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 virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses prevalence of influenza a virus in live-captured north atlantic gray seals: a possible wild reservoir. emerging microbes & infections 5 bats and their virome: an important source of emerging viruses capable of infecting humans. current opinion in virology middle east respiratory syndrome coronavirus in bats, saudi arabia bats as 'special' reservoirs for emerging zoonotic pathogens bats are a major natural reservoir for hepaciviruses and pegiviruses bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? memórias do instituto oswaldo cruz new world bats harbor diverse influenza a viruses a distinct lineage of influenza a virus from bats a distinct lineage of influenza a virus from bats serological evidence of influenza a viruses in frugivorous bats from africa differences in influenza virus receptors in chickens and ducks: implications for interspecies transmission endocytosis of influenza viruses. microbes and infection influenza a virus polymerase is a site for adaptive changes during experimental evolution in bat cells comparative distribution of human and avian type sialic acid influenza receptors in the pig sialic acid tissue distribution and influenza virus tropism. influenza and other respiratory viruses 2 bat cells from pteropus alecto are susceptible to influenza a virus infection and reassortment expression and distribution of sialic acid influenza virus receptors in wild birds host-range barrier of influenza a viruses quail carry sialic acid receptors compatible with binding of avian and human influenza viruses comparative intestinal histomorphology of five species of phyllostomid bats (phyllostomidae, microchiroptera): ecomorphological relations with alimentary habits bat flight and zoonotic viruses mass extinctions, biodiversity and mitochondrial function: are bats 'special' as reservoirs for emerging 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 investigators whose relevant work was not included due to space limitations. regulatory roles of c-jun in h5n1 influenza virus replication and host inflammation inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal h5n1 infection the mek-inhibitor ci-1040 displays a broad anti-influenza virus activity in vitro and provides a prolonged treatment window compared to standard of care in vivo the nf-kappab inhibitor sc75741 protects mice against highly pathogenic avian influenza a virus phosphoproteomic-based kinase profiling early in influenza virus infection identifies grk2 as antiviral drug target transient inhibition of sphingosine kinases confers protection to influenza a virus infected mice par1 contributes to influenza a virus pathogenicity in mice increased survival after gemfibrozil treatment of severe mouse influenza tnf/inos-producing dendritic cells are the necessary evil of 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 mice innate immune responses to influenza a h5n1: friend or foe? new fronts emerge in the influenza cytokine storm the role of antimicrobial peptides in influenza virus infection and their potential as antiviral and immunomodulatory therapy therapeutic peptides cathelicidins, multifunctional peptides of the innate immunity flip-mediated autophagy regulation in cell death control the tyrosine kinase inhibitor tyrphostin blocks the cellular actions of nerve growth factor role of protein kinase c betaii in influenza virus entry via late endosomes from virus entry to release: the diverse functions of pi3k during rna virus infections influenza a virus-induced early activation of erk and pi3k mediates v-atpase-dependent intracellular ph change required for fusion development of cellular signaling pathway inhibitors as new antivirals against influenza novel roles of focal adhesion kinase in cytoplasmic entry and replication of influenza a viruses a novel p38 mitogen activated protein kinase (mapk) specific inhibitor suppresses respiratory syncytial virus and influenza a virus replication by inhibiting virus-induced p38 mapk activation focal adhesion kinase (fak) regulates polymerase activity of multiple influenza a virus subtypes role of c-jun terminal kinase (jnk) activation in influenza a virus-induced autophagy and replication influenza virus infections and cellular kinases toll-like receptor 4-mediated activation of p38 mitogen-activated protein kinase is a determinant of respiratory virus entry and tropism antiviral activity of the mekinhibitor u0126 against pandemic h1n1v and highly pathogenic avian influenza virus in vitro and in vivo combination of mek inhibitors and oseltamivir leads to synergistic antiviral effects after influenza a virus infection in vitro jnk2 and ikkbeta are required for activating the innate response to viral infection influenza a virus-encoded ns1 virulence factor protein inhibits innate immune response by targeting ikk a novel class of potent nf-kappab signaling inhibitors the nf-kappab inhibitor sc75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance grk2 in the heart: a gpcr kinase and beyond regulation of sphingosine kinase and sphingolipid signaling sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases sphingosine kinase 1 serves as a pro-viral factor by regulating viral rna synthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection participation in inflammation altered expression and in vivo lung function of protease-activated receptors during influenza a virus infection in mice pandemic influenza: a potential role for statins in treatment and prophylaxis treating influenza with statins and other immunomodulatory agents old drugs losing effectiveness against flu; could statins fill gap? influenza and copd mortality protection as pleiotropic, dose-dependent effects of statins experience in the management of the severe form of human influenza a h1n1 association between use of statins and mortality among patients hospitalized with laboratory-confirmed influenza virus infections: a multistate study influenza morbidity and mortality in elderly patients receiving statins: a cohort study an assessment of the effect of statin use on the incidence of acute respiratory infections in england during winters drosophila rnai screen identifies host genes important for influenza virus replication the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection the use of random homozygous gene perturbation to identify novel host-oriented targets for influenza genome-wide rnai screen identifies human host factors crucial for influenza virus replication human host factors required for influenza virus replication antiviral effects of inhibiting host gene expression influenza virus-host interactomes as a basis for antiviral drug development genomewide crispr/cas9 screen identifies host factors essential for influenza virus replication repurposing host-based therapeutics to control coronavirus and influenza virus repurposing of drugs as novel influenza inhibitors from clinical gene expression infection signatures comparative influenza protein interactomes identify the role of plakophilin 2 in virus restriction network-guided discovery of influenza virus replication host factors cellular networks involved in the influenza virus life cycle genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery meta-and orthogonal integration of influenza "omics" data defines a role for ubr4 in virus budding the magnitude of the t cell response to a clinically significant dose of influenza virus is regulated by trail pathogenic potential of interferon alphabeta in acute influenza infection anticancer compound 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 from national surveillance epidemiology and seasonality of respiratory tract virus infections in the tropics viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city influenza and other respiratory viruses in three central american countries contribution of common and recently described respiratory viruses to annual hospitalizations in children in south africa identification of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in hong kong by multiplex pcr assays molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai three years surveillance of viral etiology of acute lower respiratory tract infection in children from detection for respiratory viruses in shanghai with multiplex pcr from guidelines for management of childhood community acquired pneumonia (for trial implementation) (i) cloning of a human parvovirus by molecular screening of respiratory tract samples wu polyomavirus infection among children in south china principles of the xtag respiratory viral panel assay (rvp assay) respiratory virus multiplex rt-pcr assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections viral pathogens of acute lower respiratory tract infection in hospitalized children from east guangdong of china detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy epidemiological and clinical features of respiratory viral infections in hospitalized children during the circulation of influenza virus a(h1n1) viral pathogens of acute lower respiratory tract infection in china viral etiology of 1165 hospitalized children with acute lower respiratory tract infection comparative analysis of clinical features of common respiratory tract viral infection and its trends in hospitalized children of 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 definition-as highly abnormal hosts. 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). in the context of any of these susceptibility mechanisms, failure to clear the virus is an expected consequence, indicating that a full course of effective antiviral therapy is likely to benefit this population. in the future, understanding the biological mechanisms of susceptibility to severe iav may yield therapeutic targets to modify the biology of the susceptible hosts in critical care and render them resilient. comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study the swine flu triage (swift) study: development and ongoing refinement of a triage tool to provide regular information to guide immediate policy and practice for the use of critical care services during the h1n1 swine influenza pandemic genetics of infectious diseases targeting the host immune response to fight infection influenza to target the host? type-a influenza viruses in the feces of migratory waterfowl initial infectious dose dictates the innate, adaptive, and memory responses to influenza in the respiratory tract validation of the wild-type influenza a human challenge model h1n1pdmist: an a(h1n1)pdm09 dose-finding investigational new drug study pathogenesis of avian influenza a (h5n1) viruses in ferrets h7n9 influenza viruses interact preferentially with 2,3-linked sialic acids and bind weakly to 2,6-linked sialic acids altered receptor specificity and cell tropism of d222g hemagglutinin mutants isolated from fatal cases of pandemic a(h1n1) 2009 influenza virus measurement of the mutation rates of animal viruses: influenza a virus and poliovirus type 1 heterogeneity of the mutation rates of influenza a viruses: isolation of mutator mutants viral quasispecies characteristics of patients with oseltamivir-resistant pandemic (h1n1) 2009, united states rapid selection of oseltamivir and peramivir resistant pandemic h1n1 during therapy in two immunocompromised hosts emergence of multidrugresistant influenza a(h1n1)pdm09 virus variants in an immunocompromised child treated with oseltamivir and zanamivir accumulation of humanadapting mutations during circulation of a(h1n1)pdm09 influenza virus in humans in the united kingdom antiviral b cell and t cell immunity in the lungs influenza: the new acquayantance potent protection against h5n1 and h7n9 influenza via childhood hemagglutinin imprinting global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study preexisting influenza-specific cd4+ t cells correlate with disease protection