Janus kinase (JAK) inhibitors have been developed as novel immunomodulatory drugs and primarily used for treating rheumatoid arthritis and other inflammatory diseases. Recent studies have suggested that this category of anti-inflammatory drugs could be potentially useful for the control of inflammation “storms” in respiratory virus infections. In addition to their role in regulating immune cell functions, JAK1 and JAK2 have been recently identified as crucial cellular factors involved in influenza A virus (IAV) replication and could be potentially targeted for antiviral therapy. Gingerenone A (Gin A) is a compound derived from ginger roots and a dual inhibitor of JAK2 and p70 S6 kinase (S6K1). Our present study aimed to determine the antiviral activity of Gin A on influenza A virus (IAV) and to understand its mechanisms of action. Here, we reported that Gin A suppressed the replication of three IAV subtypes  in four cell lines. IAV replication was also inhibited by Ruxolitinib (Rux), a JAK inhibitor, but not by PF-4708671, an S6K1 inhibitor. JAK2 overexpression enhanced H5N1 virus replication and attenuated Gin A-mediated antiviral activity. In vivo experiments revealed that Gin A treatment suppressed IAV replication in the lungs of H5N1 virus-infected mice, alleviated their body weight loss, and prolonged their survival. Our study suggests that Gin A restricts IAV replication by inhibiting JAK2 activity; Gin A could be potentially useful for the control of influenza virus infections.  Orthomyxoviridae family. Its genome contains eight negative-sense, single stranded RNA segments that encode 11 proteins. Influenza virus can be divided into the A, B, C, and D types that vary in host ranges and pathogenicity [Influenza is a highly contagious acute respiratory illness . The disgenicity . Influengenicity . Influengenicity . Influengenicity . Based ogenicity . Vaccination and antiviral drugs such as M2 ion channel blockers and neuraminidase inhibitors are the mainstays of influenza prevention and treatment . FavipirZingiber species belong to the Zingiberaceae (ginger) family and have been widely used as spice additives and plant medicines [edicines . Ginger edicines . Ginger edicines . Ginger edicines ,18. The edicines . JAK1 anedicines ,15. Our The experiments were approved by the Institutional Biosafety Committee of Yangzhou University. All experiments involving live virulent H5N1 viruses and animals were carried out in a P3-level biosafety lab certified by the Ministry of Agriculture, China. The protocols for the animal experiments were approved by the Jiangsu Administrative Committee for Laboratory Animals , and complied with the guidelines of Jiangsu laboratory animal welfare and ethics of Jiangsu Administrative Committee of Laboratory Animals.Ruxolitinib and PF-4708671 were purchased from Selleck Inc. . Gin A and antibodies against phospho-JAK2Y1007/Y1008 (sc-16566-R), β-actin (sc-47778), and GAPDH (sc-166574) were purchased from Santa Cruz Biotechnology, Inc. . Antibodies against phospho-tyrosine , STAT3 (#9139), phospho-STAT3Y705 (#9145), JAK2 (#3230), AKT (#4691), phospho-AKTS473 (#4060), S6K1 (#2708), phospho-S6K1T389 (#9234), S6 (#2217), and phospho-S6S235/236 (#4858) were purchased from Cell Signaling Technology . An anti-nucleoprotein (NP) polyclonal antibody was prepared by immunizing mice with purified recombinant NP protein. An anti-M1 monoclonal antibody was prepared in our lab. An anti-PB2 antibody (#GTX125926) was purchased from GeneTex Inc. .1 to 109), and each dilution (105–109) was used to infect chicken embryonic fibroblast (CEF) or MDCK cells. The 50% tissue culture infection dose (TCID50/mL) was calculated according to the Reed and Muench method. The IC50 values of Gin A required to inhibit IAV replication were calculated by using GraphPad Prism software .Madin–Darby canine kidney (MDCK), A549 , 293T (a human embryonic kidney cell line), and DF1 (a chicken fibroblast cell line) cells were purchased from the American Tissue Culture Collection . Cells were grown in DMEM containing 10% fetal bovine serum (FBS). The A/mallard/Huadong/S/2005 H5N1 virus (SY strain), a moderately pathogenic avian influenza strain, was isolated from poultry . All IAV50 values were determined on MDCK cells according to our previous publications [To determine the effect of Gin A on virus growth, four cell lines  were infected with 0.01 multiplicity of infection (MOI) of H1N1, H5N1, or H9N2 virus and then incubated in the presence of various concentrations of Gin A . For H1N1 and H9N2 virus infection, tosyl-phenylalanyl-chloromethyl-ketone-treated trypsin  (2 μg/mL) was added into the media during and after virus infection. The conditioned media were collected at 18, 24, or 36 hours post-infection (hpi). The TCIDications . Data reThe 293T cells seeded in 6-well plates were infected with 0.01 MOI H5N1 virus and then incubated in the absence or presence of Gin A  for 16 h. Total RNA was extracted from H5N1 virus-infected cells by using a Qiagen RNA extraction kit. Viral RNA levels were measured by using a one-step reverse-transcription FRET-PCR to amplify the M gene according to our recent publication . PB2, PB1, PA, and NP genes from the A/mallard/Huadong/S/2005 H5N1 virus (200 ng each), and 20 ng internal control Renilla plasmid using Polyfect Transfection Reagent . After incubation for 24 h, the cells were treated with dimethyl sulphoxide (DMSO) (0.5%) or with the indicated concentrations of Gin A  for another 24 h. The cells were harvested and analyzed for luciferase activity by using the Dual-Luciferase Reporter Assay System .The 293T cells were transfected with 100 ng of luciferase reporter plasmid  and the mixture of pcDNA3.1(+) plasmid constructs expressing the polymerase subunits The 293T, DF1, MDCK, and A549 cells seeded in 6-well plates were infected with H5N1, H1N1, or H9N2 (0.1 MOI each), and then incubated in the absence or presence of the indicated concentrations of various inhibitors for 8 h in the media containing 1% FBS. The cells were harvested and lysed in NP-40 lysis buffer  , 2 mM sodium pervanadate). Cell lysates were prepared and analyzed for the expression of viral proteins  or cellular proteins with their specific antibodies, followed by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) and SuperSignal Western Pico-enhanced chemiluminescence substrate . The density of the bands was analyzed by using an NIH Image-J software and normalized by the arbitrary units of their corresponding total proteins or β-actin. Quantified results were presented as the mean ± standard deviation (SD) from three experiments in bar graphs. 5 pfu in 50 μL PBS) by instillation. The H5N1 virus is a moderately pathogenic strain in mice. The dose was determined based on a previous publication from our group [2 inhalation. Lung tissues were homogenized in a radioimmunoprecipitation assay (RIPA) lysis buffer  for Western blot or in PBS for analyzing virus loads. To determine the therapeutic effect of Gin A, mice (10 mice per group) infected with H5N1 virus were treated daily with Gin A (5 mg/kg body weight in 20 μL PEG 200) by intranasal instillation for 7 days. To avoid physical irritation caused by gavage and poor drug absorbance in the gastrointestinal tract of the H5N1 virus-infected mice, Gin A was given intranasally at a low dose in this experiment, since the drug, administered this way, directly goes to the lung where the virus replicates. Mice in control groups were treated with PEG 200. Mice were monitored daily for body weights and survival for 2–3 weeks and were humanely sacrificed by CO2 inhalation when they became moribund or when the loss of body weight decreased by >25%. The body weights of a small fraction of mice dropped beyond 25% in the last day before they were sacrificed. The last-day data of these mice were not included. The use of animals was approved by the Institutional Animal Care and Use Committee of the College of Veterinary Medicine, Yangzhou University. Female C57BL/6 mice were purchased from the Center of Experimental Animals of the College of Veterinary Medicine, Yangzhou University. All mice were maintained on a 12 h light/dark cycle and housed in ventilated cages at an ambient temperature of 23 °C. Mice were fed ad libitum on a normal chow diet (NCD). Gin A was first dissolved in dimethyl sulfoxide (DMSO) and then diluted in polyethylene glycol (PEG) 200 . Mice (8 weeks old) (5 mice per group) were treated with Gin A (20 mg/kg/day) by gavage. Twelve hours after the first treatment, mice under diethyl ether anesthesia were intranasally mock-infected with PBS or infected with the H5N1 virus   . After i50 values for cytotoxicity (C.C.) were calculated. The S.I. values were calculated by dividing the C.C. values with the IC50 values of Gin A required to inhibit virus replication. To exclude the possibility that the inhibitory effect of Gin A on virus replication was due to inhibition of cell proliferation, 293T cells were incubated with various concentrations of Gin A for 12 h and then analyzed for cell proliferation by using a CellTiter-Glo kit . To determine how selectively Gin A inhibited virus replication versus its cytotoxicity in the cells infected with IAV, 293T, A549, MDCK, and DF-1 cells seeded in 96-well plates with an approximately 80% confluence were incubated in the presence of various concentrations  of Gin A for 24 h. Cell viability was measured by using a CellTiter-Glo kit as described above. The ICt test. Differences in the body weights of untreated and Gin A-treated mice were analyzed using a repeated measures analysis of variance (ANOVA) test. Differences in the survival of untreated and Gin A-treated mice were statistically analyzed by using a Log-Rank test. A p value of < 0.05 was considered statistically significant. All statistics were analyzed with SigmaPlot 11 software .Differences in virus titers, viral mRNA levels, the arbitrary units of scanned Western blot band density, and cell proliferation index were statistically analyzed by using an unpaired Student 50 values of Gin A to inhibit H5N1 virus replication in 293 T cells are between 10.2 and 24.5 μM. Of note, the baseline titers of IAV at 4 hours post-inoculation (hpi) were below the limit of detection. We first assessed the ability of Gin A to inhibit