior to ifnβ stimulation (columns 8-11) for 4 h. ifn-β-pretreated cells were infected with iav in the presence or absence of β-sitosterol (150-450 μg/ml). iav-infected cells were stimulated with ifn-β (500 ng/ml) in the presence or absence of β-sitosterol (150-450 μg/ml). the cells were harvested and subjected to a luciferase assay at 24 h p.i. # p < 0.05 versus the iav control group (column 3). *p < 0.05 versus the ifn-β-pretreated group (column 4). § p < 0.05 versus the group infected with iav before being stimulated with ifn-β (500 ng/ml) stimulation group (column 8). e the effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory cytokines and chemokines at the mrna levels was determined by real-time pcr. ### p < 0.001 versus the iav control group (column 2). *p < 0.05, **p < 0.01, ***p < 0.001 versus ifn-β-pretreated group (column 3). § p < 0.05, § § p < 0.01 versus the group infected with iav before being stimulated with ifn-β (500 ng/ml) (column 7). f the effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory cytokines and chemokines at the protein level was determined by a multiplex luminex assay. # p < 0.05, ## p < 0.01 versus the iav control group (column 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus ifn-β-pretreated group (column 4). § p < 0.05, § § p < 0.01, § § § p < 0.001 versus the group infected with iav before being stimulated with ifn-β (500 ng/ml) (column 8). g the effect of ifn-β neutralization on the ifn-β-mediated amplification of the iav-induced proinflammatory response. a549 cells prestimulated with ifn-β (500 ng/ml) for 4 h were treated with an ifn-β neutralizing antibody (2.5 μg/ml) prior to infection with iav. after 24 h, the culture supernatants were collected to measure proinflammatory mediator levels by a multiplex luminex assay. ***p < 0.001 versus iav control group. ns, not significant. h the effect of β-sitosterol on the ifn-β-mediated activation of stat1. lanes 1-3: a549 cells were treated with either ifn-β (500 ng/ml) or with iav for 24 h. lanes 4-7: a549 cells were pretreated with ifn-β (500 ng/ml) for 4 h prior to iav infection. lanes 8-11: a549 cells were infected with iav for 4 h and then stimulated with 500 ng/ml ifn-β. after the indicated treatments, the cells were incubated with or without β-sitosterol for 24 h. the cell lysates were analyzed by immunoblotting for the expression of phospho-stat1 and phospho-stat2. i, j the effect of β-sitosterol on the expression of rig-i. a549 cells were pretreated with ifnβ (500 ng/ml) for 4 h and infected with iav in the presence or absence of β-sitosterol (150-450 μg/ml) (columns 3-6, lanes 4-7) . meanwhile, a549 cells were infected with iav for 4 h prior to ifn-β (500 ng/ml) stimulation (columns 7-10, lanes [8] [9] [10] [11] . i the expression of rig-i was determined by quantitative real-time pcr. ## p < 0.01 versus the iav control group (column 2). *p < 0.05, **p < 0.01, ***p < 0.001 versus ifn-βpretreated group (column 3). j the expression of rig-i was determined by immunoblotting at 24 h p.i. the band intensities of rig-i were semiquantified using imagej (normalized to the loading control gapdh). *p < 0.05 versus ifn-β-pretreated group (column 4). § p < 0.05 versus the group infected with iav before being stimulated with ifn-β (500 ng/ml) (column 8). (fig. 5j) (lane 4) . notably, the dephosphorylation of the 5ʹtriphosphate of rna led to the inactivation of p65 nf-κb and p38 mapk but not stat1 (lane 5). moreover, the vrna-triggered activation of p65 nf-κb, p38 mapk, and stat1 was inhibited by βsitosterol (lanes 6-8). the stimulation of rig-i with vrna initiated signaling events that ultimately led to the expression of proinflammatory cytokines. we detected increased expression of proinflammatory mediators after 24 h of vrna stimulation, and observed that β-sitosterol treatment blocked this effect (fig. 5k) . meanwhile, these proinflammatory mediators induced by iav were effectively reduced by specific rig-i sirnas (fig. 5l) . the levels of il-8, mcp-1, rantes, and gm-csf were further reduced by the combination of rig-i sirnas and β-sitosterol (fig. 5l) . furthermore, the further increased levels of il-6, tnf-α, and ip-10 in flag-rig-i overexpression plasmid-transfected cells with iav infection is dose-dependently decreased by β-sitosterol treatment (fig. 5m) . interestingly, the viral rna-induced upregulation of cox-2 expression was decreased following β-sitosterol treatment (fig. 5n) . to further confirm that β-sitosterol suppresses viral rna-mediated cox-2 upregulation, we quantified the level of its downstream product pge2 in the culture medium using elisa. our results showed that treatment with β-sitosterol dosedependently suppressed the viral rna-induced production of pge2 (fig. 5o) . collectively, these data suggest that β-sitosterol inhibits the activation of rig-i signaling and downstream apoptosis in response to vrna. considering that β-sitosterol exhibited immunomodulatory properties in vitro, we next sought to investigate whether β-sitosterol has a protective effect in a mouse model of influenza. balb/c mice were pretreated with β-sitosterol for 2 days prior to intranasal challenge with 5 ld 50 of iav. at 5 days p.i., histological analysis of lung sections showed that infection with iav resulted in extensive inflammation characterized by massive leukocyte recruitment to the lung parenchyma (fig. 6a, upper panel) . pre-treatment with 200 mg·kg −1 ·d −1 β-sitosterol decreased leukocyte infiltration, and as a result, relatively few inflammatory cells were observed surrounding the bronchioles (fig. 6a, upper panel) . we next performed immunohistochemical staining for cd3 (pan t lymphocytes) and observed that the infiltration of cd3 + t lymphocytes was more pronounced in the lungs of mice with iav analysis of active caspase-3 and parp cleavage in a549 cells transfected with vrna at 24 h. c luminex assay for trail and sfas ligand levels in the supernatants of a549 cells transfected with vrna. *p < 0.05, **p < 0.01 versus the vrna transfection group (column 4). d flow cytometry analysis of the effect of β-sitosterol on apoptotic a549 cells infected with iav at 24 h. *p < 0.05, **p < 0.01 versus iav control group. e analysis of active caspase-3 and parp cleavage in a549 cells infected with iav at 24 h. f luminex assay for trail and sfas ligand levels in the supernatants of a549 cells infected with iav at 24 h. *p < 0.05 versus the iav control group. g flow cytometry analysis of the effect of rig-i knockdown by specific sirnas on iav-induced apoptosis. h luminex assay for trail in the supernatants of a549 cells with rig-i knockdown. ***p < 0.001 versus iav control group (column 2). i luminex assay for ifn-β and ifn-λ1 levels in the supernatants of a549 cells transfected with vrna at 24 h. *p < 0.05, **p < 0.01, ***p < 0.001 versus the vrna-transfected group (column 4). j a549 cells were transfected with vrna in the presence or absence of the indicated concentration of β-sitosterol; 24 h later, the cells were lysed and analyzed by immunoblotting with the indicated antibodies. gapdh was used as a control for equal loading. k the effect of β-sitosterol on the protein expression of cytokines and chemokines in the supernatants of vrna-transfected cells. *p < 0.05, **p < 0.01, ***p < 0.001 versus the vrna transfection group (column 4). l luminex assay for proinflammatory mediators in the supernatants of a549 cells with knockdown of rig-i or in combination with β-sitosterol treatment. ***p < 0.001 versus the iav control group (column 2). # p < 0.05, ### p < 0.001 versus the rig-i #1 sirna transfection group (column 3). § p < 0.05, § § p < 0.01, § § § p < 0.001 versus the rig-i #2 sirna transfection group (column 7). m luminex assay for cytokines and chemokines in the culture supernatants of iav-infected a549 cells transfected with flag-rig-i overexpression plasmid with or without β-sitosterol treatment. ## p < 0.01 versus the iav control group (column 2). *p < 0.05, ***p < 0.001 versus the flag-rig-i overexpression plasmid transfection control group (column 3). n the effect of β-sitosterol on the expression of cox-2 in vrna-transfected cells. the cells were transfected with vrna in the presence or absence of the indicated concentration of β-sitosterol; 24 h later, the cells were lysed and analyzed by immunoblotting using a specific antibody to cox-2. o quantification of pge2 in the culture supernatants of vrna-transfected cells in the presence or absence of the indicated concentrations of β-sitosterol at 24 h. ***p < 0.001 versus the vrna-transfected group (column 4). β-sitosterol ameliorates iav-induced inflammation and ali bx zhou infection alone compared with those that received β-sitosterol treatment (fig. 6a, lower panel) . consistent with the immunohistochemical results, a significantly greater percentage of cd3 + cd8 + cytotoxic t lymphocytes (ctls) was detected in balf from iav-infected mice compared with balf from mice treated with β-sitosterol (fig. 6b) . moreover, the quantification of granzyme b, a pro-apoptotic enzyme secreted by ctl, in lung homogenates revealed a significant increase in its expression in iav-infected mice that was significantly reduced by β-sitosterol administration (fig. 6c) . similar results were obtained for the levels β-sitosterol ameliorates iav-induced inflammation and ali bx zhou of active caspase-3 measured by immunoblotting (fig. 6c) . although influenza antigen-specific cd8 + t cells are recruited to the sites of infection and contribute to viral clearance, immunemediated lung injury can be elicited by aberrant t-cell responses. lung index and total protein levels in balf can be used as an assessment of lung damage. our results showed that compared with iav infection alone, β-sitosterol treatment produced a significantly lower lung index and protein levels in balf (fig. 6d , e), suggesting that β-sitosterol treatment alleviated lung injury likely through the suppression of cd8 + t-cell recruitment. last, iav infection resulted in 100% (13/13) mortality by 12 days p.i. and rapid and continuous weight loss (fig. 6f, g) . remarkably, the survival rate of mice that were treated with 50 and 200 mg/kg βsitosterol was significantly increased to 61.5% (8/13) and 84.6% (11/13), respectively. furthermore, β-sitosterol-treated mice exhibited less initial weight loss after viral challenge and a gradual recovery of body weight (fig. 6g) . together, these data reveal that β-sitosterol attenuates iav-induced lung injury and reduces mortality. β-sitosterol protects against iav by abrogating the iav-mediated activation of multiple signaling cascades to investigate the mechanisms underlying the protective effect of β-sitosterol against iav in vivo, we focused on inflammationassociated signal transduction in the lung. the phosphorylation levels of stat1, stat3, p38, and erk1/2 in lung homogenates were significantly increased in mice challenged with iav relative