against influenza challenge in humans populations at risk for severe or complicated influenza illness: systematic review and meta-analysis pandemic influenza a (h1n1) in pregnant women: impact of early diagnosis and antiviral treatment impact of influenza on acute cardiopulmonary hospitalizations in pregnant women pandemic 2009 influenza a(h1n1) virus illness among pregnant women in the united states mechanisms and clinical consequences of acute lung injury pregnancy and infection factors associated with death or hospitalization due to pandemic 2009 influenza a(h1n1) infection in california excess deaths associated with underweight, overweight, and obesity a novel risk factor for a novel virus: obesity and 2009 pandemic influenza a (h1n1) obesity increases the duration of influenza a virus shedding in adults epidemic inflammation: pondering obesity increased risk of influenza among vaccinated adults who are obese obesity is associated with impaired immune response to influenza vaccination in humans overweight and obese adult humans have a defective cellular immune response to pandemic h1n1 influenza a virus influenzaassociated pediatric deaths in the united states the burden of influenza in england by age and clinical risk group: a statistical analysis to inform vaccine policy the burden of seasonal and pandemic influenza in infants and children mortality associated with influenza and respiratory syncytial virus in the united states estimates of global seasonal influenza-associated respiratory mortality: a modelling study age-related inflammatory cytokines and disease the effect of age on thymic function impaired immune responses in the lungs of aged mice following influenza infection cytomegalovirus seropositivity drives the cd8 t cell repertoire toward greater clonality in healthy elderly individuals cytomegalovirus infection enhances the immune response to influenza shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease genetic and environmental influences on premature death in adult adoptees the role of host genetics in susceptibility to influenza: a systematic review an updated systematic review of the role of host genetics in susceptibility to influenza. influenza other respir viruses the role of host genetic factors in respiratory tract infectious diseases: systematic review, meta-analyses and field synopsis evidence for a heritable predisposition to death due to influenza influenza pathogenesis: the effect of host factors on severity of disease host genetics of severe influenza: from mouse mx1 to human irf7 life-threatening influenza and impaired interferon amplification in human irf7 deficiency life-threatening influenza pneumonitis in a child with inherited irf9 deficiency lethal influenza in two related adults with inherited gata2 deficiency a global reference for human genetic variation potential etiologic and functional implications of genome-wide association loci for human diseases and traits the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion the interferon-stimulated gene ifitm3 restricts west nile virus infection and pathogenesis kellam p. ifitm3 restricts the morbidity and mortality associated with influenza interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals interferon-inducible transmembrane protein 3 genetic variant rs12252 and influenza susceptibility and severity: a meta-analysis snp-mediated disruption of ctcf binding at the ifitm3 promoter is associated with risk of severe influenza in humans pathologic anatomy and bacteriology of influenza: epidemic of autumn acute myocardial infarction after laboratory-confirmed influenza infection antiviral protection by ifitm3 in vivo a functional variation in cd55 increases the severity of 2009 pandemic h1n1 influenza a virus infection ifitm3, tlr3, and cd55 gene snps and cumulative genetic risks for severe outcomes in chinese patients with h7n9/h1n1pdm09 influenza identification of tmprs22 as a susceptibility gene for severe 2009 pandemic a(h1n1) influenza and a(h7n9) influenza tmprs22 is a host factor that is essential for pneumotropism and pathogenicity of h7n9 influenza a virus in mice tmprs22 is essential for influenza h1n1 virus pathogenesis in mice the host protease tmprs22 plays a major role in in vivo replication of emerging h7n9 and seasonal influenza viruses surfactant protein b gene polymorphism is associated with severe influenza surfactant protein-a-deficient mice display an exaggerated early inflammatory response to a beta-resistant strain of influenza a virus the genetics of chronic obstructive pulmonary disease identification and characterization of gldc as host susceptibility gene to severe influenza the glycine cleavage system: composition, reaction mechanism, and physiological significance functional variants regulating lgals1 (galectin 1) expression affect human susceptibility to influenza a(h7n9) delayed clearance of viral load and marked cytokine activation in severe cases of pandemic h1n1 2009 influenza virus infection genetic variants in il1a and il1b contribute to the susceptibility to 2009 pandemic h1n1 influenza a virus tnf, il6, and il1b polymorphisms are associated with severe influenza a (h1n1) virus infection in the mexican population ifn-λ prevents influenza virus spread from the upper airways to the lungs and limits virus transmission seasonal influenza a/h3n2 virus infection and il-1β, il-10, il-17, and il-28 polymorphisms in iranian population il6 and crp haplotypes are associated with copd risk and systemic inflammation: a case-control study surfactant protein a genetic variants associate with severe respiratory insufficiency in pandemic influenza a virus infection toll-like receptor 3 gene polymorphisms and severity of pandemic a/h1n1/2009 influenza in 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 experimental system for production of dips without standard virus and for quantification of dip infectivity, which should promote efforts to develop dips for antiviral therapy. winkler. drug resistance in influenza a virus: the epidemiology and management defective interfering influenza virus rnas: time to reevaluate their clinical potential as broad-spectrum antivirals? cloned defective interfering influenza rna and a possible pan-specific treatment of respiratory virus diseases dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza a defective interfering influenza rna inhibits infectious influenza virus replication in human respiratory tract cells: a potential new human antiviral attempts to detect homologous autointerference in vivo with influenza virus and vesicular stomatitis virus continuous influenza virus production in cell culture shows a periodic accumulation of defective interfering particles cloned defective interfering influenza virus protects ferrets from pandemic 2009 influenza a virus and allows protective immunity to be established the influence of defective-interfering particles of the pr-8 strain of influenza a virus on the pathogenesis of pulmonary infection in mice interfering vaccine (defective interfering influenza a virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection in vivo antiviral activity: defective interfering virus protects better against virulent influenza a virus than avirulent virus defective interfering virus protects elderly mice from influenza a novel broad-spectrum treatment for respiratory virus infections: influenza-based defective interfering virus provides protection against pneumovirus infection in vivo defective interfering influenza virus confers only short-lived protection against influenza virus disease: evidence for a role for adaptive immunity in di virus-mediated protection in vivo defective interfering 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 signaling pathways. although further studies of the antiviral effects of pa against other iav strains (h3n2 or h5n1) will be required to advance it for drug development, pa has the potential to be developed into a novel nasal drop for influenza therapy and prophylaxis in the future. orthomyxoviridae . the viruses and their replication influenza virus . 100 years on, are we prepared against the next influenza pandemic? structural basis of influenza virus fusion inhibition by the antiviral drug arbidol safety and efficacy of antiinfluenza drugs, intravenous peramivir against influenza virus infection in elderly patients with underlying disease oseltamivir for treatment and prophylaxis of influenza infection drug resistance in influenza a virus: the epidemiology and management global transmission of oseltamivir-resistant influenza the pharmacological management of severe influenza infection -'existing and emerging therapies availability, pharmaceutics, security, pharmacokinetics, and pharmacological activities of patchouli alcohol anti-emetic principles of pogostemon cablin (blanco) benth analgesic and anti-inflammatory activities of the methanol extract from pogostemon cablin. evid based selective antibacterial activity of patchouli alcohol against helicobacter pylori based on inhibition of urease patchouli alcohol: in vitro direct anti-influenza virus sesquiterpene in pogostemon cablin benth oral administration of patchouli alcohol isolated from pogostemonis herba augments protection against influenza viral infection in mice inhibitory effect and possible mechanism of action of patchouli alcohol against influenza a (h2n2) virus in vitro inhibitory effect of carrageenan oligosaccharide on influenza a h1n1 virus inhibition of influenza a virus infection by fucoidan targeting viral neuraminidase and cellular egfr pathway analysis of relative gene expression data using real-time quantitative pcr and the 2(−delta delta c(t)) method serial histopathological examination of the lungs of mice infected with influenza a virus pr8 strain boronic acid modifications enhance the anti-influenza a virus activities of novel quindoline derivatives antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade receptor tyrosine kinase inhibitors block multiple steps of influenza a virus replication inhibition of influenza virus-induced nf-kappab and raf/ mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo a new player in a deadly game: influenza viruses and the pi3k/akt signalling pathway a central role for pi3k-akt signaling pathway in linking samhd1-deficiency to the type i interferon signature animal models for the study of influenza pathogenesis and therapy publisher's note springer nature remains neutral 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. this comparative landscaping of acute mouse pneumonia histology provides a comprehensive framework for future studies on the role of individual pathogen or host factors, complex disease mechanisms, and novel therapeutic strategies that could help to treat pneumonia in human patients. managing community-acquired pneumonia: a european perspective. respiratory medicine fact sheet n˚331 respiratory viral infection predisposing for bacterial disease: a concise review. fems immunology and medical microbiology evidence on measures for the prevention of ventilator-associated pneumonia immunostimulation is a rational therapeutic strategy in sepsis. novartis foundation symposium recent advances in our understanding of streptococcus pneumoniae infection the burden of pneumococcal pneumonia-experience of the german competence network capnetz pneumococcal conjugate vaccine for childhood immunization-who position paper. releve epidemiologique hebdomadaire mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995-1997 hospitalized patients with 2009 h1n1 influenza in the united states neuraminidase inhibitors for preventing and treating influenza in healthy adults and children. the cochrane database of systematic reviews epidemiology, microbiology, and treatment considerations for bacterial pneumonia complicating influenza legionnaires' disease: update on epidemiology and management options legionella as a cause of severe pneumonia clinical