IAV replication in 293T cells, a human embryonic kidney cell line. The antiviral activity of Gin A was largely studied on the avian A/mallard/Huadong/S/2005 (SY) strain, a zoonotic virus of the H5N1 subtype with modest pathogenicity. The 293T cells infected with H5N1 virus (0.01 MOI) were incubated in the absence or presence of the indicated concentrations of Gin A (10–50 μM) for 18, 24, or 36 h. As shown in 50 values of Gin A to inhibit H5N1 virus replication in DF-1, MDCK, and A549 cells are approximately 21.1, 12.7, and 20.7 μM, respectively. Gin A dose-dependently lowered the titers of H1N1  values of Gin A to inhibit H5N1, H1N1, and H9N2 virus replication in 293T cells are 15.6, 15.7, and 13.2, respectively. The S.I. values of Gin A to inhibit H5N1 virus replication in A549, MDCK, and DF1 cells are 5.5, 5.7, and 19.4, respectively. RT-PCR revealed that Gin A dose-dependently lowered the levels of the M1 gene of the H5N1 virus  (p < 0.01). Finally, we investigated the antiviral effect of Gin A in mice infected with the H5N1 virus. Gin A treatment significantly decreased the levels of PB2, NP, and M1 proteins, and lowered the virus titers in the lungs on day 3 A,C and 5 < 0.01) F. We als < 0.01) . Twenty  < 0.01) . The medNatural products, particularly those derived from edible vegetables, tend to be more tolerable to the human body and to have the low levels of side effects ,32. Our 50 value of approximately 10 μM [Protein tyrosine kinases have been increasingly recognized as potential molecular targets for antiviral therapy. There has been great interest in repurposing clinically approved PTK inhibitors as potential antiviral drugs for treating lethal virus infection. For example, c-Abl1 tyrosine kinase is involved in the productive replication of the Ebola virus. The inhibitor of c-Abl1 such as nilotinib suppresses the replication of the Ebola virus . JAK inhly 10 μM . Our preIFNGR-/- mice than in wild-type mice. How JAK1 and JAK2 promote IAV replication is incompletely understood. JAK1 binds multiple viral proteins and is required for vRNP incorporation into virions . Wang etSTAT3 is a transcription factor that plays a central role in regulating inflammation . STAT3 iGinger is a medicinal plant used in ancient China and an ingredient in Ge-Gen-Tang, a traditional Chinese medicine prescribed for treating human respiratory tract infections . The wat50 value of a candidate drug. Many studies have used a low MOI of IAV (0.01 or 0.001), which will lead to a much higher S.I. value. In our study, we used a 10-fold higher MOI (0.1) of IAV to infect four cell lines. In addition, the length of time used to measure the CC50 values also affects the S.I. values. The CC50 values in many studies are calculated based on the cytotoxicity data in which cells are incubated with antiviral agents for 24 h. In addition, Byun et al. reported that Gin A selectively induced apoptosis in cancer cell lines but not in normal cells [In our study, we also determined the S.I. value to assess the specificity of Gin A on virus replication. We found that the S.I. values were modestly above 10 in 293T but below 10 in A549 and MDCK cells. We postulated that the S.I. values could be affected by multiple factors. For example, the dose of IAV used to infect cells will likely affect the ICal cells . This sual cells  and a tual cells  for longIn the past decade, JAK inhibitors have been developed as novel anti-inflammatory drugs for treating rheumatoid arthritis and other inflammatory diseases . SeveralIn summary, our present study provides evidence that Gin A inhibits the replication of three IAV subtypes in vitro and controls IAV virus infection in vivo. Our mechanistic study suggests that Gin A suppresses IAV replication by inhibiting JAK2 activity. Gin A and other JAK inhibitors could be potentially developed as novel antiviral agents for the control of IAV infection in humans. Our study has validated the antiviral activity of a specific compound from ginger, a medicinal plant with antimicrobial activity.


The P2X7 receptor is a ligand-gated, cation-selective channel whose main physiological ligand is ATP. P2X7 receptor activation may also be triggered by ARTC2.2-dependent ADP ribosylation in the presence of extracellular NAD. Upon activation, this receptor induces several responses, including the influx of calcium and sodium ions, phosphatidylserine externalization, the formation of a non-selective membrane pore, and ultimately cell death. P2X7 receptor activation depends on the availability of extracellular nucleotides, whose concentrations are regulated by the action of extracellular nucleotidases such as CD39 and CD38. The P2X7 receptor has been extensively studied in the context of the immune response, and it has been reported to be involved in inflammasome activation, cytokine production, and the migration of different innate immune cells in response to ATP. In adaptive immune responses, the P2X7 receptor has been linked to T cell activation, differentiation, and apoptosis induction. In this review, we will discuss the evidence of the role of the P2X7 receptor on T cell differentiation and in the control of T cell responses in inflammatory conditions. Purinergic signaling is mediated by purine nucleotides and nucleosides such as adenosine triphosphate (ATP), nicotinamide adenine dinucleotide , and adenosine . These r+, K+, or Ca+2. Its main physiological ligand is ATP, but its function can be modulated by non-nucleotide ligands such as polymyxin B, LL-37, and serum amyloid A [Purinergic receptors are divided into three major families based on their structural properties: the P2X receptors, the P2Y receptors, and the P1 receptors. P1 receptors are G-protein coupled receptors that are activated by adenosine. P2YRs are also G protein-coupled receptors that are activated by different nucleotides such as ATP, ADP, uridine triphosphate (UTP), uridine diphosphate (UDP), and UDP-glucose . P2X recmyloid A ,15,16. Pmyloid A .50 >100 μm), it may be activated during inflammation. Moreover, this receptor triggers different responses depending on ATP concentrations; in low ATP concentrations, P2X7R can function as an ion channel, whereas in higher ATP concentrations or after sustained ATP signaling, P2X7R can form large-conductance pores, leading to apoptosis [The P2X7R is unique among P2X receptors since, as it presents low affinities for ATP , which may also be released from damaged cells or intact T cells following T cell receptor activation [+ substrate, ARTC2.2 can catalyze ADP-ribosylation of the P2X7R in Arg125, which is close to ATP binding in the extracellular loop. ADP-ribosylation induces receptor activation, which is reflected in Ca+2, Na+ input, the exposure of phosphatidylserine (PS), DNA fragmentation, propidium iodide uptake, the breakdown of mitochondrial membrane potential, cells shrinkage and finally, NAD+-induced cell death (NICD) [+ may be degraded by the CD38 ecto-NAD+ glycohydrolase mainly into ADP ribose (ADPR), cyclic ADP ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP) [In addition to ATP, P2X7R is activated by nicotinamide adenine dinucleotide  ,23. As i (NAADP) ,25  .+, thus functioning as regulators of P2X7R-induced responses [+ and ATP, affecting other cells in trans. For instance, it has been shown that in murine splenocytes, B cells express the highest levels of CD38, and the depletion of B cells increases ADP ribosylation of several proteins on T cells, indicating that CD38 exerts this control not only in cis on the same cell surface as ARTC2.2 but also in trans on the surface of other cells [We and others have postulated that CD39 and CD38 expression may provide T cells with the capacity to reduce the extracellular concentrations of ATP and NADesponses ,27. Inteer cells . Anotherer cells .+2, both of which can regulate the response of different cells of the immune system [+2 in particular, can alter the affinity of ATP binding to the P2X7 receptor, acting as allosteric inhibitors [During inflammation, several danger signals can be released into the extracellular space, including not only ATP but also Cae system ,31. It hhibitors ,33,34. Tp2rx7 gene and is the largest receptor within the P2X family of receptors, with a length of 595 amino acids for human, rat, and mouse receptors [+2.The P2X7R is encoded by the eceptors ,37,38,39eceptors . Its moneceptors . It has eceptors . Localizeceptors . In contIn response to high concentrations or prolonged exposure to ATP, P2X7R generates macropores, induces membrane blebbing , and ultIt has been proposed that the macropore formation requires molecules extrinsic to P2X7R, such as connexin-43 and pannexin-1 channels ,47,48. IOn the other hand, recent evidence supports an intrinsic role of P2X7R in the macropore formation, since it has been demonstrated that liposomes reconstituted with P2X7R show YO-PRO1 uptake in a dose-dependent manner . In addiIn addition to its function as an ionotropic receptor and macropore formation, P2X7R has been linked to the activation of numerous signaling pathways that include phospholipases A2, D, and C, neutral sphingomyelinase, MAP kinases  ,55, p38 p2rx7 gene within different murine strains [+ CD25+ T cells express higher levels of P2X7R on their surface than CD4+ T cells and CD8+ T cells (being lower in CD8+ T cells), which correlates with their higher sensitivity to ATP and NAD+ [+ and CD8+ T cells is higher than in C57BL/6 mice, and so is their sensitivity to ATP and death induced by this molecule [The functionality of P2X7R can be affected by various factors within cells of the immune system. An essential factor to consider is the alternative splicing of the P2X7 transcript ,60. In m strains . C57BL/6 strains . Finallyand NAD+ ,65. Intemolecule . This dimolecule ,66.