to uninfected mice (figs. 7a), whereas the phosphorylation levels of these molecules were decreased in mice treated with β-sitosterol. to address whether β-sitosterol inhibits the signaling events mediating the iav-induced expression of proinflammatory cytokines, we quantified cytokine and chemokine levels using luminex. the expression of cytokines and chemokines in balf (il-6, tnf-α, rantes, kc, mcp-1 and mip-1α) and lung homogenates (il-6, tnf-α, ip-10, and rantes) was reduced in mice treated with β-sitosterol (fig. 7b, c) . the iav-induced elevation of serum cytokines including ifn-γ, ip-10, and rantes, was blocked in β-sitosterol-treated mice (fig. 7d) . given that β-sitosterol inhibited iav-mediated stat1/3 activation in vivo and decreased the expression of rig-i and ifn in vitro (fig. 3b, f) , it was necessary to examine the impact of β-sitosterol treatment on the expression of rig-i and ifns in vivo. as expected, iav-induced expression of rig-i in lung homogenates was decreased by β-sitosterol treatment (fig. 7e) . similarly, the expression of ifns in balf (ifn-α, ifn-β, and ifn-γ) (fig. 7f) and lung homogenates (ifn-β) (fig. 7g) was also decreased. collectively, these data provide evidence regarding the mechanism by which β-sitosterol modulates dysregulated signaling cascades and proinflammatory responses linked to severe influenza. patients with influenza are frequently afflicted with severe pneumonia characterized by excessive infiltration of leukocytes and proinflammatory cytokine production [58] [59] [60] , leading to a high risk of death. recent studies have revealed that iav-mediated ifns play a disease-promoting role in the pathogenesis of influenza [31] . chinese herbal medicines, including l. japonica [35] , c. morifolium [36] , t. mongolicum [37] , f. suspense [38] , and i. indigotica [39] , have a long history of being used for the treatment of the common cold and for heat-clearing. in the current study, we demonstrated that β-sitosterol derived from these herbal medicines has the ability to block iav-mediated ifn and proinflammatory mediator production through the inhibition of rig-i signaling. furthermore, our data showed that β-sitosterol attenuates the amplification of the iav-mediated proinflammatory response in ifn-sensitized cells by disrupting rig-i-mediated stat1 activation. furthermore, we showed that β-sitosterol abrogates the recruitment of cytotoxic t lymphocytes (ctls) in the lung, thereby significantly improving lung injury and survival in mice challenged with iav (fig. 8) . rig-i detects viral rna (5′ppp-rna) in the cytoplasm, which is then ubiquitinated by trim25 (tripartite motif-containing protein 25) and subsequently interacts with ips-1 (ifn-β promotor stimulator 1) to initiate the activation of nf-κb, p38, and irf3/7 [61, 62] . we asked whether rig-i signaling is affected by βsitosterol during iav infection or in cells transfected with viral rna. we found that rig-i expression was increased following iav infection or prestimulation with ifn-β prior to iav infection, and that this increase in expression was significantly reduced in the presence of β-sitosterol. furthermore, the activation of nf-κb and p38 in both iav-infected and viral rna-transfected cells was inhibited by β-sitosterol treatment. these results suggest that βsitosterol treatment antagonizes the rig-i signaling cascades. the activation of rig-i and its downstream targets nf-κb and p38 contributes to the induction of proinflammatory cytokines in response to influenza virus infection [63] . in previous studies, the upregulation of rig-i during fatal h5n1 infection was shown to cause an amplification of inflammatory responses [18] . the hyperinduction of proinflammatory cytokines via rig-i, nf-κb, and p38 signaling has been suggested to contribute to severity of symptoms in patients with h5n1 infection [64, 65] . we measured nf-κb activation using an nf-κb luciferase reporter system and found that β-sitosterol treatment downregulated the transcriptional activation of nf-κb following the administration of tnf-α and iav infection. consistent with these findings, the inhibitory effects of β-sitosterol on rig-i signaling led to reduced production of cytokines, such as il-6 and tnf-α, and chemokines such as il-8 and ip-10 in iav-infected and viral rna-transfected cells. rig-i or nf-κb and p38 activation in response to viral or bacterial infection, respectively, has been reported to induce cox-2 expression [46, 66] . furthermore, it has been shown that decreased expression of cox-2 and its derivative pge2 has beneficial effects during influenza virus infection that lead to reduced hypothermia and enhanced type i ifn antiviral immunity [51] . we observed that iav infection and viral rna stimulation were associated with increased expression of cox-2 and pge2 and that β-sitosterol treatment reversed the increase in a dosedependent manner. fig. 6 β-sitosterol prevents iav-induced lung pathology in mice. two days prior to infection with 5 ld 50 of a/fm1/h1n1 virus, pbs or β-sitosterol (50 mg·kg −1 ·d −1 or 200 mg·kg −1 ·d −1 ) was intragastrically administered to mice for 7 consecutive days. a on day 5 p.i., the lungs were harvested and subsequently subjected to histological analysis by h&e staining (original magnification, ×100) or cd3 antigen (t-cell marker) staining (original magnification, ×200). b representative flow cytometry quantification of cd3 + cd8 + t cells in balf on day 4 p.i. the histograms represent the percentage of cd3 + cd8 + t cells in balf (right panel). the data are representative of three independent experiments using 5-7 mice per group. *p < 0.05, **p < 0.01 versus the iav-infected group. c on day 5 p.i., the lungs were removed and homogenized. lung homogenates were subjected to immunoblot analysis of granzyme b and active caspase-3. the ratios of the relative band intensities of granzyme b and active caspase-3 normalized to gapdh are shown (n = 3-5 mice per group) (right panel). *p < 0.05, ***p < 0.001 versus the iav-infected mice group. d the lung index (lung/body weight ratios) of mice treated with pbs (n = 6) or β-sitosterol (n = 6) on day 5 p.i. *p < 0.05, **p < 0.01 versus the iav-infected group. e on day 7 p.i., the total protein concentrations in balf was measured by bca assay (n = 3-11 mice per group). *p < 0.05, **p < 0.01 versus the iav-infected group. f, g survival rate (f) and weight curves (g) of iav-infected mice treated with or without β-sitosterol (n = 13 mice per group). β-sitosterol ameliorates iav-induced inflammation and ali bx zhou nf-κb, atf2 (a downstream target of p38), and irf3 form a transcriptional complex that drives the expression of the antiviral factor ifn-β [10] . in addition, the viral-induced expression of type iii ifn requires the involvement of rig-i, ips-1, tbk1, and p38 signaling [67] [68] [69] , suggesting that the expression of type i and iii ifns is promoted via a common mechanism. interestingly, some studies have indicated that nf-κb is crucial for ifn-β production when irf3 activation is weak but not when irf3 activation is strong [70] . although we did not detect significant activation of irf3 at 24 h, we were able to detect an inhibitory effect of β-sitosterol on the expression of ifns, including ifn-β and ifn-λ1, in cells infected with iav or in those subjected to viral rna transfection. these findings may be attributable to the inactivation of rig-i, nf-κb, and p38 signaling. a clear link between rig-i expression and stat1 activation has been established by previous studies. experiments involving rig-i overexpression or knockdown have suggested that rig-i is essential for stat1 activation in leukemia cell lines [54, 71] . in accordance with these findings, our results show that the augmentation of stat1 activation by rig-i overexpression was suppressed by β-sitosterol or the inhibition of iav-mediated isre transcriptional activity by specific rig-i sirnas. in addition, we observed that the activation of jaks was not affected by β-sitosterol. therefore, the inhibition of stat1 phosphorylation in response to ifn-β treatment for 15 min or 24 h can be attributed to the downregulation of rig-i by βsitosterol. moreover, the activation of rig-i, but not of mda-5, has been shown to involve double-stranded rna (dsrna)-induced stat1 phosphorylation [72] . our data showed that β-sitosterol treatment abrogated stat1 phosphorylation in cells stimulated with vrna (5′ppp-rna), which binds to and activates rig-i. however, the dephosphorylation of viral rna with ciap did not reduce stat1 phosphorylation. a possible explanation for these findings is that the dsrna that is generated following viral rna dephosphorylation is also a ligand for rig-i and induces stat1 phosphorylation in an ifn-dependent or ifn-independent manner, as described previously [72] . the important role of ifns in the defense against viral infection is widely recognized [73, 74] . however, ifn receptor deficiency does not lead to a detrimental outcome, which is perhaps due to decreased ifn-induced immune injury [31, 33, 34] . recent studies have clearly revealed the pathogenic potential of ifn-β-and ifn-λ1-mediated immunopathology in viral infectious diseases and autoimmune diseases [31, 33, 34, 75] . here, we have proposed a model in which ifns (including type i and iii ifn) secreted from iav-infected cells bind to their receptors and sensitize uninfected neighboring cells, leading to the amplification of proinflammatory responses driven by isgf3 following infection by progeny viruses. β-sitosterol treatment blocked the amplification of this proinflammatory response and the concomitant expression of proinflammatory cytokines through the inhibition of isgf3 complexes. the inhibitory effect on isgf3 complexes was due to the failure of downregulated rig-i to exert a converse effect on stat1 activation in β-sitosterol-treated cells (fig. 8) . the activation of rig-i-mediated apoptosis via type i ifn-dependent and type i ifnindependent mechanisms has been considered a promising strategy for cancer therapeutics [57, 76] . the ifn-induced activation of isgf3 leads to trail expression, resulting in substantial alveolar epithelial cell (aec) apoptosis and lung injury [32, 77] . aec apoptosis has been found to play a critical role in the pathogenesis of h5n1 and pandemic h1n1 in patients with ards [59, 78] . our data suggest that β-sitosterol prevents iav-induced apoptosis associated with decreased ifn-driven expression of trail. the loss of rig-i signaling has been correlated with a reduction in antigen presentation in bone marrow derived dendritic cells (bmdcs), and in the antiviral function of cd8 + cytotoxic t cells [79] . thus, it is clear that the rig-i pathway is important for mediating the production of ifns during antiviral responses. in contrast, the rig-i-mediated expression of inflammatory mediators has been shown to induce the recruitment of monocyte-derived