infectious diseases: an official publication of the infectious diseases society of america american journal of respiratory and critical care medicine ventilator-associated pneumonia in a tertiary care hospital in india: incidence and risk factors incidence, bacteriology, and clinical outcome of ventilator-associated pneumonia at tertiary care hospital development of immunization trials against klebsiella pneumoniae the epidemiology of nosocomial infections caused by klebsiella pneumoniae current epidemiology and growing resistance of gram-negative pathogens. the korean journal of internal medicine association between staphylococcus aureus strains carrying gene for panton-valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients archives de pediatrie: organe officiel de la societe francaise de pediatrie european respiratory society: standards for quantitative assessment of lung structure. american journal of respiratory and critical care medicine an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals multiparametric and semiquantitative scoring systems for the evaluation of mouse model histopathology-a systematic review the european college of veterinary pathologists (ecvp): the professional body for european veterinary pathologists a minimum core outcome dataset for the reporting of preclinical chemotherapeutic drug studies: lessons learned from multiple discordant methodologies in the setting of colorectal cancer. critical reviews in oncology/hematology assessment of reproductive toxicity under reach. regulatory toxicology and pharmacology: rtp the virulence variability of different acinetobacter baumannii strains in experimental pneumonia. the journal of infection the role of tlr2 in the host response to pneumococcal pneumonia in absence of the spleen ifns modify the proteome of legionella-containing vacuoles and restrict infection via irg1-derived itaconic acid a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform moxifloxacin is not anti-inflammatory in experimental pneumococcal pneumonia. the journal of antimicrobial chemotherapy protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein flagellin-deficient legionella mutants evade caspase-1-and naip5-mediated macrophage immunity differential roles of cd14 and toll-like receptors 4 and 2 in murine acinetobacter pneumonia. american journal of respiratory and critical care medicine correlation of klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model intubation-mediated intratracheal (imit) instillation: a noninvasive, lung-specific delivery system distinct contributions of neutrophils and ccr2+ monocytes to pulmonary clearance of different klebsiella pneumoniae strains rapid generation of a mouse model for middle east respiratory syndrome streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines. microbiological reviews lipoteichoic acid synthesis and function in gram-positive bacteria. annual review of microbiology role of lipoteichoic acid in infection and inflammation. the lancet infectious diseases interaction between the legionnaires' disease bacterium (legionella pneumophila) and human alveolar macrophages. influence of antibody, lymphokines, and hydrocortisone legionella pneumophila pathogenesis and immunity. seminars in pediatric infectious diseases early recruitment of neutrophils determines subsequent t1/t2 host responses in a murine model of legionella pneumophila pneumonia immunostimulation with macrophage-activating lipopeptide-2 increased survival in murine pneumonia american journal of physiology lung cellular and molecular physiology emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs. american journal of respiratory and critical care medicine differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels avian flu: influenza virus receptors in the human airway influenza virus-induced lung injury: pathogenesis and implications for treatment dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv. critical reviews in clinical laboratory sciences attenuation of immunemediated influenza pneumonia by targeting the inducible co-stimulator (icos) molecule on t cells rnai-mediated suppression of constitutive pulmonary gene expression by small interfering rna in mice moraxella catarrhalis induces an immune response in the murine lung that is independent of human ceacam5 expression and longterm smoke exposure. american journal of physiology lung cellular and molecular physiology multiple myd88-dependent responses contribute to pulmonary clearance of legionella pneumophila chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses surface proteins and exotoxins are required for the pathogenesis of staphylococcus aureus pneumonia cell-specific interleukin-15 and interleukin-15 receptor 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, activation, and function of different effector cells has been studied extensively [68] [69] [70] [71] . lung function and organ dysfunctions in 178 patients requiring mechanical ventilation during the 2009 influenza a (h1n1) pandemic characterization of the human cd8(+) t cell response following infection with 2009 pandemic influenza h1n1 virus gm-csf in the lung protects against lethal influenza infection gm-csf modulates pulmonary resistance to influenza a infection alveolar epithelial cells orchestrate dc function in murine viral pneumonia delivery of gm-csf to protect against influenza pneumonia hsv-1 infected cell proteins influence tetracycline-regulated transgene expression effects of influenza a virus on human neutrophil calcium metabolism. in: pavlotsky and a i tauber information about subscribing to the journal of immunology is online at : metabolism pulse-oximetry accurately predicts lung pathology and the immune response during influenza infection comparative study of three flexivent system configurations using mechanical test loads sp-r210 (myo18a) isoforms as intrinsic modulators of macrophage priming and activation how to map billions of short reads onto genomes transcript assembly and quantification by rna-seq reveals unannotated transcripts and isoform switching during cell differentiation differential expression analysis for sequence count data the tetracycline-responsive promoter contains functional interferon-inducible response elements influenza causes prolonged disruption of the alveolar-capillary barrier in mice unresponsive to mesenchymal stem cell therapy exponential analysis of the pressure-volume curve. correlation with mean linear intercept and emphysema in human lungs measuring the lung function in the mouse: the challenge of size changes in the mechanical properties of the respiratory system during the development of interstitial lung edema the relative balance of gm-csf and tgf-β1 regulates lung epithelial barrier function alveolar macrophages prevent lethal influenza pneumonia by inhibiting infection of type-1 alveolar epithelial cells lethal influenza infection: is a macrophage to blame? pyogenic bacterial infections in humans with myd88 deficiency. science (80-) hyporeactivity of alveolar macrophages and higher respiratory cell permissivity alveolar macrophages are essential for protection from respiratory failure and associated morbidity following influenza virus infection transient ablation of alveolar macrophages leads to massive pathology of influenza infection without affecting cellular adaptive immunity alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs □ depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed tnf-related apoptosis-inducing ligand tissue macrophage proliferation gm-csf regulates alveolar macrophage differentiation and innate immunity in the lung through novel markers to delineate murine m1 and m2 macrophages novel markers to delineate murine m1 and m2 m1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza -primed macrophages present similar resolving but distinct inflammatory lipid mediator signatures defining gm-csf-and macrophage-csf-dependent macrophage responses by in vitro models polarization profiles of human m-csf-generated macrophages and comparison of m1-markers in classically activated macrophages from gm-csf and m-csf origin alveolar macrophages of gm-csf knockout mice exhibit mixed m1 and m2 phenotypes macrophage polarization : tumor-associated macrophages as a paradigm for polarized m2 mononuclear phagocytes mphage_m1-m2_rev_jci2012 the m1 and m2 paradigm of macrophage activation: time for reassessment the role of macrophage polarization in infectious and inflammatory diseases the role of alpha / beta and gamma interferons in development of immunity to influenza a virus in mice rapid interferon g -dependent clearance of influenza a virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection restoring cigarette smoke-induced impairment of efferocytosis in alveolar macrophages interleukin-3, granulocytemacrophage colony-stimulating factor, and interleukin-5 transduce signals through two forms of stat5 a novel tnf receptor-associated factor 6 binding domain mediates nf-kappa b signaling by the common cytokine receptor beta subunit traf6 is required for the gm-csf-induced jnk, p38 and akt activation tumor necrosis factor receptor associated factor 6 (traf6) regulation of development, function, and homeostasis of the immune system tripartite motif 24 (trim24/tif1α) tumor suppressor protein is a novel negative regulator of interferon (ifn)/signal transducers and activators of transcription (stat) signaling pathway acting through retinoic acid receptor α (rarα) inhibition regulation of p53: trim24 enters the ring traf6 restricts p53 mitochondrial translocation, apoptosis, and tumor suppression implication of inflammatory macrophages, nuclear receptors, and interferon regulatory factors in increased virulence of pandemic 2009 h1n1 influenza a virus after host adaptation trim16 acts as an e3 ubiquitin ligase and can heterodimerize with other trim family members requirement for macrophage elastase for cigarette smoke -induced emphysema in mice macrophagespecific metalloelastase ( mmp-12 ) truncates and inactivates elr ϩ cxc chemokines and generates ccl2, −7, −8, and −13 antagonists : potential role of the macrophage in terminating polymorphonuclear leukocyte influx a new transcriptional role for matrix metalloproteinase-12 in antiviral immunity macrophage matrix metalloproteinase-12 dampens inflammation and neutrophil influx in arthritis 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 treatment option. interfering with these pathological pathways constitutes a valuable approach, with the advantage that it is independent of the appearance of drug resistant viruses. targeting exacerbated inflammatory responses, such as those triggered by ic activity leading to inflammasome activation in various respiratory infections, may reduce disease worsening and progression [23] . novel specific inhibitors of nlrp3 inflammasome showing promising results for inflammatory diseases could be applied for these purposes [119] . viroporins have been known for a long time as key contributors to virus propagation and stimulators of pathogenesis. several reports have dissected the crucial impact that ic activity has in these processes. it is remarkable how these small channels have high implications at the cell level favoring virus production and, at the same time, causing disturbances at a tissue or organism level eventually leading to pathology. understanding the molecular and physicochemical structure of these ion pores may constitute the basis for rational design of specific ic activity inhibitors and strategies to counteract the pathology mediated by ic activity. ion homeostasis, channels, and transporters: an update on cellular mechanisms viroporins: structure and biological functions the coxsackievirus 2b protein increases efflux of ions from the endoplasmic reticulum and golgi, thereby inhibiting protein trafficking through the golgi the vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels the proapoptotic influenza