+ and CD8+ T cells express the common transcript P2X7A and the P2X7B variant [Mycobacterium tuberculosis [A human equivalent to P2X7k has not been described ,62, but  variant . P2X7B i variant . P2X7B a variant ,67,68,69 variant ,71. One rculosis  and lymprculosis . It is arculosis  and cytorculosis  in respo+ T cells to Th1, Th2, Th17, and Treg cells [Dendritic cells (DCs) are specialized antigen-presenting cells that efficiently activate naïve T cells. Dendritic cells are poised in epithelial surfaces that are in contact with pathogens. These cells express P2X7R and respond to ATP released during tissue damage in an ongoing immune response. Following pathogen recognition, DCs become activated and migrate to draining lymph nodes to present the antigens derived from the pathogens to T cells. In the lymph node, T cell fate is mainly influenced by cytokines secreted during T cell activation by DCs. It is well recognized that cytokines such as interleukin (IL)-12, IL-4, IL-6, IL-23, IL-1β, and transforming growth factor beta (TGF-β) trigger the polarization of CD4eg cells ,77. In t+ T cells and has been shown to influence the growth and differentiation of T helper subsets such as Th17 cells [Perhaps the most well-recognized effect of P2X7R activation induced by ATP on DCs is IL-1β production through the inflammasome assembly. The inflammasome consists of a large multiprotein complex composed of an NLR , the ASC adaptor protein, and procaspase 1. Its assembly leads to the activation of caspase 1, which results in the cleavage of pro-IL-1β and pro-IL-18 to generate the active pro-inflammatory cytokines. Numerous inflammasomes have been identified; however, the best characterized is the NLRP3 inflammasome ,79,80. T17 cells ,82.A putative role of P2X7R on inflammasome activation in human DCs was first suggested in 2000. In this report, the authors showed that human DCs respond to ATP by increasing intracellular calcium and that LPS matured DCs secrete IL-1β and TNF-α upon incubation with ATP . Later sDanger signals such as ATP can stimulate DC migration to the draining lymph node, where they can activate naïve lymphocytes and enhance the immune response . In thisIt has also been demonstrated that signaling through the pannexin-1 channel and the P2X7R is responsible for the fast ATP-dependent migration of DCs. This signaling is required for the actin cytoskeleton reorganization associated with DC migration . In agre+ T cells. In addition, the in vivo depletion of ATP with apyrase reduced CD80 and CD86 expression in lymph node–CD11c+ cells in an acute graft-versus-host disease murine model [Normal DC maturation involves an increase in the surface expression of co-stimulatory molecules such as CD80, CD86, CD40, and MHC molecules ,70. In tne model . Furtherne model .+ T cells [11 receptor might mediate this effect through cAMP signaling [Although there is evidence of a possible role for ATP on DCs reducing Th1 responses in vitro, there are few reports on the specific effects of P2X7R on DCs during Th1 differentiation. In vitro experiments have shown that DCs stimulated with micromolar concentrations of non-hydrolyzable ATP inhibit the production and secretion of IL-12 and TNF-α . Accordi T cells . Howeverignaling ,96. On tignaling .The ATP/P2X7R axis has been described as a modulator in Th17 polarization in different tissues and models ,103,104.The effect of P2X7R activation on Th17 cell differentiation has been studied in autoimmune diseases as well. Reports in lupus-prone mice, where P2X7R is upregulated compared to control mice, showed that the renal expression of NLRP3, ASC, and caspase 1p20 was enhanced and correlated with higher renal and serum levels of IL-1β . In thisEvidence of the direct effect of P2X7R activation in DCs driving Th17 polarization arises from the study of the Atarashi’s group . In 2008Additionally, it has recently been described that the ATP/P2X7R axis DCs activates the NLRP3 inflammasome, promoting the differentiation to Th17 cells in an ovalbumin-induced murine asthmatic model . The rolAll these works suggest that the activation of P2X7R in DCs plays a role inducing Th17 cell responses by generating a cytokine microenvironment that favors the differentiation of these cells .+2 influx, IL-2 production, and proliferation by the activation of NFAT (activated T cell nuclear factor) along with an increased expression of the p2rx7 gene [Purinergic signaling at the immune synapse plays a vital role in the amplification of T cell receptor (TCR) signaling. Following antigenic recognition, signal transduction from the TCR and co-stimulatory molecules induces the rapid release of micromolar concentrations of ATP through pannexin channels by T cells ,107,108.rx7 gene ,108. Fol+ T cells and Jurkat cells by the group of Junger. In this study, the authors demonstrated that P2X7R inhibition through different strategies such as the removal of extracellular ATP by apyrase, inhibition of ATP release with gadolinium chloride, pharmacological P2X7R blockade, or siRNA silencing of P2X7R all result in calcium entry blockade and the inhibition of T cell activation [The role of P2X7R on calcium influx and downstream signaling required for T cell activation was reported on human CD4tivation . In additivation . This daL-selectin (CD62L) is fundamental for T cell migration, as it allows the entry of lymphocytes to secondary lymphoid tissues via high endothelial venules . L-selecIn addition to its effects on T cell migration, new evidence has demonstrated that P2XR activation during T cell activation in the lymph node may have an impact on T cell motility. This report demonstrated that ATP released by a stimulated T cell acts in a paracrine fashion to induce calcium waves in the neighboring T cells through P2X4/P2X7 activation. This calcium wave reduces T cells’ motility, allowing the stability of DC–T cell conjugates .Intracellular ATP and NAD nucleotides have been considered essential energy sources. However, during inflammatory processes and tissue injury, both nucleotides are released into the extracellular space ,21 where+ is considered a substrate for the post-transcriptional modification of proteins such as phosphorylation and the ADP-ribosylation. Importantly, NAD+ serves as a substrate for ARTC2.2, which catalyzes the ADP-ribosylation of the P2X7R and other cell surface proteins on T cells. ADP ribosylation of P2X7R in the presence of extracellular NAD+ (at low micromolar concentrations) induces sustained P2X7R activation, resulting in cell death by apoptosis [+ induced cell death (NIDC).Extracellular NADpoptosis . This prSustained P2X7R activation either by ATP or NAD-dependent ADP ribosylation ,27,117 lProlonged triggering of P2X7R results in the formation of non-selective membrane pores dependent on the cytoplasmic C-terminal domain of P2X7R ,52. PoreOn the other hand, P2X7R induced cell lysis or necrosis has been observed independent of pore formation and calcium influx. This pathway was found to require signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation ,123. Alt+ released during early inflammation facilitates the expansion of primed T cells, through the ARTC2.2-driven death of resting cells [The delicate regulation of the P2X7R and ARTC2.2 expression is one of the primary mechanisms that may explain the differential susceptibility of different cellular populations to ATP and NAD-induced cell death in lymph nodes. Adriouch and collaborators demonstrated that P2X7R and ARTC2.2 are decreased at early stages following the activation of T cells compared to naive T cells, suggesting that naive T cells and memory cells are susceptible to NICD mediated by P2X7R. They proposed that NADng cells .+ or ATP in vitro [Besides being tightly regulated following activation, P2X7R expression is differentially expressed in different T cell populations. For instance, reports from the literature indicate that regulatory T cells (Tregs) exhibit the highest expression of P2X7R among T cells and are more susceptible to cell death by NADin vitro ,125,126.in vitro   cells also express high levels of P2X7R, rendering this population very susceptible to NICD  against gut commensals [FH cell population in the context of Plasmodium chabaudi infection [+ T cells is sufficient to increase TFH cell frequency. An explanation of this phenomenon relies on the high susceptibility of TFH cells to ATP-induced cell death, since as mentioned earlier, they express high levels of P2X7R and express relatively low levels of CD39 ectonucleotidase, which may be insufficient to degrade eATP rapidly and thus prevent ATP-induced cell death. In addition, it was demonstrated that P2X7R deletion enhanced the generation of pathogenic TFH cells in a murine model of systemic lupus erythematosus. In this setting, P2X7R-mediated cell death was associated with the induction of pyroptosis as it was executed by gasdermin D [FH CD4+ T cells comprised a large proportion of the total antigen-specific memory CD4+ T cells in the spleen of infected mice [Other reports have demonstrated that follicular helper T  induced cell death in vitro. These data suggest that the P2X7R expression is not only regulated during T cell differentiation but also by tissue-derived specific factors [+ T cells in the presence of RA upregulated the expression of P2X7R and ARTC2.2 [In contrast to peripheral lymph nodes, the intestine requires additional checkpoints to avoid excessive immune responses. A report shows that intestinal CD8 factors . Retinoi ARTC2.2 .+ T cells, Hashimoto-Hill and collaborators have recently demonstrated that RA also induced P2X7R expression on effector T cells, and this allows the depletion of Th1 and Th17 cells in the intestine  constitute a subset of memory cells that reside in the tissues without recirculating [RM) also may escape cell death by increasing the levels of ectonucleotidases upon T cell activation. It was demonstrated that following sterile injury, liver TRM cells were highly susceptible to ATP and NAD+ induced cell death. However, the concomitant T cell receptor activation induced CD38 and CD39 nucleotidases and reduced TRM susceptibility to P2X7R induced cell death [A fundamental characteristic of adaptative immunity is the generation of memory, which allows a faster and more effective response to reinfection. Three classic memory CD8culating . Residenll death  /mammalian target of rapamycin (mTOR) axis might be involved in the P2X7R induction of memory T cell differentiation and maintenance. AMPK promotes memory differentiation and can be regulated by ATP’s intracellular concentration . Borges ntiation .