dcs (modcs) to support viral replication [80] . antiviral effector cd8 + t cells and nk cells eliminate invading pathogens through fig. 7 β-sitosterol effectively abrogates iav-triggered signaling in vivo. a mice were infected with 5 ld 50 of a/fm1/h1n1 virus and treated with pbs or β-sitosterol (intragastrically administered for 7 consecutive days beginning 2 days prior to viral infection). the lungs were harvested and homogenized on day 5 p.i. and the processed for immunoblotting with the indicated antibodies. the relative band intensities of the indicated proteins were normalized to that of gapdh (n = 3-5 mice per group) (right panel). *p < 0.05, **p < 0.01 versus the iav-infected group. b-d detection of cytokine and chemokine production in balf (b), lung homogenates (c), and serum (d) by luminex analysis. *p < 0.05, **p < 0.01 versus iav-infected group. e on day 5 p.i., lung homogenates were subjected to immunoblot analysis of rig-i. the rig-i band intensity normalized to that of gapdh is shown (n = 4-6 mice per group) (right panel). *p < 0.05 versus iav-infected group. f, g detection of ifns (ifn-α, ifn-β, and ifn-γ) production in balf (f) and lung homogenates (g) by luminex analysis. *p < 0.05, **p < 0.01 versus the iavinfected group. β-sitosterol ameliorates iav-induced inflammation and ali bx zhou several mechanisms, including the expression of pro-apoptotic proteins such as trail and fas and the secretion of granzyme b (grb) and perforin. through these mechanisms, the apoptotic caspase cascade is activated in virus-infected cells [81] [82] [83] . studies have reported that ifn-γ plays an important role in modulating cd8 + t-cell recruitment and that the production of granzyme b during the recruitment of cd8 + t cells is dependent on the ifn-βinduced activation of stat1 [81, 84, 85] . accordingly, our data show that β-sitosterol administration decreases the levels of ifn-γ and ifn-β and concomitantly induces a low level of cd8 + t-cell recruitment and granzyme b secretion in the lungs. cytotoxic cd8 + t lymphocytes (ctls) seem to be essential for viral clearance. however, severe pneumonia in patients with pandemic influenza a (h1n1) virus infection is apparently related to high levels of cd8 + t cells [86] . interestingly, one study showed that ha-transgenic mice develop lethal lung injury following treatment with influenza ha-specific cd8 + cytotoxic t cells [87] . deficiency of a20 (tnf alpha-induced protein 3, tnfaip3), which is a negative feedback ubiquitin-editing protein that inhibits nf-κb signaling, protects mice against viral challenge by reducing the population of grb + cd8 + t cells [88] . consistent with these studies, our data showed that iav-induced acute lung injury and mortality are attenuated in mice treated with β-sitosterol and that this attenuation is associated with decreased cd8 + t-cell recruitment and granzyme b secretion in the lung. furthermore, we observed the inhibition of stat1/3 and p38 phosphorylation by β-sitosterol in mouse lung tissue and made a similar observation in iavinfected a549 cells. the expression of cytokines driven by stat1/3 and p38, which exacerbate iav-induced immunopathology, in balf and lung tissue was decreased following β-sitosterol administration in balf and lung tissue. in contrast to our in vitro results, the phosphorylation of erk1/2 was attenuated in the lung tissues of β-sitosterol-treated mice. the inhibition of erk1/2 signaling is involved in the retention of viral rnp in the nucleus, but its activation also correlates with cytokine expression [89] . it is likely that the decrease in phosphorylated erk1/2 expression in vivo was a result of the immunoregulatory effects of β-sitosterol. fig. 8 schematic diagram showing the mechanism by which βsitosterol attenuates iav-induced proinflammatory responses and injury. invading viruses are sensed by rig-i, leading to the activation and that of rig-i, nf-κb, and p38, which initiates the expression of proinflammatory mediators and ifns. secreted ifns, including type i and iii ifns, bind to their receptors via an autocrine or paracrine mechanism and then exert their antiviral effects. subsequently, previously uninfected ifn-sensitized neighboring cells become infected by progeny viruses, 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nf-kappab and raf/mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo zfy and nsz conceived the study; bxz, zfy, and nsz designed the study; bxz and xll conducted the in vitro experiments; jl and xpp isolated and analyzed the compound β-sitosterol; bxz, xll, hmj, ybh, and pfx performed animal experiments; and bxz and jl wrote the paper. the online version of this article (https://doi.org/10.1038/s41401-020-0403-9) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord-344093-3bniy5b5 authors: peteranderl, christin; herold, susanne title: the impact of the interferon/tnf-related apoptosis-inducing ligand signaling axis on disease progression in respiratory viral infection and beyond date: 2017-03-22 journal: front immunol doi: 10.3389/fimmu.2017.00313 sha: doc_id: 344093 cord_uid: 3bniy5b5 interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in—but not limited to—respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in-but not limited to-respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/ trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. (73) cell death induction, e.g., bcl-2-associated x protein, caspase-8, fas-associated protein with death domain, fas ligand, and tnf-related apoptosis-inducing ligand (trail) dsrna, polyi:c (4, 110) iav (4, 5, 10, 115) sendai virus (110) trail virus control by apoptosis induction in infected cells iav (6, 170, 171) tissue injury by apoptosis of both infected and non-infected alveolar epithelial cells, lung macrophages iav (5, 7, 10) rsv (137) necrosis of fibroblasts, dendritic cells, and epithelial cells iav (146, 147, 168) increased cellular infiltration cov (175) decreased expression of na,k-atpase, impaired epithelial fluid reabsorption iav (11) introduction in 1957, isaacs and lindenmann (1) first recognized the potential of a soluble and probably cell-derived factor to combat influenza virus infection and named this factor interferon [(ifn) from latin interferre, to interfere]. since then, three subgroups of ifns have been defined, primarily by their differential receptor usage. while the groups of type i ifn and type iii ifn comprise largely agents directly limiting pathogen spread by improving cellular counter measurements, ifn-γ, the sole type ii ifn, has been mainly implicated in the modulation of innate and also adaptive immune responses (2, 3) . accordingly, type i and iii ifns are key signaling molecules in viral control, and lack of both signaling pathways results in increased viral loads and disease severity. still, there is accumulating evidence that not only lack of an antiviral response but that also an unbalanced overshooting activation of ifns contributes to an exaggerated inflammatory reaction, tissue injury, reduced proliferative capacity, and thus enhanced disease severity ( table 1) . especially in viral infections, this effect has not only been tracked down to ifn signaling in general but specifically to the exaggerated production of key effector ifn-stimulated genes (isgs) (4) . a prominent example is the tnf-related apoptosis-inducing ligand (trail) that displays an ambivalent role in viral infection (5-7) ( table 1) . whereas first identified as factor produced by immune cells in non-respiratory infection (8, 9) , trail is now especially well studied in influenza a virus (iav) infection, where it is released in high amounts from bone marrow-derived macrophages upon pathogenassociated molecular patterns (pamps) recognition and type i ifn production (10) . macrophage-released soluble trail, but also membrane-bound cell-associated trail, acts via distinct receptors on infected but also on non-infected, neighboring cells. in viral infection, its preliminary role is to drive infected cells into apoptosis to limit virus spread. however, studies performed within the last decade demonstrate that trail's antiviral activity seems to be outweighed by the functional and structural damage it induces not only in infected but also in bystander cells such as uninfected cells of the alveolar epithelium (10, 11) . this process is not only relevant in promoting viral disease progression but has further implications in bacterial superinfection and probably also in chronic diseases. the recognition of the ambivalent role of ifn-driven signaling in vivo is a first important step to better understand disease progression and to envision novel treatment options for primary viral respiratory infection targeting distinct host-derived signaling mediators such as trail. it is a commonly accepted concept that-as janeway (12) already proposed in 1989-immune activation toward invading pathogens is mounted upon recognition of pamps. pamps are evolutionary conserved biomolecules such as proteins, lipids, nitrogen bases, sugars, and complexed biomolecules such as lipoglycans that are essential to the survival of a given pathogen (13) . pamps are recognized by distinct pattern recognition receptors (prrs) that are germ-line encoded and-similar to pamps-usually show a high evolutionary conservation. the first recognized and probably most intensely studied family of prrs are the toll-like receptors [tlrs; reviewed in mogensen (14) ; leifer and medvedev (15) ]. in viral infection, both host cell membrane-localized tlrs (tlr2, tlr4, detecting viral envelope proteins) and endosomal tlrs (tlr3, tlr7, tlr8, and tlr9 , nucleic acid sensors) initiate signal transduction cascades leading to ifn production (figure 1 ). tlr activation results in either myeloid differentiation factor 88 (myd88) or tir-domaincontaining adaptor protein-inducing ifn-β (trif) recruitment that both trigger various downstream signaling events, eventually leading to ifn regulatory factor (irf)3, irf7, and nfκb nuclear translocation as well as map kinase and activator protein 1 (ap-1) activation (16, 17) . similar to endosomal tlrs, the cytosolic retinoic acidinducible gene (rig)-i-like receptors (rlrs) are specialized to recognize viral nucleic acid contents and are central prrs relevant to mount an antiviral response, providing resistance to most rna (e.g., orthomyxoviruses) and some dna (e.g., reoviruses) viruses [reviewed in ref. (18, 19) ]. both melanoma differentiation-associated gene 5 (mda-5) and rig-i recognize dsrna, 5′-triphosphate rna, or the synthetic analog to dsrna, polyi:c (20, 21) . both drive the dimerization of the mitochondriaassociated adaptor protein ifn-β promoter stimulation 1 (ips-1) (also named mavs, visa, cardif). a subsequently activated cascade including tradd (tnf receptor type 1-associated death domain protein), traf3 (tnf receptor-associated factor 3), and tank (traf family member-associated