a virus protein pb1-f2 forms a nonselective ion channel the hepatitis c virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives influenza virus m2 protein has ion channel activity sars coronavirus e protein forms cation-selective ion channels mers coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels coronavirus e protein forms ion channels with functionally and structurally-involved membrane lipids analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge intracellular proton conductance of the hepatitis c virus p7 protein and its contribution to infectious virus production hepatitis c virus p7-a viroporin crucial for virus assembly and an emerging target for antiviral therapy viroporins from rna viruses induce caspase-dependent apoptosis rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection influenza virus activates inflammasomes via its intracellular m2 ion channel encephalomyocarditis virus viroporin 2b activates nlrp3 inflammasome human respiratory syncytial virus viroporin sh: a viral recognition pathway used by the host to signal inflammasome activation rhinovirus-induced calcium flux triggers nlrp3 and nlrc5 activation in bronchial cells activation of the nlrp3 inflammasome by iav virulence protein pb1-f2 contributes to severe pathophysiology and disease porcine reproductive and respiratory syndrome virus activates inflammasomes of porcine alveolar macrophages via its small envelope protein e severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis ion flux in the lung: virus-induced inflammasome activation pedv orf3 encodes an ion channel protein and regulates virus production the orf4a protein of human coronavirus 229e functions as a viroporin that regulates viral production severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release structure and mechanism of proton transport through the transmembrane tetrameric m2 protein bundle of the influenza a virus viroporin-mediated membrane permeabilization. pore formation by nonstructural poliovirus 2b protein a potassium channel protein encoded by chlorella virus pbcv-1 in vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel kcv structural flexibility of the pentameric sars coronavirus envelope protein ion channel structure and inhibition of the sars coronavirus envelope protein ion channel three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (vpu) from hiv-1 structure and ion channel activity of the human respiratory syncytial virus (hrsv) small hydrophobic protein transmembrane domain the 3-dimensional structure of a hepatitis c virus p7 ion channel by electron microscopy high-risk human papillomavirus e5 oncoprotein displays channel-forming activity sensitive to small-molecule inhibitors reconstitution of the influenza virus m2 ion channel in lipid bilayers correlation of the structural and functional domains in the membrane protein vpu from hiv-1 direct measurement of the influenza a virus m2 protein ion channel activity in mammalian cells proton conductance of influenza virus m2 protein in planar lipid bilayers definitive assignment of proton selectivity and attoampere unitary current to the m2 ion channel protein of influenza a virus cation-selective ion channels formed by p7 of hepatitis c virus are blocked by hexamethylene amiloride inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment syringomycin e channel: a lipidic pore stabilized by lipopeptide? effect of lipids with different spontaneous curvature on the channel activity of colicin e1: evidence in favor of a toroidal pore activation of the m2 ion channel of influenza virus: a role for the transmembrane domain histidine residue the e5 oncoprotein of bovine papillomavirus binds to a 16 kda cellular protein atpase mutants transform cells and define a binding site for the papillomavirus e5 oncoprotein subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein na(+)/k (+)-atpase beta1 subunit interacts with m2 proteins of influenza a and b viruses and affects the virus replication function of fxyd proteins, regulators of na, k-atpase phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump the gamma subunit of the na, k-atpase induces cation channel activity mutual functional destruction of hiv-1 vpu and host task-1 channel influenza virus m2 protein inhibits epithelial sodium channels by increasing reactive oxygen species sars-cov proteins decrease levels and activity of human enac via activation of distinct pkc isoforms hepatitis c virus p7 protein is crucial for assembly and release of infectious virions generation of recombinant influenza a virus without m2 ion-channel protein by introduction of a point mutation at the 5 1 end of the viral intron a novel gene of hiv-1, vpu, and its 16-kilodalton product generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo the small envelope protein e is not essential for murine coronavirus replication inhibition of sars-cov replication cycle by small interference rnas silencing specific sars proteins, 7a/7b, 3a/3b and s recombinant respiratory syncytial virus from which the entire sh gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse recombinant respiratory syncytial virus bearing a deletion of either the ns2 or sh gene is attenuated in chimpanzees the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis the influenza virus m2 protein cytoplasmic tail interacts with the m1 protein and influences virus assembly at the site of virus budding influenza virus m2 protein mediates escrt-independent membrane scission tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu the interferon-induced protein bst-2 restricts hiv-1 release and is downregulated from the cell surface by the viral vpu protein ion channel activity of hiv-1 vpu is dispensable for counteraction of cd317 influenza a virus m2 ion channel activity is essential for efficient replication in tissue culture influenza a virus can undergo multiple cycles of replication without m2 ion channel activity identification of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from hiv-1-infected cells influence of amantadine resistance mutations on the ph regulatory function of the m2 protein of influenza a viruses hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication influenza a virus m2 protein: monoclonal antibody restriction of virus growth and detection of m2 in virions membrane topology of coronavirus e protein characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e role of virion m2 protein in influenza virus uncoating: specific reduction in the rate of membrane fusion between virus and liposomes by amantadine structure of influenza haemagglutinin at the ph of membrane fusion stepwise priming by acidic ph and a high k+ concentration is required for efficient uncoating of influenza a virus cores after penetration four na+/h+ exchanger isoforms are distributed to golgi and post-golgi compartments and are involved in organelle ph regulation the ion channel activity of the influenza virus m2 protein affects transport through the golgi apparatus a single polar residue and distinct membrane topologies impact the function of the infectious bronchitis coronavirus e protein functional analysis of picornavirus 2b proteins: effects on calcium homeostasis and intracellular protein trafficking the open reading frame 3a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-beta signaling is required for rotavirus replication a view of acidic intracellular compartments influenza virus m2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport influenza a virus lacking m2 protein as a live attenuated vaccine classical swine fever virus p7 protein is a viroporin involved in virulence in swine severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis inhibition of nf-kappab mediated inflammation in severe acute respiratory syndome coronavirus-infected mice increases survival sars coronaviruses with mutations in e protein are attenuated and promising vaccine candidates potential enhancement of osteoclastogenesis by severe acute respiratory syndrome coronavirus 3a/x1 protein augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/x1 and 7a/x4 proteins through nf-kappab activation sars coronavirus accessory proteins in vivo functional characterization of the sars-coronavirus 3a protein in drosophila coxsackievirus b3 proteins directionally complement each other to downregulate surface major histocompatibility complex class i the acute respiratory distress syndrome: pathogenesis and treatment proinflammatory activity in bronchoalveolar lavage fluids from patients with ards, a prominent role for interleukin-1 contribution of neutrophils to acute lung injury the inflammasome in lung diseases inflammasomes in health and disease critical role for calcium mobilization in activation of the nlrp3 inflammasome k(+) efflux is the common trigger of nlrp3 inflammasome activation by bacterial toxins and particulate matter initiation and perpetuation of nlrp3 inflammasome activation and assembly hepatitis c virus induces interleukin-1beta (il-1beta)/il-18 in circulatory and resident liver macrophages the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug, amantadine antivirals for the treatment and prevention of epidemic and pandemic influenza. influenza other respir amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv-1 protein vpu structure of the amantadine binding site of influenza m2 proton channels in lipid bilayers combination chemotherapy for influenza genotype-dependent sensitivity of hepatitis c virus to inhibitors of the p7 ion channel a novel hepatitis c virus p7 ion channel inhibitor, bit225, inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-alpha-2b and nucleoside analogues a 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 review global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial viral infection in children with compromised immune function respiratory syncytial virus and influenza a infections in the hospitalized elderly respiratory syncytial virus infection in elderly and high-risk adults influenza-and respiratory syncytial virus-associated mortality and hospitalisations epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children < 5 years old parainfluenza viruses estimates of us influenza-associated deaths made using four different methods. influenza other respir viruses global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study viruses and bacteria in the etiology of the common cold human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children clinical disease in children associated with newly described coronavirus subtypes a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia risk of primary infection and reinfection with respiratory syncytial virus the role of human metapneumovirus in upper respiratory tract infec tions in children: a 20-year experience prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness changes in iga and igg concentrations in nasal secretions prior to the appearance of antibody during viral respiratory infection in man impaired antibody-mediated protection and defective iga b-cell memory in experimental infection of adults with respiratory syncytial virus antibody and th1-type cell-mediated immune responses in elderly and young adults immunized with the standard or a high dose influenza vaccine influenza nucleoprotein-specific cytotoxic t-cell clones are protective in vivo role of t lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice mapping and characterization of the primary and anamnestic h-2d-restricted cytotoxic t-lymphocyte response in mice against human meta pneumovirus lung airway-surveilling cxcr3(hi) memory cd8+ t cells are critical for protection against influenza a virus virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection memory cd8 t cells mediate severe immunopathology following respiratory syncytial virus infection central role of dendritic cells in shaping the adaptive immune response during respiratory syncytial virus infection