+ T cells could be refractory to the effect of high extracellular ATP concentration, although they express similar levels of P2X7R as other CD8+ T cell population [+ memory T cells do not suffer the effects of high extracellular ATP, such as CD62L shedding, pore formation, and cell death, lower concentrations of this nucleotide might be beneficial to the long-term maintenance of memory cells. It has been proposed that the positive feedback loop between P2X7R and pannexin-1 channels in memory T cells maintains a constant efflux of intracellular ATP that could favor their long-term survival, perhaps through AMPK signaling [2+ that ultimately influence the activity of AMPK and impact memory T cell maintenance.A later study showed that a subpopulation of long-lived memory CD8pulation ,148. Howignaling ,149. TheRegulatory T cells are identified by the expression of a key transcription factor, known as forkhead box P3 (Foxp3), that is required for their development, maintenance, and function ,151. TheAs previously stated, the effect of ATP on T cell function is entirely dependent on its concentration. Trabanelli and collaborators showed that ATP at low physiological concentrations did not modulate either the proliferation or cell death of activated Tregs. Conversely, high ATP concentrations (1 mM) can “turn on” Treg cells, making them capable of exerting their immunosuppressive functions . HoweverAnother type of regulatory T cells is the Tr1 cells, which are characterized by the high production of IL-10 and the absence of Foxp3 . EvidencIn 2016, our group reported further evidence for ATP inhibition of Tr1 responses. In this report, we demonstrated that Th17 cells expressing high levels of CD39 present a plastic phenotype, allowing their differentiation into Tr1 cells in a model of experimental colitis. We reported that Th17 cells differentiated in the presence of TGF- β1 express high levels of CD39 and produce IL-10 . These c+ may also activate the P2X7 receptor, as it serves as a substrate for ARTC2.2-mediated ADP-ribosylation of this receptor. Several cellular components of the immune system, such as T cells and dendritic cells, express the P2X7 receptor. ATP-mediated P2X7 receptor activation on dendritic cells triggers several responses, such as dendritic cell maturation, migration, and inflammasome assembly, all of which impact T cell activation, promoting the differentiation of inflammatory T helper subsets. On T cells, P2X7 receptor activation may have different outcomes, including the induction of T cell activation/differentiation, T cell migration, and ultimately T cell death through ATP and NAD+ induced cell death (+ and ATP-induced cell death is context-dependent and may serve to allow the expansion of primed T cells in the lymph node, reduce the overt activation of inflammatory T cell responses in the gut, and allow the expansion of activated resident memory T cells in inflamed tissues. Thus, understanding the delicate regulation of ectonucleotidases and P2X7 receptor expression in different conditions may unravel the impact of purinergic signaling in the adaptive immune response.The P2X7 receptor is a ligand-gated cation-selective channel whose main physiological ligand is ATP. NADll death . Importa


AR and MAP3K1 plasmid. Cell lysates were used to analyze androgen receptor (AR) production. A novel hemizygous AR variant  and a rare heterozygous MAP3K1 variant  were identified by WES in the proband and her mother. Bioinformatic analysis predicted these two variants to be pathogenic. Multiple amino acid sequence alignments showed that p. His690 and p. Arg260 are conserved among various species. His690Glu is a mutation that decreased the AR production, whereas the Arg260Cys mutation increased the AR production. The novel compound variants of the AR and MAP3K1 genes also increased the production of AR protein. Thus, the phenotype of the patient may be caused by defects in both the AR and MAP3K1 signaling pathways. Compound variants of the AR and MAP3K1 genes resulted in a specific phenotype in this patient with AIS. WES might reveal genetic variants that explain the heterogeneity of AIS.Androgen insensitivity syndrome  is the most frequent cause of 46, XY disorders of sex development (DSD). However, the correlation between genotype and phenotype has not been determined. We conducted a systematic analysis of the clinical characteristics, hormone levels, ultrasonography data and histopathology of a 46, XY Chinese patient with AIS. The family was followed up for nearly 8 years. We applied whole-exome sequencing (WES) for genetic analysis of the pedigree and performed bioinformatic analysis of the identified variants. Human embryonic kidney 293T/17 (HEK293T/17) cells were transiently transfected with wild-type or mutant  Furthermore, the exact cause of this variation is not well understood. With the development of next-generation sequencing, useful information for explaining this heterogeneity, such as unidentified coactivator proteins, is expected to be forthcoming.Androgen insensitivity syndrome  is the most frequent cause of 46, XY disorders of sex development (DSD). AIS is characterized by a patient exhibiting a 46, XY karyotype and a normal amount of testosterone secretion but with symptoms and signs of androgen deficiency or reduction, such as female external genitalia, blind vagina, and uterine absence ,2. It isAR) gene . More thpatients . The samAR gene, there are many other genes that cause disorders of sex development, such as the MAP3K1 gene. MAP3K1 variants were first described in two large families and two of 11 sporadic cases of 46, XY DSD in 2010 [MAP3K1 is one of the more common genes mutated in 46, XY DSD, with 13–18% of these patients showing variants of this gene [MAP3K1 gene is located at 5q11.2 and encodes a key serine/threonine protein kinase (MAP3K1) of approximately 196 kD in the MAPK pathway that activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) pathways and plays an important role in cell proliferation, differentiation, and apoptosis [MAP3K1 and AR variants can cause 46, XY DSD, the question of whether a correlation exists between the two molecules needs to be addressed.AIS exhibits overlapping clinical phenotypes with other DSDs, such as ovotesticular DSD and 5α-reductase 2 deficiency ,9. Mülle in 2010 . Recent his gene . The MAPpoptosis . As bothMAP3K1 and AR in a 46, XY Chinese patient with AIS. The patient presented with bilateral inguinal masses , female external genitalia (underdeveloped labia and clitoris), and the presence of a uterus, which was indicative of incomplete Müllerian regression and did not match the typical characteristics of AIS. A novel hemizygous AR variant  and a rare heterozygous MAP3K1 variant  were identified by whole-exome sequencing (WES) in the proband and her mother. Bioinformatic analysis and in vitro experiments showed that these two variants are pathogenic. Our study emphasizes the heterogeneity of AIS, which has significance for research on the heterogeneity of diseases.Interestingly, we detected a compound variant of The Chinese patient with AIS in our study, in whom bilateral inguinal hernias were discovered, was 2.6 years of age in November 2011. We collected her clinical characteristics, physical examination results, hormone levels, ultrasonography data, and pathological examination results to determine the location and nature of the gonads and the appropriate surgical intervention. Peripheral blood samples were obtained from the patient and her parents.We followed this family for nearly 8 years. We closely tracked the patient’s urogenital development, intellectual development, drug treatment regimens and potential complications, and developments such as psychological and cognitive impairment, gender anxiety disorder, and the presence of gonadal tumors.Genomic DNA was isolated from peripheral blood leukocytes using QIAamp DNA Mini Kit  following the manufacturer’s instructions. WES was performed using DNA from peripheral blood. After genomic DNA fragmentation, paired-end adaptor ligation, amplification and purification, human exons were captured by using SeqCap EZ Med Exome Enrichment Kit . A DNA library was generated by postcapture amplification and purification and then sequenced with the Illumina HiSeq sequencing platform. Sequence data alignment to the human genome reference (hg19) and variant calling were performed with NextGene V2.3.4 software to obtain the coverage and mean read depth of target regions. The average coverage of the exome was >100×, which permitted examination of the target region with sufficient depth to exactly match >99% of the target exome. To ensure the accuracy of data analysis, mutations with low coverage in the target area were filtered.http://www.ncbi.nlm.nih.gov/snp/), in the exome variant server (http://evs.gs.washington.edu/EVS/), in the ensemble database and in 500 Chinese controls or when the allele frequency was found to be <0.001 in the database. Pathogenic variants were determined according to Standards and Guidelines for the Interpretation of Sequence Variants published by the American College of Medical Genetics and Genomics (ACMG) in 2015 with Human Genome Variation Society (HGVS) nomenclature.Additionally, annotation information, including the conservation of nucleotide bases and amino acids, predictions of biological functions, frequency in normal populations (Genome Aggregation Database (GnomAD), Trans-Omics for Precision Medicine (TOPMED), Exome Aggregation Consortium (ExAC), and data from Human Gene Mutation Database (HGMD), Clinvar and Online Mendelian Inheritance in Man (OMIM) databases, was performed by using NextGene V2.3.4 and our in-house scripts. A variant was recognized as a mutation when it was not found in dbSNP (AR and MAP3K1 were designed using Primer3 version 1.1.4 (http://www.sourceforge.net) and GeneDistiller 2014 (http://www.genedistiller.org/), as follows: Forward primer (AR): 5′-GCATTCAGGCAGAGCTGGAT-3′; Reverse primer (AR): 5′-CTGCTGTGCATCAATGTGGG-3′; Forward primer (MAP3K1): 5′-GTTGATATATTGTCTGGTATACTGG-3′; Reverse primer (MAP3K1): 5′-AGGGGTCTGCAGATCATATTTTG-3′. Polymerase chain reaction (PCR) was performed in a 50-μl reaction system including 4 μl genomic DNA, 1 μl forward and reverse primers, 5 μl 10 × PCR buffer, 4 μl dNTPs, and 0.3 μl Taq Hot Start . The PCR conditions were as follows: an initial denaturation step (95°C for 5 min), followed by 40 cycles of denaturation (95°C for 