nf-κb activator) induces the phosphorylation of irf3 and irf7, resulting in type i ifn production (22) . the third rlr, lgp2, so far has primarily been implicated to regulate rig-i or mda-5 as a cofactor; however, a recent study by stone et al. (23) demonstrated a novel, non-redundant, and independent role of lgp2 in west nile virus infection. another class of prr, the nucleotide oligomerization domain (nod)-like receptors (nlrs), has mainly been implicated in bacterial recognition (24) , still several nlrs are activated as well upon virus infection. especially, nlrp3 is known to recognize rna of different viruses including hepatitis c virus, measles virus, influenza, and vesicular stomatitis virus (vsv) (25) (26) (27) (28) , resulting in inflammasome formation and caspase-1-dependent activation of il-1β and il-18 (29) (30) (31) . in addition, virus infections are sensed also by a structurally diverse group of viral rna and dna sensors residing in the cytoplasm. these include the cyclic gmp-amp synthase that synthesizes the second messenger cgamp. cgamp in turn activates stimulator of ifn genes (sting), tank-binding kinase 1, and irf3, triggering ifn production (32) (33) (34) . moreover, sting itself acts as a prr and has been implicated in dna virus recognition including hsv, adenovirus, vaccinia virus, and papilloma virus and in sensing of retroviral rna-dna hybrids (35) and rna viruses after being activated by rig-i (36, 37) . another cytosolic nucleic acid sensor, pkr, is well known for its phosphorylation of eukaryotic initiation factor 2 α (eif2α) in response to viral dsrna. phosphorylation of eif2α results in its deactivation, host translational shut-off, and the limitation of viral replication. of note, the pkr-eif2α-driven inhibition of protein synthesis can contribute to an ips-1-dependent ifn-β induction (38) . furthermore, pkr has been implicated in efficient type i ifn activation by tlr3 in response to dsrna (39) and can mediate-at least partially-activities of irf1 (40) . in addition to type i ifns, also type iii ifns exert antiviral activity and are widely expressed after viral recognition, being produced by most cell types including epithelial, endothelial, fibroblast, and polymorphonuclear cells [reviewed in ref. (41, 42) ]. like type i ifns, type iii ifns are induced in viral infection by the prr rig-i as well as tlr3 and tlr9 and rely on the activation of the same transcriptional activators, including irf3, irf7, and nfκb. these observations initially led to the conclusion that type i and type iii ifn comprised two completely redundant systems to induce isgs in response to pamp recognition. however, more recent data suggest distinct selection mechanisms for either type i or type iii ifn expression. as such, ips-1 specifically induces ifn-λ, but not type i ifn, when located at the peroxisomal membrane instead of the mitochondrial membrane in response to rig-i activation by reovirus, sendai virus, or dengue virus challenge (43) . interestingly, type iii ifn induction is largely independent toward ap-1 translocation, which facilitates an instantaneous induction of ifn-λ after viral recognition, highlighting it as an important immediate factor driving innate microbial defense mechanisms. release of ifns upon pathogen recognition is a highly conserved mechanism-found from teleost fish to insects and mammals-to prepare the surrounding cells as well as the host defense against the invading threat (44, 45). whereas often high-level ifn production relies on specialized sentinel cells such as macrophages or dendritic cells (dcs), mostly all cells of the multicellular organisms are able to respond to at least one type of ifn by expression of respective receptors. receptor binding then induces a signal transduction cascade relying on the janus kinase (jak) and signal transducer and activator of transcription (stat), which results in efficient transcription of a plethora of different isgs in infected as well as bystander cells (46, 47) . ifns engage a classical canonical signal transduction cascade employing jak/stat molecules after binding to their respective receptors. herein, type i ifns ligate to their common heterodimeric receptor consisting of the ifn-α receptor (ifnar)1 and ifnar2 subunits, whereas type iii ifns act via a interleukin-10 receptor 2 (il-10r2)/ifn-λ receptor 1 (ifnlr1) heterodimer that to date has been reported to be restricted in its expression to epithelial cells (48) . in type i ifn signaling, ifnar engagement leads to the activation of the receptor-associated protein tyrosine kinases jak1 and tyrosine kinase 2 followed by the recruitment or repositioning of already associated but elsewise latent cytoplasmatic transcription factors stat1 and stat2. consequently, stat1/stat2 are phosphorylated on conserved tyrosine residues, they disassemble, undergo conformational changes enabling their heterodimerization as well as the exposure of a nuclear localization sequence. subsequently, the stat1/stat2 heterodimer translocates into the nucleus where it interacts with the irf9 to form the trimeric ifn-stimulated factor 3 (isgf3). isgf3 binds cognate dna sequences, the ifn-stimulated response elements (isre), finally leading to isg induction. also type iii ifn interaction with the il-10r2/ifnlr1 receptor complex triggers stat1/stat2 heterodimerization, nuclear translocation, and isgf3 assembly (49) . interferon signaling results in the induction of isgs evoking different cellular responses against viral infection, both in infected as well as in non-infected cells, including direct antiviral, immune-modulatory, or cell death-inducing effects to enable an immediate and robust response to a pathogen challenge. many isgs directly interfere with viral replication on an intracellular level. well-studied examples of antiviral isgs comprise ifn-induced transmembrane proteins (ifitms) effective in iav, west nile virus, and dengue virus infection (50, 51) , the myxovirus resistance protein a (mxa) that interferes with vsv mrna production and binds the iav nucleocapsid to prevent nuclear translocation of viral genetic material (52) (53) (54) , the 2-5-oligoadenylate synthase (oas), which activates rnase l triggering viral rna degradation, or the prr pkr, which besides activating the ifn response has a major impact on viral protein translation by inhibiting the eif2α (55) . more recently identified isgs include the plasminogen activator inhibitor 1 that blocks iav infection by inhibiting glycoprotein cleavage executed by extracellular airway proteases (56) or the antiviral isg20 that limits iav viral replication via its exonuclease activity most likely by interfering with the viral np (57) . accordingly, ifn pretreatment usually results in the establishment of an antiviral state that limits viral replication and spread from the start of infection and thus favors milder disease outcomes. ifn-α pretreatment has been demonstrated to limit viral spreading of seasonal iav strains and thus decrease morbidity and mortality in mice, guinea pigs as well as ferrets (58) (59) (60) . as shown by a study by tumpey et al. (61) , this effect can be attributed to the early induction of antiviral isgs including mxa. importantly, type i ifn pretreatment also dampens early replication of highly pathogenic avian influenza in ferrets (58) . also in respiratory syncytial virus (rsv) infection, treatment with recombinant ifn-α results in significantly decreased lung viral titers, alveolar inflammatory cell accumulation, and clinical disease in rsv-infected mice (62) . in addition, respiratory infections caused by emerging coronaviruses (cov) can be ameliorated by type i ifn pretreatment strategies. in an in vivo macaque model (macaca fascicularis) of severe acute respiratory syndrome (sars)-cov infection, it could be demonstrated that pretreatment with pegylated ifn-α significantly diminished cov replication and excretion and resulted in reduced pulmonary damage (63) . macaques also serve as a preclinical model for middle east respiratory syndrome (mers)-cov, and similar to sars-cov, ifn-α in combination therapy with ribavirin reduces viral replication and severe histopathological changes (64) . in line, genetic alteration leading to an enhanced type i ifn signaling has been demonstrated to limit iav-induced disease outcomes, as a recent study by xing et al. (65) reported that deletion of trim29, a negative regulator of nemo, which leads to nfκb induction and therefore enhanced type i ifn production, is protective in vivo in iav-infected mice. conversely, the genetic depletion of ifn signaling in ifn receptor-deficient mice can result in a lack of viral control, resulting in enhanced viral titers in different viral infections including rsv or iav (66, 67) . still, it must be noted that this effect is often mild in ifnar-or ifnlr-deficient animals, which is probably related to a certain redundancy between type i and type iii ifn signaling in limiting viral spreading in epithelial cells (68) . in contrast, ifnar/ ifnlr-double knockout or stat1 knockout animals that are deficient in both type i and type iii ifn signal transduction succumb more readily to infection due to excessive viral replication (69) (70) (71) . vice versa, mutations in key isgs such as ifitm3 are associated with increased iav disease severity in mice and humans (72) . however, ifn pretreatment and genetic loss-of-function approaches generally are not relevant to human respiratory virus-induced hospitalizations, where patients already present with ongoing respiratory infection and inflammation, and preclinical studies underline that type i ifn signaling in an already inflamed organ is rather detrimental and enhances tissue injury, and lack of type i ifn in vivo may even ameliorate disease outcome. accordingly, in cases where the antiviral defense was not compromised (e.g., in animals with efficient type iii ifn signaling) ifnar-deficient mice infected with sendai virus or iav were reported to be more resistant to infection-induced morbidity and mortality (73, 74) . similarly, in sendai virus in vivo infection, wetzel et al. (75) showed that increased ifn-β levels in the lung homogenate correlates to increased morbidity and mortality, and also for sars-cov, a recent study demonstrates that high type i ifn induction in an already ongoing viral infection contributes to mortality in sars-cov-infected mice (76) . also for iav infection, type i ifn application after infection has been proven to drive disease severity (74) . of note, the detrimental effects of type i ifns were especially pronounced in mice lacking central antiviral factors, namely the ifit protein in sendai virus infection and mxa in iav. interestingly, beilharz et al. (77) demonstrated that application of low doses of ifn-α reduces viral load, which to a certain degree led to attenuated disease progression, whereas high dose application of type i ifn