differential roles of lung dendritic cell subsets against respiratory virus infection the effector t cell response to influenza infection visualization and characterization of respiratory syncytial virus f-specific cd8(+) t cells during experimental virus infection characterization of the cd8+ t cell responses directed against respiratory syncytial virus during primary and secondary infection in c57bl/6 mice regulatory t cells promote early influx of cd8+ t cells in the lungs of respiratory syncytial virus-infected mice and diminish immunodominance disparities the pulmonary localization of virus-specific t lymphocytes is governed by the tissue tropism of infection virus-specific cd8+ t cells in primary and secondary influenza pneumonia respiratory syncytial virus-specific cd8+ memory t cell responses in elderly persons nonspecific recruitment of memory cd8+ t cells to the lung airways during respiratory virus infections the chemokine receptor ccr5 plays a key role in the early memory cd8+ t cell response to respiratory virus infections airway-resident memory cd8 t cells provide antigen-specific protection against respiratory virus challenge through rapid ifn-gamma production cutting edge: effector memory cd8+ t cells in the lung airways retain the potential to mediate recall responses type i interferons regulate cytolytic activity of memory cd8+ t cells in the lung airways during respiratory virus challenge the who's who of t-cell differentiation: human memory t-cell subsets memory t cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus lung niches for the generation and maintenance of tissue-resident memory t cells lung-resident memory cd8 t cells (trm) are indispensable for optimal cross-protection against pulmonary virus infection specific niches for lung-resident memory cd8+ t cells at the site of tissue regeneration enable cd69-independent maintenance long-term persistence and reactivation of t cell memory in the lung of mice infected with respiratory syncytial virus long-term maintenance of virus-specific effector memory cd8+ t cells in the lung airways depends on proliferation memory t cell populations in the lung airways are maintained by continual recruitment cutting edge: antigen is not required for the activation and maintenance of virus-specific memory cd8+ t cells in the lung airways in situ imaging reveals different responses by naive and memory cd8 t cells to late antigen presentation by lymph node dc after influenza virus infection smad4 promotes differentiation of effector and circulating memory cd8 t cells but is dispensable for tissue-resident memory cd8 t cells environmental and antigen receptor-derived signals support sustained surveillance of the lungs by pathogen-specific cytotoxic t lymphocytes enhanced survival of lung tissue-resident memory cd8+ t cells during infection with influenza virus due to selective expression of ifitm3 airway t cells protect against rsv infection in the absence of antibody vaccine-generated lung tissue-resident memory t cells provide heterosubtypic protection to influenza infection intranasal administration of rsv antigen-expressing mcmv elicits robust tissue-resident effector and effector memory cd8+ t cells in the lung reduced generation of lung tissue-resident memory t cells during infancy dynamics of influenza-induced lung-resident memory t cells underlie waning heterosubtypic immunity in vivo effector function of influenza virus-specific cytotoxic t lymphocyte clones is highly specific cytotoxic t cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus cd4+ t cells clear virus but augment disease in mice infected with respiratory syncytial virus. comparison with the effects of cd8+ t cells distinct types of lung disease caused by functional subsets of antiviral t cells the recovery of mice from influenza virus infection: adoptive transfer of immunity with immune t lymphocytes transfer of specific cytotoxic t lymphocytes protects mice inoculated with influenza virus recovery from a viral respiratory infection. ii. passive transfer of immune spleen cells to mice with influenza pneumonia clearance of persistent respiratory syncytial virus infections in immunodeficient mice following trans fer of primed t cells virus clearance and immunopathology by cd8(+) t cells during infection with respiratory syncytial virus are mediated by ifn-gamma cytotoxic lymphocytes in the lungs of mice infected with respiratory syncytial virus cellular response to respiratory viruses with particular reference to children with disorders of cell-mediated immunity quantitative effects of palivizumab and donor-derived t cells on chronic respiratory syncytial virus infection, lung disease, and fusion glycoprotein amino acid sequences in a patient before and after bone marrow transplantation cytotoxic t-cell immunity to influenza recovery from severe h7n9 disease is associated with diverse response mechanisms dominated by cd8(+) t cells cd8+ t cell effector mechanisms in resistance to infection cd8+ t cells clear influenza virus by perforin or fas-dependent processes multiple redundant effector mechanisms of cd8+ t cells protect against influenza infection role of tumor necrosis factor-related apoptosis-inducing ligand in immune response to influenza virus infection in mice cd8 t cells utilize trail to control influenza virus infection alternative mechanisms of respira tory syncytial virus clearance in perforin knockout mice lead to enhanced disease prolonged production of tnf-alpha exacerbates illness during respiratory syncytial virus infection inhibition of tumor necrosis factor reduces the severity of virus-specific lung immunopathology role of t cells in virus control and disease after infection with pneumonia virus of mice production of interferon-gamma by influenza hemagglutinin-specific cd8 effector t cells influences the development of pulmonary immunopathology the role of ifn in respiratory syncytial virus pathogenesis recombinant baculovirus-based vaccine expressing m2 protein induces protective cd8+ t-cell immunity against respiratory syncytial virus infection lung cd8+ t cell impairment occurs during human metapneumovirus infection despite virus-like particle induction of functional cd8+ t cells cytotoxic t-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice vaccine-elicited cd8+ t cells protect against respiratory syncytial virus strain a2-line19f-induced pathogenesis in balb/c mice induction of partial specific heterotypic immunity in mice by a single infection with influenza a virus heterosubtypic immunity to influenza type a virus in mice. effector mechanisms and their longevity profound protection against respiratory challenge with a lethal h7n7 influenza a virus by increasing the magnitude of cd8+ t-cell memory protective cross-reactive cellular immunity to lethal a/goose/guangdong/1/96-like h5n1 influenza virus is correlated with the proportion of pulmonary cd8+ t cells expressing gamma interferon cross-reactive t cells are involved in rapid clearance of 2009 pandemic h1n1 influenza virus in nonhuman primates natural t cell-mediated protection against seasonal and pandemic influenza. results of the flu watch cohort study natural reinfection with respiratory syncytial virus does not boost virus-specific t-cell immunity transgenic mice lacking class i major histocompatibility complex-restricted t cells have delayed viral clearance and increased mortality after influenza virus challenge the chemokine mip1alpha/ccl3 determines pathology in primary rsv infection by regulating the balance of t cell populations in the murine lung animal model of respiratory syncytial virus: cd8+ t cells cause a cytokine storm that is chemically tractable by sphingosine-1-phosphate 1 receptor agonist therapy activation of the granzyme pathway in children with severe respiratory syncytial virus infection severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses respiratory syncytial virus and influenza virus infections: observations from tissues of fatal infant cases whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection respiratory syncytial virus infection suppresses lung cd8+ t-cell effector activity and peripheral cd8+ t-cell memory in the respiratory tract functional impairment of cytotoxic t cells in the lung airways following respiratory virus infections impairment of the cd8+ t cell response in lungs following infection with human respiratory syncytial virus is specific to the anatomical site rather than the virus, antigen, or route of infection regulation of cytokine production by virus-specific cd8 t cells in the lungs foxp3+ cd4 regulatory t cells limit pulmonary immunopathology by modulating the cd8 t cell response during respiratory syncytial virus infection influenza a virus infection results in a robust, antigen-responsive, and widely disseminated foxp3+ regulatory t cell response antigen-specific memory regulatory cd4+foxp3+ t cells control memory responses to influenza virus infection epitope-specific regulatory cd4 t cells reduce virus-induced illness while preserving cd8 t-cell effector function at the site of infection effector t cells control lung inflammation during acute influenza virus infection by producing il-10 multiple cd4+ t cell subsets produce immunomodulatory il-10 during respiratory syncytial virus infection il-10 regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice cd8+ treg cells suppress cd8+ t cell-responses by il-10-dependent mechanism during h5n1 influenza virus infection local blockade of epithelial pdl-1 in the airways enhances t cell function and viral clearance during influenza virus 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 : expression of cytokines, chemokines and antiviral genes in wdnhbe cells in response to pandemic influenza infection, table s4 : gene copy numbers for iav h1n1pdm09 vs. mock-infected wdnhbe cells, figure s8 : poly(i:c) stimulation of innate immune response genes in wdnhbe cells, figure s9 : the basolateral secretion of cytokines and chemokines by wdnhbe cells in response to infection with iav h1n1pdm09, figure s10 global rise in human infectious disease outbreaks china novel coronavirus investigating research team, a novel coronavirus from patients with pneumonia in china preparedness for a high.