30 s), annealing (65°C for 30 s), and elongation (72°C for 30 s). Amplicons were sequenced using an ABI 3730 system , and sequence analysis was performed using the autoassembler software Chromas 2.6.6 , Mutation Taster (http://www.mutationtaster.org/) and PROVEAN (http://provean.jcvi.org/index.php). Modeling of wild-type and mutant proteins was performed with I-TASSER software (https://zhanglab.ccmb.med.umich.edu/I-TASSER/), and PyMOL Viewer was employed to observe the effect of the two variants on the protein.We applied Clustal W  software to compare 20 species to confirm the conservation of amino acids at mutated positions. To predict the potential pathogenic effects of the two variants, we used online software such as PolyPhen-2 . AR variants (pcDNA3.1-AR-MU) and MAP3K1 variants (pcDNA3.1-MAP3K1-MU) were generated by site-directed mutagenesis. To obtain the AR His690Glu amino acidic substitution (from cytosine to adenine at nucleotide 2070), we used the corresponding primers: primer forward ACGGGCCCTCTAGACTCGAGCGCCACCATGGAAGTGCAGTTAGGGCTGGGAAGG, primer reverse AGTCACTTAAGCTTGGTACCGACTGGGTGTGGAAATAGATGGGCTTGACTTTC. To obtain the MAP3K1 Arg260Cys amino acidic substitution (from cytosine to thymine at nucleotide 778), the following primers were used: primer forward ACGGGCCCTCTAGACTCGAGCGCCACCATGGCGGCGGCGGCGGGGAATCGCGCC, primer reverse ACGGGCCCTCTAGACTCGAGCGCCACCATGGCGGCGGCGGCGGGGAATCGCGCC. The protocol used for site-directed mutagenesis is available upon request. All the primers were purchased from GenScript . The presence and the construct fidelity of pcDNA3.1-AR-MU and pcDNA3.1-MAP3K1-MU were confirmed by an ABI 3730 system .Wild-type AR (pcDNA3.1-AR-WT) and MAP3K1(pcDNA3.1-MAP3K1-WT) or pcDNA3.1-AR-MU and pcDNA3.1-MAP3K1-MU using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). The transfection concentration of a single plasmid was 2.5 μg/ml. When the cotransfection of the two plasmids was conducted, the concentration of each plasmid is 1.5 μg/ml.Then, we used the high glucose Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum (FBS), 5% penicillin-streptomycin and 2 mM L-glutamine to culture the human embryonic kidney 293T/17 (HEK293T/17) cells . HEK293T/17 cells were transfected with plasmids containing the wild-type After 48 h, cells were collected and lysed by mammalian protein extraction reagent (ThermoFisher Scientific) and protease inhibitor cocktail (Sigma Aldrich). The cells lysates were used to analyze the AR protein expression by Western blot.The cells lysates were mixed with Laemmli buffer containing 2-mercaptoethanol. Then, the mixtures were boiled at 95°C for 5 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes . After membranes were incubated overnight with primary antibodies, they were washed two times for 10 min with 0.2% tris-buffered saline Tween. Next, membranes had been incubated with secondary antibodies for 1 h. Before 5-min incubation with chemiluminescent HRP substrate , membranes were washed three times for 10 min with 0.2% tris-buffered saline Tween. Finally, bands were visualized by Chemidoc XRS System . The following antibodies were used: rabbit anti-AR (Abcam) and mouse anti-Actin (Servicebio).The patient’s lineage is shown in The patient’s last follow-up was at 10.3 years of age, and the patient identified as female. Gynecological examination confirmed mild clitoromegaly, an existing vagina, no significant labia majora, and the absence of pubic hair or crest for the female external genitalia E. The paHer hormone levels at the last follow-up, including testosterone, 17α-hydroxyprogesterone, follicle stimulating hormone, adrenocorticotropic hormone and cortisol, were normal for a prepubertal patient. Conversely, the patient’s estradiol, prolactin and luteinizing hormone levels were low for a prepubertal patient .AR gene (c.2070C>A) of I-2 and II-1, resulting in a change in the 690th amino acid of the encoded protein from histidine to glutamine  that results in a change in the 260th amino acid of the encoded protein from arginine to cysteine , T = 0.00001  and T = 0.00002 . The serine/threonine kinase domain is located at the C-terminus of MAP3K1, upstream of which there is a conserved caspase-3 cleavage site and a ubiquitin interaction motif (UIM). The N-terminus of MAP3K1 contains two zinc finger structures, including one RING structure and one SWIM region. The heterozygous variant occurs at position 260 (Arg260Cys)  B. Polyphof 0.791 C, and PR species D. HoweveAR variant and the rare heterozygous MAP3K1 variant, we did not find any other DSD gene variants.Other than the novel hemizygous AR and MAP3K1 variants at the protein level, plasmids containing the wild-type AR and MAP3K1 cDNA or carrying AR and MAP3K1 variants were transfected into HEK293T/17 cells. The AR production of HEK293T/17 cells transfected with the pcDNA3.1-AR-MU decreased significantly by about 50% compared with that of cells transfected with pcDNA3.1-AR-WT. Cells transfected with both wild-type plasmids showed a significantly increase in AR protein production, compared to cells with pcDNA3.1-AR-WT or pcDNA3.1- MAP3K1-WT . This result indicates that pcDNA3.1- MAP3K1-WT can increase the expression of AR. The AR production of HEK293T/17 cells transfected with pcDNA3.1- MAP3K1-MU was 6.73 times higher than that of cells with pcDNA3.1- MAP3K1-WT transfection, indicating that the effect of increasing AR production became stronger after MAP3K1 was mutated. Compared to cells transfected with the pcDNA3.1-AR-MU, cells transfected with both mutant plasmids showed a significantly increase in AR protein production (approximately 14 times), which also indicated that the effect of increasing AR production became stronger after MAP3K1 was mutated  and a rare heterozygous variant in the MAP3K1 gene . Bioinformatics analysis predicted these two variants to be pathogenic. And we characterized at a molecular level the pathogenic variants in the AR and MAP3K1 genes that affect the production of AR protein. The rare heterozygous variant in the MAP3K1 gene might act as a genetic modifier of the phenotype, as the patient has rudimentary Müllerian structures, which is uncommon in AIS. Therefore, we consider that the two pathogenic variants are the cause of AIS and that they are the exclusive causative agents of the phenotype of the described patient. Moreover, the confirmation of an AIS diagnosis by WES can reveal additional genetic variants to explain the heterogeneity of the disease.In the present study, we describe a patient with AIS with a novel hemizygous variant in the AIS is highly heterogeneous and divided into three categories according to the degree of genital masculinization: mild androgen insensitivity syndrome (MAIS), partial androgen insensitivity syndrome (PAIS) and complete androgen insensitivity syndrome (CAIS). The clinical phenotypes range from a typical male habitus with mild spermatogenic defects and reduced secondary terminal hair to a full female habitus despite the presence of a Y chromosome. The correlation of genotype and phenotype has not yet been established.AR gene leads to cellular resistance to androgens. More than 1000 AIS-causing mutations in the AR gene have been identified [AR gene variants include gene deletions, splice site mutations, premature stop codons and missense mutations, among others. The most common genetic abnormalities are missense mutations, which often occur in two important segments of the receptor protein: the DBD and LDB regions. Missense mutations that result in a single amino acid substitution are known to produce the most phenotypic diversity.AIS is a congenital disorder in which a defect in the entified . The typAR variants reduce or abolish AR function  and a ubiquitin interaction motif (UIM). As mentioned above, two zinc finger structures, including one RING structure and one SWIM region, are present at the N-terminus of MAP3K1 [MAP3K1 gene variants result in altered binding of cofactors and increase phosphorylation of the downstream MAP kinase pathway targets MAPK11, MAP3K and MAPK1, thereby promoting ERR1/2 and p38 phosphorylation and ultimately leading to 46, XY DSD. Increased phosphorylation of ERK1/2 and p38 may result in decreased expression of SOX9 and increased activity of β-catenin, respectively, which are important signaling molecules in testicular and ovarian-promoting pathways  and a rare heterozygous variant of MAP3K1 . A missense mutation in the AR gene  has been reported in the same codon [MAP3K1 gene might be related to the clinical heterogeneity of AIS.Interestingly, we detected a compound variant of the with AIS ,50–54. Tme codon . Rosa etin vitro experiments of the AR and MAP3K1 variants. His690Glu is an AR mutation that decreased the AR production, whereas the Arg260Cys mutation of MAP3K1 increased the AR production. The novel compound variants of the AR and MAP3K1 genes also increased the production of AR protein. Taken together, these findings suggest that the AR and MAP3K1 variants severely affect the AR protein production, explaining the uncommon phenotype in the patient carrying these variants. Furthermore, we predicted the relationship between AR and MAP3K1. MAP3K1 is expected to activate the transcription factor AP-1 [AR cistrome [MAP3K1 promoter regulation [To explain the molecular mechanism underlying our genetic findings, we carried out tor AP-1 , which mcistrome , leadingcistrome , other sgulation . Casalingulation . Overallgulation . Furthergulation . These rAR and MAP3K1 gene. Our findings extend the gene variant profile of AR and MAP3K1. However, the association between the phenotype and genotype of AIS remains unclear, and more patients need to be studied to provide more comprehensive data.It is undeniable that the present study has some limitations. The study included a 46, XY Chinese female patient with AIS whose grandparents had died, and we were unable to trace potential genetic variation. In addition, there is a lack of basic experiments to validate regulatory mechanism between the In summary, our study contributes to the known heterogeneity of AIS, which has significance for research on the heterogeneity of diseases. The widespread use of WES may reveal further genetic variants, and some genetic variants may help to explain genotype–phenotype associations.