contributed to morbidity (77) . in line, high expression levels of isgs have been shown to correlate to worse outcomes in ards patients (78) . this observation corresponds to reports stating that the ifn threshold needed to induce antiviral isgs-showing a beneficial effect in acute respiratory viral infection-is by at least 10-fold lower than the ifn dose necessary to trigger isgs that show immunomodulatory, death-inducing, or anti-proliferative effects and thus can contribute to disease progression (79) (80) (81) (82) . altogether, these data demonstrate that ifns may significantly contribute to unbalanced inflammation and tissue injury during respiratory viral infection depending on expression levels and duration of ifn-related signaling events. to date, the underlying mechanisms leading to the ifndependent enhanced disease progression are not fully understood but often result from a dysregulated ifn signaling response. one mode of action of ifn and ifn-stimulated isgs is to stimulate negative feedback loops on ifn signaling. for example, suppression of jak1 or stat1 via specific phosphatases, expression of suppressor of cytokine signaling (socs)1 and socs3, or ubiquitination and endocytosis of the ifn receptors (83) (84) (85) (86) desensitize cells to ifn signaling and allow recovery and the return to homeostasis after microbial challenge. as demonstrated by bhattacharya et al. (87) , the lack of ifnar downregulation and thus the failure to initiate ifn-desensitization contributes to increased inflammatory signaling, extensive lung injury and, importantly, also impaired tissue regeneration (87) . moreover, ifns are immunomodulatory and shape the specific responses of cells of the immune system, which has been implied to influence disease progression both positively and negatively. in a recent study, type i ifns have been associated in the regulation of innate lymphoid immune cells (ilc)2 in iav infection, where they-in concert with ifn-γ and il-27-promote an ilc2-dependent restriction of immunopathology (88) . moreover, type i ifns play an important role in stimulating the immune response driven by dcs; they stimulate the expression of mhc molecules as well as the co-stimulatory ligands cd80 and cd86 and thus activate t cell responses (89, 90) . additionally, ligand-driven activation of ifnar enhances the proliferation of cd8 positive t cells, especially early in infection. however, late in infection, type i ifns were also implied in decreasing t cell expansion upon sars-cov and arenavirus infection (76, 91) , which might potentially be related to the above described desensitization upon prolonged ifn signaling and might be detrimental if initiated too early in infection. in line, pinto et al. (92) reported an impairment of t cell responses upon type ifn induction in west nile virus infection. in b cells, the lack of ifnar has been demonstrated to result in enhanced release of neutralizing antibodies in iav infection (93) implying a repressive role for type i ifn in b cell antibody production. however, immunization studies by le bon et al. (94) reported the necessity of ifnar on b cells for efficient igm and igg production, underlining the need for further studies to understand the detailed effects of ifn-dosage and timing adaptive immunity activity upon respiratory viral infection. type i ifns additionally induce the production of high levels of pro-inflammatory cytokines that have been closely linked to worsened outcomes of acute respiratory viral infection. especially in iav, disease severity and disease progression are linked with an overshooting, ifn-driven inflammatory response, in which further exogenous supplementation with type i ifn in fact correlates with increased morbidity and mortality (74, 95) . in non-human primates, iav infection with a highly pathogenic h5n1 isolate evokes a strong induction of type i ifn, resulting in severe lung injury by a necrotizing bronchiolitis and alveolotis (96) . ifn levels in turn have been demonstrated to cause elevated pro-inflammatory cytokine levels after in vivo iav infection and additionally, in human alveolar macrophages, the release of pro-inflammatory cytokines (e.g., mcp-1) are preceded by a robust type i ifn response (97) . importantly, also in human infection with h5n1, levels of pro-inflammatory cytokines are strongly elevated in bronchoalveolar lavage fluid, and cytokine levels have been associated with organ damage and worsened disease outcomes (98, 99) . still, it should be noted that due to strain differences in virus-elicited prr activation and, importantly, ifn antagonism by the iav non-structural (ns)1 protein, ifn levels and disease severity do not always directly correlate; actually, the extent to which ns1 can suppress the ifn response relates to prolonged viremia and thus can also be a determinant of virus pathogenicity both in human bronchial epithelial cells and in an in vivo model of iav infection (100, 101) . alongside iav, also in rsv infection the induction of high levels of pro-inflammatory cytokines has been directly related to type ifn, as rsv-infected but ifnar-deficient mice presented with significantly diminished pro-inflammatory cytokine release, which translated into an attenuated disease course (67) . also in sars-cov, the late phase type i ifn induction relates to accumulation of inflammatory macrophage populations and elevated lung cytokine levels (76) . in addition to antiviral, immunomodulatory, and pro-inflammatory isgs, ifn signaling results in the transcription and translation of cell death-inducing isgs. in the context of viral infection, these factors provide a mode to block viral spreading and reinfection by killing those infected cells, in which the internal activation of antiviral isgs is not sufficient to restrict viral replication. thus, the infected cell is sacrificed to prevent the release of infectious progeny virions to limit viral spreading. however, especially in the lung, the disruption of the alveolar epithelial barrier by cell death of infected cells, but importantly also non-infected bystander cells induced by factors such as trail, significantly contributes to worsened disease outcomes. controlled cell death or apoptosis can be induced by intrinsic and extrinsic signals. the intrinsic apoptosis pathway is initiated by diverse intracellular stimuli that influence the expression and activation of b cell lymphoma (bcl)-2 family proteins that govern the permeabilization status of the outer mitochondrial membrane. once cytochrome c is released from the mitochondria, it binds to the intracellular adaptor protein, apoptotic peptidase activating factor 1, forming the so-called apoptosome that in turn recruits pro-caspase-9 (102). caspases (cysteine-aspartic proteases) exert their action by cleaving other proteins and substrates. herein, initiator caspases such as caspase-8 and caspase-9 target other downstream caspases, whereas effector caspases, including caspase-3, -6, and -7, directly cause apoptosis by cleaving and thus inactivating or disassembling a vast array of cellular integral proteins and complexes (103) . the extrinsic apoptosis pathway relies on an extracellular signal exerted by ligands of the tumor necrosis factor (tnf) receptor (tnfr) superfamily, including trail, tnf-α, and fas ligand (fasl) (104) . their ligation to their respective cell surface-expressed death receptors (dr) leads via the signal transmission by fas-associated protein with death domain (fadd) to the activation of the initiator caspases-8 or -10, finally stimulating effector caspases including caspase-3 (105) . to date, several type i and type iii ifn-induced, proapoptotic factors have been identified (106) . both caspase-4 and caspase-8 have been shown to be upregulated upon type i ifn signaling (4, 107); caspase-8 enhances the fadd-driven extrinsic apoptosis pathway, whereas the less-studied caspase-4 may promote pro-il-1β cleavage and inflammasome-driven cell death (pyroptosis) in macrophages (108, 109) . chattopadhyay et al. (110) demonstrated that sendai virus infection and polyi:c treatment resulted in bcl-2-associated x protein (bax) activation and apoptosis induction via one of the key transcription factors of ifn genes, irf3. in addition irf5 was reported to enhance trail-dependent extrinsic apoptosis by nuclear translocation resulting in the translation of to date undefined factors that increase cell death upstream of caspase-8 activation (111) . furthermore, both rlrs, rig-i and mda-5, trigger the proteins puma and noxa that induce bcl and the ifn/trail signaling axis frontiers in immunology | www.frontiersin.org march 2017 | volume 8 | article 313 thus activate the intrinsic mitochondrial apoptotic cascade (112) . also, pkr influences a cell's susceptibility to apoptotic signals, as it was demonstrated to sensitize to the fadd/caspase-8 apoptosis pathway upon type i ifn signaling after challenge with iav or dsrna (4) and the oas-rnasel system has been suggested to contribute to ifn-α-related cell death induction, but the exact mechanisms remain to be elucidated (113) . finally, also two classical initiators of the extrinsic apoptosis cascade are induced as isgs. both fasl and its receptor fas are upregulated on mrna levels by ifn-α (114) , and fasl was reported to be induced by type i ifn in iav infection in the murine lung in vivo (115) . also, the proapoptotic factor trail (or tnfsf10, apo2l) is induced by ifn-mediated and isgf3-executed transcriptional activation, as has been shown by sato et al. (116) , who revealed the presence of the isre sequence within the trail promoter region (116) . in iav infection, trail is released in high amounts from infected alveolar macrophages depending on a pkr-and ifn-β-driven autocrine signaling loop. binding of ifn-β to macrophageexpressed ifnar activates a jak/stat-dependent release of trail, which then acts through its receptor dr5 on the alveolar epithelial cells (5, 10) . however, certain prerequisites may decrease the ability of a cell to undergo apoptosis, including a shortage in pro-caspase-8 availability, expression of cellular fadd-like il-1β-converting enzyme-inhibitory proteins (c-flips) that block fadd-driven caspase activation, inactivation, or degradation of fadd itself, or expression of cyld, which acts as a receptor-interacting serine/threonine-protein (rip)1 kinase de-ubiquitinase and thus stabilizes rip1. however, in these cases ifn signaling can still promote a caspase-independent, programmed inflammatory cell death by activating the necroptosis pathway (117, 118) . necroptosis is induced by a complex formation by rip1 and rip3 kinases that activate both poly-adp-ribose (par) polymerase 1 (parp-1) and/or mixed lineage kinase domain-like (mlkl), leading to atp depletion, calpain activation, par polymer accumulation or cell membrane permeabilization, and release of damage-associated molecular patterns, respectively [reviewed