-impact respiratory pathogen pandemic a world at risk: annual report on global preparedness for health emergencies; world health organization spillover and pandemic properties of zoonotic viruses with high host plasticity the characteristics of pandemic pathogens characteristics of microbes most likely to cause pandemics and global catastrophes world health organization, pandemic influenza risk management: a who guide to inform and harmonize national and international pandemic preparedness and response; world health organization back to the future: lessons learned from the 1918 influenza pandemic estimation of potential global pandemic influenza mortality on the basis of vital registry data from the 1918-20 pandemic: a quantitative analysis influenza virus: a master tactician in innate immune evasion and novel therapeutic interventions in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses onward transmission of viruses: how do viruses emerge to cause epidemics after spillover? re)emergence of a(h1n1)pdm09 influenza viruses with pandemic markers in the 2018/2019 flu season in the usa emerging respiratory infections: the infectious disease pathology of sars, mers, pandemic influenza, and legionella influenza-induced innate immunity: regulators of viral replication, respiratory tract pathology & adaptive immunity role of the iinnate cytokine storm induced by the influenza a virus new fronts emerge in the influenza cytokine storm die another way: interplay between influenza a virus, inflammation and cell death estimates of global seasonal influenza-associated respiratory mortality: a modelling study regulation of influenza a virus induced cxcl-10 gene expression requires pi3k/akt pathway and irf3 transcription factor optimization of normal human bronchial epithelial (nhbe) cell 3d cultures for in vitro lung model studies barrier function of airway tract epithelium. tissue barriers the airway epithelium: soldier in the fight against respiratory viruses respiratory barrier as a safeguard and regulator of defense against influenza a virus and streptococcus pneumoniae beyond inflammation: airway epithelial cells are at the interface of innate and adaptive immunity parallel activities and interactions between antimicrobial peptides and complement in host defense at the airway epithelial surface control of local immunity by airway epithelial cells epithelium dysfunction in asthma role of airway epithelial barrier dysfunction in pathogenesis of asthma influenza virus-induced lung injury: pathogenesis and implications for treatment tight junctions in pulmonary epithelia during lung inflammation respiratory epithelial cells orchestrate pulmonary innate immunity tight junction proteins and signaling pathways in cancer and inflammation: a functional crosstalk tight junctions as regulators of tissue remodelling architecture of tight junctions and principles of molecular composition epithelial barrier assembly requires coordinated activity of multiple domains of the tight junction protein zo-1 influence of the scaffolding protein zonula occludens (zos) on membrane channels the multifarious regulation of the apical junctional complex the tight junction protein zo-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton stem cell-based lung-on-chips: the best of both worlds? reconstituting organ-level lung functions on a chip small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro long-term expanding human airway organoids for disease modeling well-differentiated human airway epithelial cell cultures the air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia human airway primary epithelial cells show distinct architectures on membrane supports under different culture conditions mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells differentiated human airway organoids to assess infectivity of emerging influenza virus avian influenza viruses infect primary human bronchial epithelial cells unconstrained by sialic acid a2,3 residues host protective immune responses against influenza a virus infection host-virus interaction: how host cells defend against influenza a virus infection innate immunity to influenza virus infection the host immune response in respiratory virus infection: balancing virus clearance and immunopathology proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells tropism and innate host responses of the 2009 pandemic h1n1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract validation of normal human bronchial epithelial cells as a model for influenza a infections in human distal trachea human and avian influenza viruses target different cell types in cultures of human airway epithelium early host responses of seasonal and pandemic influenza a viruses in primary well-differentiated human lung epithelial cells relative respiratory syncytial virus cytopathogenesis in upper and lower respiratory tract epithelium in vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo rhinovirus infection interferes with induction of tolerance to aeroantigens through ox40 ligand, thymic stromal lymphopoietin, and il-33 tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs comprehensive evaluation of poly(i:c) induced inflammatory response in an airway epithelial model polyinosinic:polycytidylic acid induces protein kinase d-dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells expression of influenza a virus internal antigens on the surface of infected p815 cells a simple method of estimating fifty per cent endpoints teer measurement techniques for in vitro barrier model systems circulating micrornas: understanding the limits for quantitative measurement by real-time pcr co-expression networks for plant biology: why and how the igraph software package for complex network research r: a language and environment for statistical computing transepithelial/endothelial electrical resistance (teer) theory and applications for microfluidic body-on-a-chip devices human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals infection of human airway epithelium by human and avian strains of influenza a virus influenza h5n1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells rhinovirus disrupts the barrier function of polarized airway epithelial cells programmed cell death in the pathogenesis of influenza the induction and consequences of influenza a virus-induced cell death a quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (tnf) activity detection of necrosis by release of lactate dehydrogenase activity tight junctions at a glance slippery when wet: airway surface liquid homeostasis and mucus hydration the interaction between respiratory pathogens and mucus muc5b is required for airway defence the frontline defence of the lung clinical aspects and cytokine response in severe h1n1 influenza a virus infection influenza h5n1 and h1n1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation intensive cytokine induction in pandemic h1n1 influenza virus infection accompanied by robust production of il-10 and il-6 influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions cytokine responses in patients with mild or severe influenza a(h1n1)pdm09 inflammatory responses in influenza a virus infection the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts genenames.org: the hgnc and vgnc resources in 2019 rhinovirus-induced barrier dysfunction in polarized airway epithelial cells is mediated by nadph oxidase 1 innate immune recognition in infectious and noninfectious diseases of the lung innate immunity in the lung: how epithelial cells fight against respiratory pathogens collectins and cationic antimicrobial peptides of the respiratory epithelia structure and function of the polymeric mucins in airways mucus influenza a induces the major secreted airway mucin muc5ac in a protease-egfr-extracellular regulated kinase-sp1-dependent pathway a review of inflammatory mechanism in airway diseases respiratory epithelial cells as master communicators during viral infections airway epithelial differentiation and mucociliary clearance identification and functions of pattern-recognition receptors pattern recognition receptors and inflammation toll-like receptors and their crosstalk with other innate receptors in infection and immunity differential roles of mda5 and rig-i helicases in the recognition of rna viruses influenza virus activation of the interferon system the innate immune function of airway epithelial cells in inflammatory lung disease respiratory epithelial cells in innate immunity to influenza virus infection a protein-interaction network of interferon-stimulated genes extends the innate immune system landscape interferon-stimulated genes: a complex web of host defenses induction and evasion of type i interferon responses by influenza viruses interferon-stimulated genes: what do they all do? delayed clearance of viral load and marked cytokine activation in severe cases of pandemic h1n1 2009 influenza virus infection cytokine and chemokine profiles in lung tissues from fatal cases of 2009 pandemic influenza a (h1n1): role of the host immune response in pathogenesis th1 and th17 hypercytokinemia as early host response signature in severe pandemic influenza integrated analysis of microrna-mrna expression in a549 cells infected with influenza a viruses (iavs) from different host species ifn-gamma-inducible protein 10 (cxcl10) contributes to airway hyperreactivity and airway inflammation in a mouse model of asthma cxcl10/ip-10 in infectious diseases pathogenesis and potential therapeutic implications synergistic up-regulation of cxcl10 by virus and ifn gamma in human airway epithelial cells innate immune response to h3n2 and h1n1 influenza virus infection in a human lung organ culture model human airway epithelial cells produce ip-10 (cxcl10) in vitro and in vivo upon rhinovirus infection modulating the innate immune response to influenza a virus: potential therapeutic use of anti-inflammatory drugs antibody and inflammatory response-mediated severity of pandemic 2009 (ph1n1) influenza virus cytokine 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 using a two-step small interfering rna screen genome-wide rnai screen identifies human host factors crucial for influenza virus replication differentiated human airway organoids to assess infectivity of emerging influenza virus n-(p-amylcinnamoyl)anthranilic acid (aca): a phospholipase a2 inhibitor and trp channel blocker anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects influenza virus-host interactome screen as a platform for antiviral drug development adaptors for clathrin coats: structure and function inhibition of the receptor-binding function of clathrin adaptor protein ap-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis tyrphostin a23 inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the ap-2 adaptor complex reticulon 3 binds the 2c protein of enterovirus 71 and is required for viral replication clathrin adaptor ap-2 is essential for early embryonal development ap-2-associated protein kinase 1 and cyclin g-associated kinase regulate hepatitis c virus entry and are potential drug targets identification and targeting of an interaction between a tyrosine motif within hepatitis c virus core protein and ap2m1 essential for viral assembly flik: a direct-binding assay for the identification and kinetic characterization of stabilizers of inactive kinase conformations molecular architecture and functional model of the endocytic ap2 complex human induced pluripotent cell-derived sensory neurons for fate commitment of bone marrow-derived schwann cells: implications for remyelination therapy comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 47 + cd4 + effector/ effector memory t cells differentiate into productively and latently infected central memory t cells by transforming growth factor 1 during hiv-1 infection srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target talaromyces marneffei mp1p is a virulence factor that binds and sequesters a key proinflammatory lipid to dampen host innate immune response a tricyclic pyrrolobenzodiazepine produced by klebsiella oxytoca is associated with cytotoxicity in antibiotic-associated hemorrhagic colitis and nanog is essential for oncogenic viral flice-inhibitory protein-induced acetylation of p65/rela, nf-b activation, and promotion of cell invasion and angiogenesis human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus the celecoxib derivative kinase inhibitor ar-12 (osu-03012) inhibits zika virus via down-regulation of the pi3k/akt pathway and protects zika virus-infected a129 mice: a host-targeting 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 could be used to better understand host factors affecting viral replication and disease severity and to develop new and more effective therapeutic strategies against viral infections. modeling the intracellular dynamics of influenza virus replication to understand the control of viral rna synthesis temperature sensitive mutations in influenza a viral ribonucleoprotein complex responsible for the attenuation of the live attenuated influenza vaccine the structure of native influenza virion ribonucleoproteins structure of influenza a polymerase bound to the viral rna promoter structural insights into rna synthesis by the influenza virus transcription-replication machine the influenza virus rna synthesis machine: advances in its structure and function downregulating viral gene expression: codon usage bias manipulation for the generation of novel influenza a virus vaccines a complicated message: identification of a novel pb1-related protein translated from influenza a virus segment 2 mrna an overlapping