Ae. longissima on gene transcriptions of the wheat landrace Chinese Spring. RNA-seq analyses demonstrated 5.37% and 4.30% of the genes were significantly differentially expressed (DEGs) in CS-Ae. longissima disomic 3Sl#2(3B) substitution line TA3575 and disomic 6Sl#3 addition line TA7548, respectively when compared to CS. In addition, 561 DEGs, including 413 up-regulated and 148 down-regulated or not transcribed genes, were simultaneously impacted by introgressed chromosomes 3Sl#2 and 6Sl#3, which accounts for 41.25% of the DEGs in TA3575 and 38.79% in TA7548. Seventeen DEGs, annotated as R genes, were shared by both introgression lines carrying chromosomes 3Sl#2 and 6Sl#3, which confer resistance to powdery mildew. This study will benefit the understanding of the wheat gene responses as result of alien gene(s) or chromosome intogression and the plant defense response initiated by powdery mildew resistance genes in chromosomes 3Sl#2 and 6Sl#3.Agronomic characteristics and tolerance to biotic and abiotic stresses in hexaploid wheat can be drastically improved through wheat-alien introgression. However, the transcriptional level interactions of introduced alien genes in the wheat genetic background is rarely investigated. In this study, we report the genome-wide impacts of introgressed chromosomes derived from  Triticum aestivum L., 2n = 6x = 42, AABBDD) is a species of the genus Triticum in the grass family Poaceae (or Gramineae). Wheat evolved from two independent natural hybridization events followed by chromosome doubling in the new hybrids. The first hybridization occurred between Triticum urartu Tumanian ex Gandilyan  and Aegilops speltoides Tausch  about 0.5 million years ago, forming a new tetraploid species T. turgidum L. 2. The domesticated T. turgidum subsp. dicoccum then hybridized with another diploid species, Ae. tauschii Coss.  about 7,000 to 12,000 years ago, producing a fertile hexaploid bread wheat 7. Although of relatively recent origin, hexaploid bread wheat is currently the most favored staple food for human beings planted on the most acres and most traded food crop in the world.Allohexaploid wheat (Puccinia graminis f. sp. tritici (Pgt), was a devastating disease during the 20th century. However, epidemics of stem rust were well under control in the past several decades largely due to the deployment of highly effective resistance genes, such as Sr31 from Secale cereale L., Sr24 from Thinopyrum elongatum (Host) D. R. Dewey, and Sr36 from T. timopheevii (Zhuk.) Zhuk. in wheat varieties worldwide. However a novel race named Ug99 (TTKSK) emerged in Uganda (1999) having virulence for Sr31 and many other stem rust resistance genes thus Ug99 has become a major threat to wheat production13. Continuous efforts are needed to identify and deploy new, effective and durable resistance genes to maintain world food security. Of the currently catalogued genes against the major diseases in wheat like leaf rust, stripe rust, and powdery mildew, at least 50% of those genes have been derived from wheat ancestors or other wild relatives including Ae. tauschii, Ae. speltoides, Ae. geniculata Roth, Ae. longissima Schweinf. & Muschl., S. cereale, Dasypyrum villosum, Th. intermedium (Host) Barkworth & D. R. Dewey, Th. elongatum, and Agropyron cristatum (L.) Gaertn15.Although common wheat is one of the earliest domesticated crop plants, it is still a young species compared with its ancestors or other wild relatives. Gene pools derived from wild relatives, which have a much longer evolutionary history, play an important role in modern wheat improvement. For example, stem rust, caused by S. cereale not only carries resistance genes against fungal pathogens, such as stripe rust resistance gene Yr9, leaf rust resistance gene Lr26, stem rust resistance gene Sr31 and Pm8 against wheat powdery mildew19, but also enhances root biomass, grain yield, protein content23 and tolerance to drought26. However, chromosome arm 1RS also carries Sec-1 loci encoding γ-secalin and ω-secalin high molecular weight proteins, which decreases the processing and baking quality of wheat containing the T1BL·1RS translocation30.Despite the importance of the improvement of genetic diversity in common wheat, most alien genes are currently under-utilized due to the limitation of deleterious linkage drag introduced with alien chromosome segments. Linkage drag could be decreased by either shortening the targeted gene segments or possibly deactivating the undesired deleterious genes. For example, the wheat-rye T1BL·1RS translocation has been widely used in wheat cultivars or advanced breeding lines from Europe, China, India, the USA, and other developing countries since the mid-1980s because of remarkable agronomic and disease-resistant characteristics. The short arm of chromosome 1 R (1RS) from 35. However, only a few investigations of chromosome-wide gene expression in alien chromosome addition lines have been reported38. Rey et al. (2018) reported that the ditelosomic addition of 7HL from Hordeum vulgare L. into the genetic background of CS could lead to 3% differentially expressed genes (DEGs) of wheat and 42% of the 7HL-derived genes in the wheat-barley 7HL addition line38. Nevertheless, the regulatory mechanism underlying the widespread gene transcription of the alien chromosome(s) is still unclear.In addition, linkage drag may also be caused by the inability of introduced alien chromosome segments to adequately compensate for the loss of their homoeologous counterparts or incompatible gene interactions between the alien donor and the recipient wheat line. Parallel with the recent rapid progress of genome sequencing techniques and ever increasing evidence from genome sequence analyses has shown that linkage drag maybe a result of gene expression/suppression caused by gene interactions between the donor and the recipient, post-translational modifications of gene products, and heteromeric complexes formed by the donor and the recipient gene productsAegilops longissima  is an S-genome diploid species belonging to the section Sitopsis3. This species contains many traits desirable for wheat improvement, such as the powdery mildew resistance gene Pm13 located on chromosome 3Sl, which has been widely used in wheat breeding programs40. In addition, we have previously reported that chromosome 6Sl#3 (TA7548) also confers resistance to wheat powdery mildew41. Several QTLs conferring resistance to wheat eyespot are located on chromosomes 1Sl, 3Sl, 5Sl and 7Sl43. Additionally, chromosome 1Sl has been reported to carry genes enhancing bread-making quality45. A few wheat-Ae. longissima amphiploids and sets of addition and substitution lines have been developed and several translocation lines, such as T3BL·3BS-3Sl and T3DL·3DS-3Sl translocations carrying Pm13, have been deployed in wheat breeding programs worldwide49.l#2 and 6Sl#3 (Ae. longissima), each of which harbors resistance genes to powdery mildew41, based on comparative transcriptome analyses of CS with a CS-Ae. longissima disomic 3Sl#2(3B) substitution line (TA3575) and a disomic 6Sl#3 addition line (TA7548).In this study, we report the genome-wide impact on gene transcription in a recipient wheat landrace CS caused by the introgression of pairs of the alien chromosomes 3Sl-specific markers  3Sl#2(3B) line, rather than the disomic addition (DA) 3Sl#2 line previously identified by the Wheat Genetic Resource Center (WGRC), Kansas State University, Manhattan, KS.Karyotype analysis of TA3575 was identified by integration analyses of PCR amplification using ten 3B-specific and 12 3Smes Fig. . Thus, TA total of 77,962,967, 77,492,142 and 76,266,826 clean reads (>100 bp) were obtained from RNA sequencing for CS, TA3575 and TA7548, respectively. Sequencing quality scores Q30, which inferred a base call accuracy of 99.9%, were greater than 93.06% for all samples, which signifies that the RNA-seq quality was appropriate for subsequent sequence assembly and analysis.Clean reads of all the samples were further assembled into a total of 158,953 unigenes with a median length of 1,247.95 bp using the short-read assembly software, Trinity. The contig N50 value was 1,725 bp in length. We mapped 91.29% clean reads back to the reference assembly.Ae. longissima DS 3Sl#2(3B) line TA3575 and CS-Ae. longissima DA 6Sl#3 line TA7548 yielded 1,991 (5.37%) and 1,592 (4.30%) genes, respectively, which were significantly differentially transcribed with a cut-off of |log2(fold