in ref. (119, 120) ]. both a type i ifn-dependent jak/stat-driven activation of pkr as well as signaling by the prr dai (dnadependent activator of irfs) initiates necroptosis via rip1/rip3 activation, respectively (117, 121) . importantly, the activation of proapoptotic and pro-necroptotic pathways in respiratory infection can result in a structural disruption of the airway and the alveolar epithelial barrier, which is a major hallmark of respiratory disease and its progression to the acute respiratory distress syndrome (122, 123) . in virusinduced lung injury, especially expression of trail, which can initiate both apoptosis as well as necroptosis has been correlated with more severe outcomes. as described earlier, trail belongs to the superfamily of tnf ligands and has been reported to be inducible by both type i and type iii ifns. trail has been found to be present in various cells of the immune system, among them natural killer (nk) cells, t cells, nk t cells, dc subsets such as ifn-γ-producing killer dcs and macrophages, and can be displayed in large amounts on the cell surface or be shed upon ifn-and/or pro-inflammatory cytokine signaling (124) (125) (126) . in addition to cells of the immune system, fibroblasts have been shown to produce trail after ifnγ treatment or viral challenge. also, club cells and the alveolar epithelium have been reported to produce trail (127) (128) (129) (130) . similar to other ligands of the tnf superfamily, trail is a homotrimeric type ii transmembrane protein with a conserved c-terminal extracellular domain that mediates receptor binding and can be cleaved by metalloproteinases to generate a soluble mediator (131) . however, trail can induce cell death also in its membrane-bound form, that is, similar to trail expression levels and trail shedding, upregulated by type i ifn (126) . direct cell-to-cell trail-dr interactions have been demonstrated to play a role in macrophage, nk as well as cd4 + t cell-mediated induction of cellular death (132, 133) . in humans, five different binding partners for trail are present: the membrane-bound dr4 (trail-r1) and dr5 (trail-r2) that both induce a proapoptotic signaling cascade, the membrane-bound anti-apoptotic decoy receptors (dcr)1 and dcr2, and the soluble interaction partner osteoprotegerin (134) . in the murine system, only dr5 has been identified to ligate to trail (135) . in the human respiratory compartment, both dr4 and dr5 have been demonstrated to be present under steady-state conditions (136, 137) . however, upon viral infection, cell-sensitivity to trail-induced apoptosis is enhanced, which has been attributed to increased trail receptor expression especially on infected cells, as dr levels are markedly increased in iav-, adenovirus-, and paramyxovirus-infected cells in contrast to non-infected bystander cells (10, 138, 139) . of note, studies on the dependency of dr upregulation upon type i ifn signaling after iav infection have yielded conflicting results in different strains of mice (10, 74) , highlighting the complex interplay of ifn-induced cascades in a host-and tissue-specific context, whereas the exact virus-and host-specific mechanisms for dr regulation remain less well defined. moreover, previous assumptions that also dcr expression would correlate with cell-sensitivity to trail-induced cell death could not be experimentally verified (125) . tumor necrosis factor-related apoptosis-inducing ligand ligation to the proapoptotic receptors dr4 or dr5 triggers a trimerization of the receptors. subsequently, depending on additional stimuli, presence or absence of adaptor molecules or inhibitory proteins, different signaling pathways can be activated (figure 2) . in the classical trail-dependent extrinsic apoptosis induction, the proteins rip, tradd, and fadd are subsequently recruited to the dr cytoplasmic domain upon trail ligation (140, 141) . these factors and the proapoptotic drs all share a cytoplasmic death domain (dd), which is lacking or truncated and thus inactive in the dcr. the dd plays a central role in the concerted formation of the death-inducing signaling complex (disc). disc formation exposes a second functional domain of fadd, the death effector domain that is directly able to recruit pro-caspase-8 figure 2 | trail/dr5-mediated cellular signaling pathways. in presence of rip1, tradd, and fadd, trail ligation to dr5 results in apoptosis induction, which is initiated by recruitment of the pro-caspase-8 or -10 to fadd. these in turn activate the effector caspases-3 and -7, which leads to dna fragmentation and apoptosis induction. in addition, tradd can trigger a traf2-and jnk-dependent activation of bax and subsequent release of mitochondrial cytochrome c, inducing the pro-caspase-9 activation. in the presence of cyld, c-flip or absence of sufficient amounts of fadd or pro-caspase-8, trail ligation to dr triggers the interaction of rip1 and rip3 kinase, which in turn cause cell death via induction of mlkl and/or parp-1. in the presence of ciaps, fadd is not recruited to dr5 upon trail ligation, and tak1 is activated by tradd/traf2 interactions. tak1 induces nemo followed by iκb degradation and nfκb activation, as well as mkk and jnk activation leading to ap-1 nuclear translocation; both events promote the production of cytoprotective factors such as xiap, ciaps, and c-flip. additionally, tak1 triggers ampk activation and thus mtorc inhibition, which results in enhanced autophagic activity. abbreviations: ap-1, activator protein 1; tnf, tumor necrosis factor; trail, tnf-related apoptosis-inducing ligand; dr5, death receptor 5; rip1, receptor-interacting serine/threonine-protein kinase 1; tradd, tnf receptor type 1-associated death protein; fadd, fas-associated protein with death domain; traf, tnf receptor-associated protein; jnk, janus kinase; bax, bcl-2-associated x protein; c-flip, cellular fadd-like il-1β-converting enzyme-inhibitory proteins; rip, receptor-interacting serine/threonine-protein; mlkl, mixed lineage kinase domain-like; parp-1, poly-adp-ribose (par) polymerase 1; ciap, cytoprotective factors including inhibition of the autophagic machinery; xiap, x-linked inhibitor of apoptosis protein; ampk, amp-activated protein kinase; mtorc, mammalian target of rapamycin complex; traf2, tnf receptor-associated protein 2; mkk, mitogen-activated protein kinase. and pro-caspase-10. how exactly disc formation induces caspase activation is still under debate. the most probable scenarios include either an autocatalytic cleavage of caspase-pro-domains enabled by the spatial proximity between pro-caspases (generated by their recruitment to disc), by pro-caspase dimerization, or by pro-caspase conformational stabilization (125) . removal of the pro-domain of caspase-8 and caspase-10 results in the activation of the effector caspases-3 and -7, which cleave dna fragmentation factor 45 and lead to apoptosis (142, 143) . moreover, trailbinding to dr4 and dr5 can induce the jnk either via caspase-8 or recruitment of tnf receptor-associated protein 2 (traf2) to the disc complex, which results in the activation of the intrinsic apoptotic cascade by bax-dependent mitochondrial cytochrome c release (144) . in addition, trail signaling is also able to induce necroptosis by both activating the rip1/rip3 kinase downstream effectors parp-1 and mlkl, contributing to epithelial cell death and tissue injury (145) (146) (147) . it has become apparent in recent years that trail signaling is closely linked to induction of autophagy, a process generally associated with the blockade of apoptosis and necrosis. indeed, autophagy has been reported to improve cellular survival in cell stress by catabolic removal of cytoplasmic long-lived proteins and damaged organelles. it also contributes to viral clearance and the transfer of viral material to endosomal-/lysosomallocated tlr7 or mhc class ii compartments for the activation of adaptive immunity (148). several studies outline that trail ligation to dr5 can result in a traf2-dependent activation of tak1 (map3k7) that has been attributed a central role in trail-induced autophagy activation (149) . tak1 modulates the ikk-dependent translocation of nfκb, and it also induces jnk activation via mitogen-activated protein kinase. both events lead to expression of autophagy-related factors including inhibition of the autophagic machinery (ciap)1, ciap2, x-linked inhibitor of apoptosis protein, and c-flip (150, 151) . especially, c-flip has been associated with desensitization of cells to trail-induced apoptosis, favoring autophagy-related cascades (152) . another study revealed that upon trail signaling the amp-activated protein kinase (ampk) is activated. ampk in turn inhibits the mammalian target of rapamycin complex 1 that itself is an inhibitor of autophagy, thus the activation of the autophagic machinery is promoted (153) . the decision if trail signaling results rather in necroptotic or apoptotic cell death or in activation of autophagy seems to be dependent on the presence of ciaps that promote rip kinase ubiquitination and degradation (146) , but also on the balance between active caspases and autophagic proteins such as beclin-1 (154, 155) . this suggests a scenario where autophagy is activated as cell protective mechanism until cell stress-as executed by enhanced trail signaling or additional viral infection-increases over a threshold to favor cell death induction. accordingly, as trail signaling is not restricted to infected cells, excessive cell death activation might be limited by autophagy induction in non-infected bystander cells. however, autophagy is not only related to cell survival but can also positively affect apoptosis and induce-even if the exact mechanisms are still under debate-autosis, the autophagy-related cell death, another mode of trail to trigger cell death (156, 157) . of note, autophagy activation needs to be placed into its virus-specific context, as some viruses, including dengue virus, poliovirus, and coxsackie b virus (158) , can exploit autophagic pathways for their own replication and thus promote apoptosis and tissue injury. as discussed above, trail is a potent activator of cell death. however, its signaling outcomes can differ largely depending on its delivered form (e.g., membrane-bound versus soluble), the availability of drs on the target cell membrane, alternate intracellular pathways that might be activated and finally the pathogen itself, as it might exploit trail-induced pathways for its own survival and replication. in acute respiratory infection, trail signaling is often part of an ifn-driven overshooting inflammatory reaction that promotes unspecific tissue injury and thus disease severity by increasing functional and structural changes in infected but also non-infected cells, as will be outlined below. the release and effects of trail have been especially well studied in iav infection in the last decade. earlier