protein-coding region in influenza a virus segment 3 modulates the host response the biology of influenza viruses the interplay between the host receptor and influenza virus hemagglutinin and neuraminidase molecular evolution of hemagglutinin gene of influenza a virus the role of receptor binding specificity in interspecies transmission of influenza viruses influenza virus neuraminidase structure and functions functional balance between neuraminidase and haemagglutinin in influenza viruses influenza virus neuraminidase (na): a target for antivirals and vaccines the influenza virus m2 protein cytoplasmic tail interacts with the m1 protein and influences virus assembly at the site of virus budding influenza virus assembly and budding influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles assembly and budding of influenza virus expression and analysis of the ns2 protein of influenza a virus molecular assembly of influenza virus: association of the ns2 protein with virion matrix ns2 protein of influenza virus is found in purified virus and phosphorylated in infected cells emerging roles for the influenza a virus nuclear export protein (nep) modulation of innate immune responses by the influenza a ns1 and pa-x proteins interplay of pa-x and ns1 proteins in replication and pathogenesis of a temperature-sensitive 2009 pandemic h1n1 influenza a virus twenty amino acids at the c-terminus of pa-x are associated with increased influenza a virus replication and pathogenicity pa-x-associated early alleviation of the acute lung injury contributes to the attenuation of a highly pathogenic h5n1 avian influenza virus in mice pa-x decreases the pathogenicity of highly pathogenic h5n1 influenza a virus in avian species by inhibiting virus replication and host response selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein epidemiological, antigenic and genetic characteristics of seasonal influenza a(h1n1), a(h3n2) and b influenza viruses: basis for the who recommendation on the composition of influenza vaccines for use in the 2009-2010 northern hemisphere season prevention and control of seasonal influenza with vaccines the annual impact of seasonal influenza in the us: measuring disease burden and costs the continual threat of influenza virus infections at the human-animal interface: what is new from a one health perspective? influenza virus reservoirs and intermediate hosts: dogs, horses, and new possibilities for influenza virus exposure of humans new world bats harbor diverse influenza a viruses evolution and ecology of influenza a viruses antigenicity of the 2015-2016 seasonal h1n1 human influenza virus ha and na proteins prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic competitive detection of influenza neutralizing antibodies using a novel bivalent fluorescence-based microneutralization assay (bifma) reverse genetics approaches for the development of influenza vaccines inactivated influenza virus vaccines: the future of tiv and qiv oseltamivir, zanamivir and amantadine in the prevention of influenza: a systematic review novel approaches for the development of live attenuated influenza vaccines host genetic determinants of influenza pathogenicity an overview of the epidemiology and emergence of influenza a infection in humans over time h5n1, a wealth of knowledge to improve pandemic preparedness innate immunity to influenza virus infection synthetic toll-like receptor 4 (tlr4) and tlr7 ligands work additively via myd88 to induce protective antiviral immunity in mice standing on three legs: antiviral activities of rig-i against influenza viruses the role of the nlrp3 inflammasome in regulation of antiviral responses to influenza a virus infection aim2 inflammasome is critical for influenza-induced lung injury and mortality influenza viruses control the vertebrate type i interferon system: factors, mechanisms, and consequences interferon-lambda in the context of viral infections: production, response and therapeutic implications molecular pathways in virus-induced cytokine production. microbiol type i and type iii interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion induction and function of type i and iii interferon in response to viral infection from interferons to cytokines multiple functions of the ikk-related kinase ikkepsilon in interferon-mediated antiviral immunity maniatis, t. i kappa b kinase epsilon (ikk epsilon) regulates the balance between type i and type ii interferon responses interferons and viruses: an evolutionary arms race of molecular interactions role of natural killer cells in the generation of influenza virus-specific cytotoxic t cells t cell-dependent production of ifn-gamma by nk cells in response to influenza a virus evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice accelerated migration of respiratory dendritic cells to the regional lymph nodes is limited to the early phase of pulmonary infection lymph node dendritic cells control cd8+ t cell responses through regulated fasl expression antiviral b cell and t cell immunity in the lungs differential expression of nlrp3 among hematopoietic cells type i ifn triggers rig-i/tlr3/nlrp3-dependent inflammasome activation in influenza a virus infected cells the expanding role of nlrs in antiviral immunity the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna activation of the nlrp3 inflammasome by iav virulence protein pb1-f2 contributes to severe pathophysiology and disease influenza virus activates inflammasomes via its intracellular m2 ion channel complement evasion strategies of viruses: an overview. front c1q: structure, function, and receptors membrane complement regulatory proteins the role of c5a in acute lung injury induced by highly pathogenic viral infections detection of single nucleotide polymorphisms functional implications of single nucleotide polymorphisms (snps) in protein-coding and non-coding rna genes in multifactorial diseases single nucleotide polymorphisms (snps): functional implications of regulatory-snp (rsnp) and structural rna (srsnps) in complex diseases single nucleotide polymorphisms of toll-like receptors and susceptibility to infectious diseases the genomics and genetics of human infectious disease susceptibility meta-analysis of the relationship between single nucleotide polymorphism of il-10-1082g/a and rheumatic heart disease single nucleotide polymorphisms in dna repair genes and putative cancer risk the association between the ubqln1 polymorphism and alzheimer's disease risk: a systematic review the drd2 rs1076560 polymorphism and schizophrenia-related intermediate phenotypes: a systematic review and meta-analysis single-nucleotide polymorphism to associate cancer risk microrna single-nucleotide polymorphisms and diabetes mellitus: a comprehensive review genetic determinants of the inflammatory response cognitive dysfunction and single nucleotide polymorphisms in hepatitis c virus-infected persons: a systematic review genetic variation in pattern recognition receptors: functional consequences and susceptibility to infectious disease human genetic determinants of viral diseases host nucleotide polymorphism in hepatitis b virus-associated hepatocellular carcinoma role of some predominant host immunomodulators' single nucleotide polymorphisms in severity of hepatitis b virus and hepatitis c virus infection a missense mutation of the toll-like receptor 3 gene in a patient with influenza-associated encephalopathy toll-like receptor 3 gene polymorphisms and severity of pandemic a/h1n1/2009 influenza in otherwise healthy children ifitm3, tlr3, and cd55 gene snps and cumulative genetic risks for severe outcomes in chinese patients with h7n9/h1n1pdm09 influenza loss of tlr3 aggravates chikv replication and pathology due to an altered virus-specific neutralizing antibody response impaired intrinsic immunity to hsv-1 in human ipsc-derived tlr3-deficient cns cells human traf3 adaptor molecule deficiency leads to impaired toll-like receptor 3 response and susceptibility to herpes simplex encephalitis herpes simplex encephalitis in children with autosomal recessive and dominant trif deficiency heterozygous tbk1 mutations impair tlr3 immunity and underlie herpes simplex encephalitis of childhood association of toll-like receptor 3 single-nucleotide polymorphisms and hepatitis c virus infection toll-like receptor 3 polymorphism and its association with hepatitis b virus infection in saudi arabian patients defective rna sensing by rig-i in severe influenza virus infection irf-7 is the master regulator of type-i interferon-dependent immune responses differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7 ifn regulatory factor family members differentially regulate the expression of type iii ifn (ifn-lambda) genes infectious disease. life-threatening influenza and impaired interferon amplification in human irf7 deficiency polymorphisms in interferon regulatory factor 7 reduce interferon-alpha responses of plasmacytoid dendritic cells to hiv-1 life-threatening influenza pneumonitis in a child with inherited irf9 deficiency impaired control of multiple viral infections in a family with complete irf9 deficiency enhanced survival of lung tissue-resident memory cd8(+) t cells during infection with influenza virus due to selective expression of ifitm3 ifitm3 restricts the morbidity and mortality associated with influenza interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals interferon-inducible transmembrane protein 3 genetic variant rs12252 and influenza susceptibility and severity: a meta-analysis association between ifitm3 rs12252 polymorphism and influenza susceptibility and severity: a meta-analysis snp-mediated disruption of ctcf binding at the ifitm3 promoter is associated with risk of severe influenza in humans calculated risk: a new single-nucleotide polymorphism linked to severe influenza disease il-1: discoveries, controversies and future directions genetic variants in il1a and il1b contribute to the susceptibility to 2009 pandemic h1n1 influenza a virus allele-specific induction of il-1beta expression by c/ebpbeta and pu.1 contributes to increased tuberculosis susceptibility differential binding of proteins to the il1b -31 t/c polymorphism in lung epithelial cells il-1 beta enhances cd40 ligand-mediated cytokine secretion by human dendritic cells (dc): a mechanism for t cell-independent dc activation role of tumor necrosis factor gene single nucleotide polymorphisms in the natural course of 2009 influenza a h1n1 virus infection tnf, il6, and il1b polymorphisms are associated with severe influenza a (h1n1) virus infection in the mexican population association of single nucleotide polymorphisms in tnfa and ccr5 genes with japanese encephalitis: a study from an endemic region of north india a study of tnf-alpha-238 and -308 polymorphisms with different outcomes of persistent hepatitis b virus infection in china ccr5 deficiency increases risk of symptomatic west nile virus infection chemokine receptor 5 big up tri, open32 allele in patients with severe pandemic (h1n1) 2009. emerg resistance to hiv-1 infection in caucasian individuals bearing mutant alleles of the ccr-5 chemokine receptor gene a functional variation in cd55 increases the severity of 2009 pandemic h1n1 influenza a virus infection identification of complement-related host genetic risk factors associated with influenza a(h1n1)pdm09 outcome: challenges ahead characterization of the decay-accelerating factor gene promoter region genetic variants associated with severe pneumonia in a/h1n1 influenza infection soluble host defense lectins in innate immunity to influenza virus absence of sp-a modulates innate and adaptive defense responses to pulmonary influenza infection surfactant protein-a-deficient mice display an exaggerated early inflammatory response to a beta-resistant strain of influenza a virus mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins surfactant protein a genetic variants associate with severe