change)| ≥ 1 and P < 0.05. These differential genes are described as differentially expressed genes (DEGs) for convenience. Expression pattern analyses revealed that the majority of the DEGs were non-expressed genes (NEGs) or down-regulated expressed genes (DRGs) in both TA3575  and TA7548 . Up-regulated expressed genes (URGs) accounted for 37.07% in TA3575 and 42.09% in TA7548  values of the sequences for further analysis of differential genes. A total of 69,808 genes were transcribed by CS at an FPKM ≥ 0.5 in two independent biological replicates, of which 37,058 (53.10%) were assigned to the wheat genome (expect < =1e-10&qcov > =75%) . Of the 21 chromosome pairs, chromosome 2B possessed a maximum number of transcribed genes , followed by 5B , 2D  and 6B , whereas the minimum number of genes were mapped on chromosome 6A   and 129 (16.52%) genes were non-transcribed and down-regulated, respectively, whereas the remaining 141 genes (18.05%) were up-regulated. In contrast, the transcription of genes in group 7 was least affected by introgression of 3Sl#2, and only 1.83%  of the genes were differentially transcribed , ranging from 1.47% (7/475) for the short arm of 5 A (5AS) to 7.23%  for the long arm (5AL) and 7.55% (52/689) for the short arm of chromosome 3D  out of the 1,839 genes mapped on chromosome 3B in CS were differentially transcribed in TA3575, with 33.43% on the short and 30.18% on the long arm of chromosome 3B Fig. . AnalysiAe. longissima chromosome 3Sl#2, whereas the other three were absent on chromosome 3Sl#2. Using the non-3B-specific primer pairs , gDNA of CS, N3B-T3D, and TA3575 produced the expected PCR amplification  were not expressed, two (CL22200Contig1 and CL27257Contig1) were down-regulated and transcribed, and three, including CL29051Contig1, CL53819Contig1, and CL51762Contig1, were up-regulated and expressed in TA3575 as compared to CS Fig. . On the Ae. longissima DA 6Sl#3 line TA7548 and CS on comparison of 37,058 mapped genes  DEGs, including 396 NEGs (24.87%), 526 DRGs (33.04%) and 670 URGs (42.09%), were identified based on a pairwise comparison of expressed genes in CS-.3% DEGs,548 Fig. .Figure 4l#3 in TA7548.Unlike in TA3575, where the highest ratio of non-transcribed to transcribed genes, were those genes that were located to the missing chromosome 3B. The percentage of DRGs mapped to chromosome 6A in TA 7548 was significantly higher than that for any other chromosomes. The DEGs included 13.76% on the short arm and 13.65% on the long arm of chromosome 6A  and 6A in TA7548 . Of the 561 shared DEGs, the overwhelming majority of genes  were consistently transcribed in both TA3575 and TA7548, with 84 (14.97%) not expressed, 60 (10.70%) down-regulated, and the majority of 413 up-regulated, accounting for 73.62% shared genes  to powdery mildew of wheat41. The impact of 3Sl#2 in TA3575 and 6Sl#3 in TA7548 on the transcription of CS-derived putative plant disease resistance (R) genes was analyzed in this study. A sequence homology search of DEGs in the PRGdb discovered 34 (1.71%) R genes in seven classes from 1,991 DRGs in TA3575, with the maximum number of R genes in the RLP class , followed by those in classes NL , CNL and T , and N, TNL and other   (CL77976Contig1 and CL29051Contig1) mapped on chromosome 3B (in CS) in TA3575 where chromosome 3B was missing, indicating that these two R genes are derived from 3Sl#2 rather than CS wheat chromosome 3B (Table Both chromosomes 3Sh) Table . Of thes3B Table .Table 2Ll#3  DEGs were annotated as R genes, which were grouped in six classes, including CNL (6), NL (6), other (1), RLP (16), T (3) and TNL (2) R genes. Of these R genes, five in class CNL and 12 in class RLP were down-regulated; the 17 other R genes were up-regulated in TA7548  R genes were transcribed with up-regulation, whereas the remaining five (29.41%), which all belong to class RLP, were down-regulated in both TA3575 and TA7548  rather than NEGs  of chromosome 6A-specific DEGs led to the suspicion that chromosomes 6A might be missing in some TA7548 individuals (progenies) used for RNA-seq in this study.As shown in TA3575, if a pair of wheat chromosomes is missing, non-transcribed genes account for the majority of DEGs located on the substituted chromosomes. In TA3575, where a pair of 3B chromosomes is replaced by a pair of 3Sl-specific markers showed that all these chromosome 6A-specific SSR markers were present in five of the seven plants ; two individuals  presented 6Sl-specific markers but lacked 6A-specific markers  analysis of these individual plants indicated that the two individuals missing 6A-specific markers had 42 chromosomes, 40 wheat chromosomes plus a 6Sl chromosome pair. Further fluorescent in situ hybridization (FISH) analysis verified that a pair of 6A chromosomes was replaced by a 6Sl#3 chromosome pair in these two plants, which resulted in a disomic 6Sl#3(6A) substitutions  plants. This novel CS-Ae. longissima DS 6Sl#3(6A) line, spontaneously formed during the long process of reproduction of the original DA 6Sl#3 plants, was named TA7548-6A. The substitution of chromosome 6A by chromosome 6Sl#3 in some individuals of TA7548 led to the unusually high ratio of DRGs of chromosome 6A in TA7548 used for RNA-seq in this study.Therefore, the TA7548 population used in this study was composed of about 71.43% DA 6S52. However, linkage drag that may be attributed to the inability of alien genes to replace missing wheat genes, or the interactions between the introduced alien genes and recipient wheat genes, has, until now, been relatively under-researched53. In this study, we verified the genome-wide impact on wheat gene expression by the presence of introduced alien chromosomes. Transcription of 1,413 (4.01%) of 35,219 genes mapped to non-3B chromosomes in CS were significantly changed by the substitution of a pair of chromosome 3B by chromosome 3Sl#2 derived from Ae. longissima in TA3575. Whereas 1,402 (3.93%) of 35,673 non-6A genes in CS were affected by introgression of chromosome 6Sl#3 in TA7548. The ratios of affected genes in this study were higher than the 3%  reported by Rey et al. (2018), investigating the effect of introgression of barley telosomic 7HL in CS38. Further analyses of DEGs on different wheat chromosomes demonstrated that the impact of introgressed chromosomes 3Sl#2 and 6Sl#3 was obviously different for wheat chromosomes. The minimum ratio of DEGs was associated with chromosome 5AS in TA3575 (1.47%) and chromosome 5BS in TA7548 (1.78%); whereas the highest ratios were on chromosomes 3DS in TA3575 (7.55%) and 6DL in TA7548 (6.35%). Those negatively impacted genes, including non-transcribed and down-regulated, accounted for 48.90%  in TA3575, and 53.42%  in TA7548. Whether these impacts may lead to linkage drag of agronomic traits, or the effects can be decreased in different recipient wheat backgrounds, are currently under investigation. The present study will benefit the understanding of gene interactions between recipient wheat and the alien donor species.Linkage drag, caused by alien chromosome segments introduced into wheat genetic backgrounds, has led to unfavorable agronomic and end-use quality traits and, thus, limited the utilization of alien genes in wheat improvement programs. The linkage drag caused by deleterious genes associated with targeted genes had been widely demonstratedl#2 and 6Sl#3 from Ae. longissima, 17 genes in six R gene classes were annotated as putative R genes from 561 DEGs shared by introgressed 3Sl#2 in TA3575 and 6Sl#3 in TA7548. Transcription analysis of these 17 R genes, based on transcriptome analyses, showed that 12 shared R genes (70.59%) were significantly up-regulated and transcribed in both TA3575 and TA7548, whereas only five genes (29.41%) in class RLP were down-regulated. Because both chromosomes 3Sl#2 and 6Sl#3 confer resistance to powdery mildew of wheat41, whether the transcription changes of these shared R genes were involved in the pathogen-defense processes deserved to be further investigated. Molecular marker could be developed from these R gene for mapping of the two powdery mildew resistance genes introgressed from Ae. longissima.Of those DEGs caused by introgressed chromosomes 3S55. In this study, the discovery of an abnormally high percentage of DRGs on both the short and the long arm of wheat chromosome 6A, based on RNA-seq analyses, implied that chromosome variation of chromosome 6A might exist in the CS-Ae. longissima DA 6Sl#3 line TA7548. We