studies reported that within 3 days after infection, bronchial, bronchiolar, and alveolar epithelial cells undergo apoptosis (159) . this early induction of cell death is mainly attributed to direct apoptosis induction by the virus itself, as iav actively promotes apoptosis for efficient viral replication (160) . herein, the viral ns1 and pb-f2 proteins not only play a crucial role (161, 162) but also the viral m2 protein has been implicated in this process as it inhibits autophagy in infected cells (163) . in addition, our own data revealed that later in iav in vivo infection, the recruitment of bone marrowderived macrophages via the cc chemokine receptor type 2 (ccr2)-cc-chemokine ligand 2 (ccl2) axis significantly contributes to alveolar cell apoptosis and structural damage of the alveolar epithelium (5) . studies by wurzer et al. (164) had previously demonstrated that iav promotes the production of proapoptotic factors in an auto-and paracrine fashion via nfκb transcriptional activation by iav (164) . subsequently, brincks et al. (6) elucidated that human peripheral blood mononuclear cell treated with iav released trail and that increased trail levels correlated with type i as well as ii ifn induction. additionally, trail sensitivity was increased in influenza virusinfected cells. in line, our investigations could elucidate that iav triggers a pkr-dependent translocation of nfκb that results the production of type i ifns. these in turn induce, via ligation to the ifnar receptor complex, expression and shedding of trail by bone marrow-derived macrophages (10) . in addition, davidson et al. (74) demonstrated that type i ifn application to iavinfected mice increased morbidity and lung injury, which could be attributed to both dr5 and trail upregulation inducing epithelial cell apoptosis. importantly, högner et al. also reported that the iav strain used in these studies, a/pr8 (h1n1), which is highly pathogenic for mice, induced an approximately 800-fold induction in macrophage trail expression, whereas the lower pathogenic virus a/x-31 (h3n2) only stimulated trail by a factor of eight. of note, the relation between trail induction and iav strain-specific pathogenicity also translates to the highly pathogenic avian h5n1 iav, causing severe pneumonia in mice as well as in humans (165, 166) . moreover, human infection with both the highly pathogenic h5n1 as well as the pandemic 1918 h1n1 iav strains are characterized by a massive influx of mononuclear phagocytes into the alveoli, which is correlated with extensive alveolar epithelial cell apoptosis (97, 167) . additionally, macrophages gained from bronchoalveolar lavages of patients presenting with ards caused by the pandemic h1n1/2009 virus strain showed high surface expression and release of trail (10) . another recent report demonstrates that in highly pathogenic avian influenza, in addition to macrophages also the alveolar epithelium might be involved in causing elevated levels of trail in the alveolar space (130) . besides its role in apoptosis, trail signaling upon iav infection has also been implicated in the induction of necroptosis in fibroblasts, dcs, and lung epithelial cells (146, 147, 168) . rodrigue-gervais et al. (146) demonstrated that lack of cipa2 promotes rip3 kinase-mediated necroptosis in response to trail-but also the proapoptotic factor faslreleased from hematopoietic cells. this contributed to severe lung epithelial degeneration and increased mortality, even though viral control was not compromised. nogusa et al. (147) further elucidated that iav-induced necroptosis depends on rip3 kinase activation of mlkl, and that rip3 kinase deficiency, similar to ciap2-deficiency, increased iav-susceptibility in vivo. in iav infection, as mentioned earlier, dr5 expression is elevated on infected alveolar epithelial cells, but not in noninfected cells in vivo, which might impact on trail susceptibility to apoptosis induction (10) . however, both infected as well as neighboring bystander cells were found to be targeted for apoptosis induction by macrophage-released trail. nonetheless, we could recently show that specifically in noninfected cells within the iav-infected lung, trail severely compromises the function of the ion channel na,k-atpase, which was mediated via induction of the stress kinase ampk (11) , thereby potentially revealing a cross-link to trailinduced autophagic cell stress pathways in bystander cells both in vitro and in vivo. the trail-induced and ampk-mediated downregulation of the na,k-atpase, a major driver of vertical ion and fluid transport from the alveolar airspace toward the interstitium, resulted in a reduced capacity of iav-infected mice to clear excessive fluid from the alveoli. thus, trail signaling contributes to intensive edema formation, a hallmark of disease in virus-induced ards (123) . notably, this effect of trail on na,k-atpase expression was induced independently of cell death pathways elicited by caspases, as treatment of cells and mice with a specific caspase-3 inhibitor diminished apoptosis in alveolar epithelial cells but still allowed for the reduction of the na,k-atpase (11) . conclusively, treatment of iav-infected mice with neutralizing antibodies directed against trail or the abrogation of recruitment of trail + bone marrow-derived macrophages inhibited apoptosis of both non-infected and bystander cells. thus, lung leakage due to loss of alveolar barrier function was reduced, whereas alveolar fluid clearance capacity was enhanced, resulting in reduced edema, improved survival, and outcome upon iav challenge in vivo. however, trail has also been shown to be upregulated on nk, dc, and on cd4 + and cd8 + t cells after iav infection (169) . studies by brincks et al. demonstrated that especially cd8 + t involved in cytotoxic t cell responses toward iav and drive iav-infected cells into apoptosis via trail, thus contributing to efficient virus clearance (6, 170) . in addition, both fasl and trail are involved in dc-mediated ctl activation and cytotoxicity against iav-infected cells (6, 171) . furthermore, studies showed delayed viral clearance upon neutralizing anti-trail antibody administration (169, 172) . our data, however, demonstrate that the transfer of trail-deficient bone marrow into irradiated wild-type mice, resulting in loss of trail production by bone marrow-derived macrophages upon iav infection, does not impact on the capacity to fully clear viral particles from the lung at day 7 after infection, suggesting that other compensatory mechanisms are recruited to guarantee viral clearance (10) . taken together, in iav infection, trail acts both as an important mediator of infected cell killing but particularly as a detrimental factor contributing to tissue injury and impaired inflammation resolution when released in excessive amounts by recruited immune cells. respiratory syncytial virus is an important cause of respiratory tract infections especially in children worldwide. generally, there seem to be virus-elicited anti-apoptotic mechanisms active in the lung epithelium, as rsv-infected primary human airway cells show a minimal cytopathic effect (173) . however, several cell lines including small airway cells, primary tracheal-bronchial cells, and a549 and hep-2 showed increased expression of trail and its ligands dr4 and dr5 in an in vitro rsv infection model (174) . moreover, soluble trail released from leukocytes was elevated in the bronchoalveolar lavage fluid of patients with rsv-associated respiratory failure, suggesting that similar to iav, trail contributes to rsv-induced epithelial injury and disease progression (137) . also in cov respiratory tract infection, trail levels, but less so fasl, have been reported to be markedly elevated (175, 176) . for sars-cov that presents with a severe damage to both the upper and lower respiratory tract (177) , especially dcs respond with a strong induction of trail production, which was suggested to correlate to increased cellular lung infiltrations present in sars-cov patients (175) . interestingly, sars-cov infection drives cells into apoptosis by a pkr-driven but eif2α-independent pathway (178) , which might-similarly as seen in iav infection-suggest a pkr-induced and autocrine/paracrine executed activation of apoptosis. also mers-cov, which causes pneumonia and respiratory failure, has been demonstrated to induce profound cell death within 24 h of infection, irrespective of viral titers produced by the infected cells. however, type i ifn expression is strongly reduced in mers-cov in comparison to seasonal human cov in in vitro infection models, including human monocyte-derived macrophages, calu-3, and human lung fibroblasts (179, 180) , which might also dampen downstream trail induction. therefore, the exact mechanism by which mers-cov promotes cell death remains to be investigated. recurrently, viral infections of the respiratory tract are followed by outgrowth of colonizing gram-positive bacteria that aggravates the course of illness. this is well documented for iav, where "super" infections with streptococcus pneumoniae and staphylococcus aureus are the most frequent and increase viral pneumonia-associated morbidity and mortality (181) . during the 1918 iav pandemic, bacterial pneumonia was evident in most cases (182) and also during the recent 2009 h1n1 pandemic, coinfections were a relevant factor for severe disease in a young patient population without comorbidities (183) . interestingly, virus-induced elevation of the type i ifn response levels might promote secondary bacterial outgrowth by several mechanisms [reviewed in ref. (184) ]. in line, it has been repeatedly demonstrated that lack of type i ifn signaling results in better bacterial clearance and increased survival rates in iav-and s. pneumoniae-superinfected mice (185) (186) (187) . herein, ifn-induced apoptosis induction as well as depletion or impaired recruitment of lymphocyte subsets necessary for bacterial control play a critical role (188, 189) . bacterial clearance from the lung has been reported to rely on sufficient phagocyte generation, recruitment, and survival. type i ifn has been demonstrated to cause apoptosis in bone marrow-derived granulocytes, affecting the numbers of recruited neutrophils (189) , but also to impair expression of the cytokines cxcl1 (or kc) and cxcl2 (or mip-2), thus inhibiting neutrophil recruitment to the lungs with severe effects on survival of superinfected mice (185) . a recent report by schliehe et al. (190) elucidated the mechanistic background for impaired cxcl1 expression and secretion and demonstrated that type i ifns activate the histone methyltransferase setdb2, which in turn represses the cxcl1 promoter and thus impairs neutrophil recruitment and bacterial clearance. moreover, type i ifn production decreases ccl2 production, thus inhibiting macrophage recruitment, which as well has been reported to