respiratory insufficiency in pandemic influenza a virus infection galectin-1 binds to influenza virus and ameliorates influenza virus pathogenesis functional variants regulating lgals1 (galectin 1) expression affect human susceptibility to influenza a(h7n9) tmprss2 is essential for influenza h1n1 virus pathogenesis in mice identification of tmprss2 as a susceptibility gene for severe 2009 pandemic a(h1n1) influenza and a(h7n9) influenza does consecutive influenza vaccination reduce protection against influenza: a systematic review and meta-analysis repeated annual influenza vaccination and vaccine effectiveness: review of evidence cost-effectiveness of adult vaccinations: a systematic review il-28b is a key regulator of b-and t-cell vaccine responses against influenza associations between human leukocyte antigens and nonresponsiveness to influenza vaccine impact of host 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 potential antivirals and important differences with sars coronavirus release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndromeassociated coronavirus infection imbalanced host response to sars-cov-2 drives development of covid-19 sars-cov-2 productively infects human gut enterocytes the signaling pathways project, an integrated'omics knowledgebase for mammalian cellular signaling pathways a comprehensive collection of systems biology data characterizing the host response to viral infection a transcriptional regulatory atlas of coronavirus infection of human cells interferon-stimulated genes: a complex web of host defenses the interferon-inducible isoform of ncoa7 inhibits endosome-mediated viral entry a partial form of recessive stat1 deficiency in humans tap, the human homolog of mex67p, mediates cte-dependent rna export from the nucleus genome-wide crispr screen reveals host genes that regulate sars-cov-2 infection test and visualize gene overlaps toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection interferon-beta 1a and sars coronavirus replication dysregulation in mtor/hif-1 signaling identified by proteo-transcriptomics of sars-cov-2 infected cells up-regulation of il-6 and tnf-alpha induced by sars-coronavirus spike protein in murine macrophages via nf-kappab pathway trials of anti-tumour necrosis factor therapy for covid-19 are urgently needed viruses exploit the function of epidermal growth factor receptor viral infection increases glucocorticoid-induced interleukin-10 production through erk-mediated phosphorylation of the glucocorticoid receptor in dendritic cells: potential clinical implications the glucocorticoid receptor (gr) stimulates herpes simplex virus 1 productive infection, in part because the infected cell protein 0 (icp0) promoter is cooperatively transactivated by the gr and kruppel-like transcription factor 15 viral interactions with the notch pathway the critical role of notch ligand delta-like 1 in the pathogenesis of influenza a virus (h1n1) infection covid-19 in the heart and the lungs: could we 'notch' the inflammatory storm? xenobiotic pregnane x receptor (pxr) regulates innate immunity via activation of nlrp3 inflammasome in vascular endothelial cells does vitamin d status impact mortality from sars-cov-2 infection? med. drug discov editorial: low population mortality from covid-19 in countries south of latitude 35 degrees north supports vitamin d as a factor determining severity coronavirus breakthrough: dexamethasone is first drug shown to save lives multi-level proteomics reveals host-perturbation strategies of sars-cov-2 and sars-cov the nf-kappab-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229e-infected cells influenza viruses and the nf-kappab signaling pathway -towards a novel concept of antiviral therapy the ns1 protein of influenza a virus blocks rig-i-mediated activation of the noncanonical nf-kappab pathway and p52/relb-dependent gene expression in lung epithelial cells the molecular basis of viral inhibition of irf-and stat-dependent immune responses severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane the role of interferon in influenza virus tissue tropism the unique role of stat2 in constitutive and ifn-induced transcription and antiviral responses stat2 signaling as double-edged sword restricting viral dissemination but driving severe pneumonia in sars-cov-2 infected hamsters viral targeting of tfiib impairs de novo polymerase ii recruitment and affects antiviral immunity a new paradigm for transcription factor tfiib functionality hepatitis b virus px targets tfiib in transcription coactivation sumo1 modification stabilizes cdk6 protein and drives the cell cycle and glioblastoma progression beyond the cell cycle: a new role for cdk6 in differentiation cak-independent activation of cdk6 by a viral cyclin cell cycle control and hiv-1 susceptibility are linked by cdk6-dependent cdk2 phosphorylation of samhd1 in myeloid and lymphoid cells cyclin-dependent kinase activity is required for type i interferon production toll-like receptor-4 (tlr4) down-regulates microrna-107, increasing macrophage adhesion via cyclin-dependent kinase 6 multiomics investigation revealing the characteristics of hiv-1-infected cells in vivo p-tefb goes viral in vitro replication of dna containing either the sv40 or the polyoma origin dna topoisomerase 1 facilitates the transcription and replication of the ebola virus genome angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the pivotal link between ace2 deficiency and sars-cov-2 infection a transcription regulatory network within the ace2 locus may promote a pro-viral environment for sars-cov-2 by modulating expression of host factors sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia pregnancy and susceptibility to infectious diseases pandemic 2009 influenza a(h1n1) virus illness among pregnant women in the united states ru486 blocks the anti-inflammatory effects of exercise in a murine model of allergen-induced pulmonary inflammation covid-19 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of new york city hospitals universal screening for sars-cov-2 in women admitted for delivery interferons and progesterone for establishment and maintenance of pregnancy: interactions among novel cell signaling pathways ovulation: new dimensions and new regulators of the inflammatory-like response progesterone for the treatment of covid-19 in hospitalized men a sars-cov-2 protein interaction map reveals targets for drug repurposing molecular mechanisms of epithelial-mesenchymal transition epithelial-mesenchymal transition contributes to pulmonary fibrosis via aberrant epithelial/fibroblastic cross-talk alveolar epithelial cells undergo epithelial-mesenchymal transition in acute interstitial pneumonia: a case report inflammatory and fibrinolytic system in acute respiratory distress syndrome pulmonary fibrosis and covid-19: the potential role for antifibrotic therapy chest ct findings of early and progressive phase covid-19 infection from a us patient a pneumonia outbreak associated with a new coronavirus of probable bat origin the transcription factor slug represses e-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with snail and e47 repressors hif-2α promotes epithelial-mesenchymal transition through regulating twist2 binding to the promoter of e-cadherin in pancreatic cancer the transcription factor lef-1 induces an epithelial-mesenchymal transition in mdck cells independent of β-catenin tgf-beta-induced epithelial to mesenchymal transition dbemt: an epithelial-mesenchymal transition associated gene resource influenza a virus infection dysregulates the expression of microrna-22 and its targets; cd147 and hdac4, in epithelium of asthmatics persistent transmissible gastroenteritis virus infection enhances enterotoxigenic escherichia coli k88 adhesion by promoting epithelial-mesenchymal transition in intestinal epithelial cells lipoxin a(4) ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial-mesenchymal transition the role of adar1 and adar2 in the regulation of mirna-21 in idiopathic pulmonary fibrosis identification and validation of differentially expressed transcripts by rna-sequencing of formalin-fixed, paraffin-embedded (ffpe) lung tissue from patients with idiopathic pulmonary fibrosis extracellular superoxide dismutase in pulmonary fibrosis basigin/cd147 promotes renal fibrosis after unilateral ureteral obstruction angiotensin ii as a morphogenic cytokine stimulating renal fibrogenesis cd147 induces epithelial-to-mesenchymal transition by disassembling cellular apoptosis susceptibility protein/ e-cadherin/beta-catenin complex in human endometriosis sars-cov-2 infection induces emt-like molecular changes, including zeb1-mediated repression of the viral receptor ace2, in lung cancer models the molecular basis of e2f-1/dp-1-induced s-phase entry and apoptosis the rb/e2f pathway: expanding roles and emerging paradigms e2f3a is critically involved in epidermal growth factor receptor-directed proliferation in ovarian cancer the retinoblastoma tumour suppressor in development and cancer cyclin-dependent kinase 1 (cdk1) is essential for cell division and suppression of dna re-replication but not for liver regeneration proliferating cell nuclear antigen is required for dna excision repair cdc6: from dna replication to cell cycle checkpoints and oncogenesis silencing cenp-f weakens centromeric cohesion, prevents chromosome alignment and activates the spindle checkpoint nusap is essential for chromatin-induced spindle formation during early embryogenesis cell cycle arrest through indirect transcriptional repression by p53: i have a dream increased expression of rrm2 by human papillomavirus e7 oncoprotein promotes angiogenesis in cervical cancer an e2f site in the 5′-promoter region contributes to serum-dependent up-regulation of the human proliferating cell nuclear antigen gene the role of cellular transcription factor e2f in the regulation of cdc2 mrna expression and cell cycle control of human hematopoietic cells sars coronavirus 7a protein blocks cell cycle progression at g0/g1 phase via the cyclin d3/prb pathway breaking bad: how viruses subvert the cell cycle the dna damage response arouses the immune system compensation and specificity of function within the e2f family transcription factor compensation during mammary gland development in e2f knockout mice the global phosphorylation landscape of sars-cov-2 infection ndex: a community resource for sharing and publishing of biological networks ndex 2.0: a clearinghouse for research on cancer pathways cytoscape: a software environment for integrated models of biomolecular interaction networks detectable serum sars-cov-2 viral load (rnaaemia) is closely correlated with drastically elevated interleukin 6 (il-6) level in critically ill covid-19 patients sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes a review of pathway-based analysis tools that visualize genetic variants a network integration approach to predict conserved regulators related to pathogenicity of influenza and sars-cov respiratory viruses a dual controllability analysis of influenza virus-host protein-protein interaction networks for antiviral drug target discovery coronaviruses hijack the complement system the niddk information network: a community portal for finding data, materials, and tools for researchers studying diabetes, digestive, and kidney diseases a simple step toward improving reproducibility through rigor and transparency of experimental methods a draft network of ligand-receptor-mediated multicellular signalling in human limma powers differential expression analyses for rna-sequencing and microarray studies edger: a bioconductor package for differential expression analysis of digital gene expression data voom: precision weights unlock linear model analysis tools for rna-seq read counts chess: a new human gene catalog curated from thousands of large-scale rna sequencing experiments 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 quant