have detrimental effects on bacterial clearance and disease progression in bacterial superinfection after viral insult in vivo (186) . in addition, type i ifns also impair γδ t cell function and il-17 release, which was shown to increase susceptibility to s. pneumoniae superinfection after iav challenge (187) . also in s. aureus pneumonia, a robust type i ifn response is correlated to excessive morbidity and tissue injury (191) . in a model of polyi:c, s. aureus (methicillinresistant strain, mrsa) superinfection, polyi:c treatment prior to bacterial infection enhanced type i ifn levels and decreased bacterial clearance and survival (192) . furthermore, shepardson et al. (193) demonstrated that late type i ifn induction rendered mice more susceptible to secondary bacterial pneumonia in a model of iav-mrsa superinfection. only limited data are available on a direct role of trail in respiratory disease progression due to bacterial superinfections. in a model of iav-haemophilus influenza infection, neither deficiency for cc chemokine receptor type 2, inhibiting bone marrow-derived macrophage recruitment, nor deficiency of fas or tnfr1 impacted outcome (194) . yet, during s. pneumoniae single infection, early cell death of macrophages is thought to limit an exuberant inflammatory reaction and accordingly, a study by steinwede et al. (195) revealed that neutrophil-derived trail limits tissue injury by inducing cell death in dr5-epressing lung macrophages in bacterial mono-infection (195) . in contrast, in the iav-s. pneumoniae superinfection mouse model, iavinduced trail has a detrimental effect on overall mortality (7), as trail-induced epithelial injury enhanced bacterial outgrowth of s. pneumoniae-administered at day 5 after iav infection-markedly. importantly, administration of anti-trail neutralizing antibodies enhanced bacterial control by the host organism. thus, the activation of ifn/trail-mediated signaling in viral infection has detrimental implication for outcome of secondary bacterial infection following viral insult, rendering the ifn/trail signaling axis an interesting therapeutic target not only in respiratory viral infections but also in complicating bacterial superinfection. an increasing number of reports connect progression of chronic respiratory disease to acute respiratory virus infection or proapoptotic signaling events. in fact, trail has been reported to be a critical determinant for promoting the development of chronic lung disease in early life (196) ; targeting trail by genetic deletion or neutralizing antibody application in early-life respiratory infections ameliorated infection-induced histopathology, inflammation, as well as emphysema-like alveolar enlargement and lung function. furthermore, trail was also shown to play a role in the development of allergy and asthma. trail is not only elevated in the sputum of asthmatic patients but has also been reported to be highly expressed in an experimental mouse model of asthma, where it induces ccl20 secretion by bronchial epithelial cells, thus promoting th2 cell responses and airway hyperreactivity (197) . in copd, acute exacerbations driven by viral and bacterial infection are a major factor increasing both mortality and morbidity, and both influenza and s. pneumoniae have been identified among the most common causes of copd exacerbations (198) . indeed, primary bronchial epithelial cells isolated from subjects with copd show an impaired production of type i ifn (199) , which has been implied in the enhanced susceptibility of copd patients to respiratory infections; however, even in absence of high ifn induction, both an abnormally elevated loss of alveolar epithelial cells due to apoptosis as well as elevated trail and dr5 levels were reported (200) , implying a possible link between viral/bacterial induction of trail and acute exacerbations in copd. trail induction has also been directly linked to cigarette-smoke exposure, a common cause of copd, and trail deficiency resulted in decreased pulmonary inflammation and emphysema-like alveolar enlargement in vivo (201) . moreover, increased levels of both trail and dr5 were associated to impaired lung function and increased systemic inflammation in human copd patients (202) . while alveolar epithelial cell death is closely connected to idiopathic pulmonary fibrosis (ipf), trail and its receptors dr4 and dr5 in aec were shown to be upregulated in ipf lungs (129) . also, in pulmonary arterial hypertension virus infection is considered to be a possible risk factor (203) , and pulmonary hypertension has been reported to be a side effect of prolonged treatment with type i ifn (204, 205) . in line, trail has been closely linked to disease progression in pulmonary hypertension. trail has been found to be increased within pulmonary vascular lesions of patients with pulmonary hypertension (206) and also in a mouse model of hypoxia-induced pulmonary hypertension, levels of soluble trail correlated with right ventricular systolic pressure, right ventricular hypertrophy, and pathologic alterations (33, 34) . importantly, neutralizing antibody-treatment against trail showed positive effects on survival while reducing pulmonary vascular remodeling (207) . notably, the extent to which infection-induced trail release causes or exacerbates chronic lung disease or in how far trail production in chronic lung diseases affects susceptibility to respiratory viral and complicating bacterial infection remains to be elucidated. respiratory viral infections are major causative agents for lung injury and ards; however, in many cases antivirals are not sufficient to limit disease (208). besides the fact that most viruses are subject to strong selective pressures that favor quickly evolving, drug-resistant virus variants, recent advances in understanding the processes that contribute to tissue injury and ards highlight a crucial role of immune-related, ifn-driven events. therefore, novel therapeutic strategies often aim to improve the outcome of severe respiratory infection by modulating host cell responses; however, to date, clinical trials trying to improve severe viral infections or ards outcomes by targeting host pathways have not resulted in approval of new drugs (122) . of note, for establishment of such therapies it has to be considered that the timing and intensity of induction and amplification as well as of dampening and termination of the ifn-driven immune response needs to precisely match the pathogen-and organ-specific requirements of a given infection. a non-controlled regulation of these processes may lead to either an unrestricted pathogen spreading or, on the other extreme, to an overshooting inflammatory response, including the increased production of pro-inflammatory and proapoptotic mediators, elevated levels of recruited immune cells, and/or aberrant repair processes. notably, both too low and too high levels of ifn-induced effects facilitate disease progression with a possible increase of fatal outcomes in ards patients (78) . accordingly, preclinical in vivo studies of ifn-directed therapies yielded seemingly adverse results, depending on the context, timing, and dosage of ifn modulation. however, in multiple settings of acute respiratory viral infection, studies demonstrate that an exaggerated signaling derived from type i ifn in an already inflamed tissue contributes to worsened outcomes, and importantly, might favor secondary bacterial superinfection [e.g., ref. (75, 76, 209) ]. interestingly, davidson et al. (209) demonstrated that type iii ifn release upon influenza challenge-in contrast to type i ifn induction-does not trigger an unbalanced inflammatory response that critically contributes to respiratory disease progression in vivo, highlighting it as a possible therapeutic option in iav-induced lung injury. most likely, this effect derives from the lack of the ifn-λr1/il-10r2 receptor complex, but presence of ifnar, on immune cells, including bone marrowderived macrophages. nonetheless, other reports identify ifn-λ as a driver of macrophage polarization to an inflammatory m1 phenotype (41) that has been attributed to further promote an overshooting inflammatory response, highlighting the need for further studies of type iii ifn biology in pathogen-associated disease progression. as generally ifn-directed therapeutic approaches target various downstream signaling events that might both act beneficially as well as detrimentally on viral replication and pathogenesis, a further approach is to address specific isgs that primarily show detrimental effects on disease progression. as outlined above, trail or its downstream signaling events might comprise a suitable target for adjunct therapies in addition to antivirals. accordingly, our own data in a preclinical mouse model of iav infection demonstrate a clear benefit of the systemic application of neutralizing antibodies against trail at days 3 and 5 postinfection for lung injury, morbidity, and mortality (10, 11) . targeting trail as a major determinant of disease severity in respiratory viral infections including iav, but also rsv and cov, may yield therapeutic approaches that are superior to ifn-directed strategies, as they seemingly do not bear the risk of compromising host defense. yet, it should be thoroughly excluded that blocking trail-induced cell death of infected cells will not lead to an overwhelming viral spreading, especially as reports on viral loads upon trail inhibition in preclinical models of iav are controversial (6, 10, 170) . accordingly, additional studies are needed to understand how and to which extent virus-infected cells can be killed or viral spreading can be controlled by other means in the absence of trail. moreover, targeting pathways and signaling hubs downstream of trail/drs, such as ampk (11), in a well-timed and lung compartment-specific way, may open new therapeutic avenues but requires more detailed preclinical studies on efficacies and side effects. a valid approach might be the use of a combination therapy of such a treatment together with a classical antiviral drug therapy limiting viral replication; however, exact dosage, timing, kinetics, and application routes remain to be defined. cp and sh performed bibliographic research and drafted the manuscript. this work was supported by the german research foundation (sfb-tr84 b2, sfb1021 c05, kfo309 p2/p8, exc147), by the german center for lung research (dzl), and by the german center for infection research (dzif). 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factor related apoptosis-inducing ligand axis in the pathogenesis of pulmonary arterial hypertension inhibition of tumor necrosis factor-related apoptosis-inducing ligand (trail) reverses experimental pulmonary hypertension luks am. ventilatory strategies and supportive care in acute respiratory distress syndrome ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. 