further verified that 28.57% of the plants in the TA7548 population were, in fact, spontaneously formed DS 6Sl#3(6A) by integration analyses of RNA-seq data, molecular markers and in situ hybridization. This novel DS 6Sl#3(6A) line (TA7548-6A) will be useful for further transfer of 6Sl#3 resistance genes to powdery mildew into wheat by inducing wheat-Ae. longissima 6Sl-6A translocations. In a similar way, we selected a spontaneous Robertsonian translocation T4Sl#2·4BL carrying the powdery mildew resistance gene Pm6656. In summary, integrated use of genome sequencing, molecular markers, and classic cytogenetic techniques can speed up the introgression of alien targeted genes, thus promoting the utilization of wild relatives of wheat in breeding programs.RNA-sequencing techniques have provided a very useful means for examining chromosome structural changesAe. longissima introgression lines carrying powdery mildew resistance gene(s) and their genetic background line wheat CS. The results showed that introgression of chromosomes 3Sl#2 and 6Sl#3 derived from Ae. longissima had genome-wide impact on gene transcription in wheat. A total of 5.37% and 4.30% genes (or 4.01% of non-3B-specific genes and 3.93% of non-6A-specific genes) were differentially transcribed due to the introduction of chromosomes 3Sl#2 in TA3575 and 6Sl#3 in TA7548, respectively. Furthermore, 17 putative R genes of wheat were significantly impacted together in both TA3575 and TA7548, which carry chromosomes 3Sl#2 and 6Sl#3. The majority of these shared R genes (70.59%) were significantly up-regulated by introgression of chromosomes 3Sl#2 and 6Sl#3. Whether these shared putative R genes were involved in the defensive processes initiated by powdery mildew resistance genes located on chromosomes 3Sl#2 and 6Sl#3 needs to be further investigated.In the current study, transcriptome analyses were performed using two CS-Ae. longissima disomic 3Sl#2(3B) substitution line TA3575, where a pair of 3Sl#2 chromosomes replaces the 3B chromosome pair in CS; and a CS-Ae. longissima disomic 6Sl#3 addition line TA7548, where a pair of 6Sl#3 chromosomes are added to the chromosome complement of CS; were used in this study. Both chromosomes 3Sl#2 and 6Sl#3 confer resistance to powdery mildew41. The number following the chromosome designation is used to distinguish between the same Ae. longissima chromosome derived from different Ae. longissima accessions57. All plant materials were kindly provided by the Wheat Genetic Resources Center (WGRC) at Kansas State University, USA, propagated and stored at the experimental station of Henan Agricultural University.Wheat landrace Chinese Spring (CS); nullisomic 3B-tetrasomic 3D , where a pair of chromosome 3B is replaced by two pairs of 3D; N6A-T6B (TA3152) where 6A is substituted by two pairs of 6B; a CS-Ae. longissima introgression lines (TA3575 and TA7548) and CS were sterilized with 5% sodium hypochlorite solution for 10 min at room temperature and then planted in 8-cm diameter pots filled with an autoclaved vermiculite potting medium. The seedlings were grown in an illuminated incubator at 18–20 °C, 18 h light and 6 h dark, and 75% relative humidity. Pooled, equal-length segments from the first leaf of 10 individuals for each line were then immediately frozen in liquid nitrogen for subsequent RNA isolation. Two independent, biological replicates were collected for subsequent cDNA library construction and RNA sequencing.Seeds of the two wheat-Total RNA was extracted from leaf samples of three plants using an mirVana miRNA Isolation Kit  following the manufacturer’s protocol. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer . The samples with an RNA Integrity Number (RIN) ≥ 7 were subjected to subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit  according to the manufacturer’s instructions. These libraries were sequenced on the Illumina sequencing platform (HiSeqTM 2500 or Illumina HiSeq X Ten) by OE Biotech (Shanghai), China, and 125 bp/150 bp paired-end reads (raw reads) were generated.58. Valid ratio, Q30 and GC content of each sample were calculated after pretreatment.Paired-end reads were pretreated using the NGS QC Toolkit software to remove sequences containing adapters or poly-N above 5%, and low-quality reads to produce valid data (clean data) for downstream sequence assemblyDe novo assembly of clean data was applied using Trinity (version: trinityrnaseq_r20131110)59, followed by the removal of abundant sequences using TGICL60. Annotation and function of these genes were assigned with a cut off e-value <1e−5 based on blastx alignment against protein sequences in public databases NR , GO  and PRGdb (Plant Resistance Gene Database)61.http://bioconductor.org/packages/release/bioc/html/DESeq.html) with a threshold FDR ≤ 0.05 & |log2(fold change)| ≥ 1. Genes with an FPKM ≥ 0.5 were considered expressed, whereas genes with FPKM = 0 were not transcribed. Genes of CS were pair-wise compared with those of TA3575 and TA7548 to identify genes present in CS and absent in TA3575 and TA7548. All the genes were assigned to chromosome arms based on blastn alignment against wheat reference genomic sequences (CS RefSeq v1.0)62 with a cutoff of expect < =1e-10&qcov > =75% at UGRI BLAST (https://urgi.versailles.inra.fr/blast/blast.php).For DEGs analysis, the read counts of genes were first normalized as FPKM. Then, the expression significance of genes of CS vs. TA3575 and CS vs. TA7548 were calculated using DESeq (Genomic DNA (gDNA) was extracted from young leaf segments with a DNeasy Plant Mini Kit  following the manufacturer’s directions. Total RNA was isolated using an RNA Prep Pure Plant Kit , and cDNA synthesized using a PrimeScript™ Double Strand cDNA Synthesis Kit .l#2-specific EST markers were used for the karyotype analysis of TA357564. Another nine PCR primer pairs of 3B-specific and three non-3B chromosomes were designed based on transcription sequences of CS and used for validation of gene expression changes in TA3575. The remaining five 6A-specific SSR and five 6Sl#3-specific markers were used for molecular marker analyses of TA754867. PCR reaction mixture preparation and amplification by “F50SSR” (for SSR markers) or “Touch-down 63” (for the remaining markers) followed Liu et al.14. The PCR products were digested with restriction enzymes followed by Liu et al.14. The PCR products or restricted fragments were resolved in 2.5% agarose gels and visualized by ethidium bromide staining under UV light.In total, 44 molecular markers were used in the study  and TAMRA (6-carboxytetramethylrhodamine) modified oligonucleotide probes followed Huang et al.68. After hybridization and slide washing, 25–30 µl of Vectashield mounting medium containing 1 µg/ml DAPI  was added to each slide and then covered with a 24 × 30 cm glass coverslip. Observation of fluorescent images was on a Zeiss Axio Scope A1 fluorescence microscope (Germany) and captured with an AxioCam MRc5 CCD camera. Images were further processed with Adobe Photoshop CS3 (Version 10.0.1) .Root tip collection, nitrous oxide treatment, fixation, and slide preparations were according to Liu Supplementary Information.Table S1.Table S2.Table S3.Table S4.Table S5.Table S6.Supplementary Figures.


H7 subtype avian influenza viruses have caused outbreaks in poultry, and even human infection, for decades in both Eurasia and North America. Although effective vaccines offer the best protection against avian influenza viruses, antigenically distinct Eurasian and North American lineage subtype H7 viruses require the development of cross-protective vaccine candidates. In this study, a methodology called computationally optimized broadly reactive antigen (COBRA) was used to develop four consensus H7 antigens . In vitro experiments confirmed the binding of monoclonal antibodies to the head and stem domains of cell surface-expressed consensus HAs, indicating display of their antigenicity. Immunization with DNA vaccines encoding the four antigens was evaluated in a mouse model. Broadly reactive antibodies against H7 viruses from Eurasian and North American lineages were elicited and detected by binding, inhibition, and neutralizing analyses. Further infection with Eurasian H7N9 and North American H7N3 virus strains confirmed that CH7-22 and CH7-24 conferred the most effective protection against hetero-lethal challenge. Our data showed that the consensus H7 vaccines elicit a broadly reactive, protective response against Eurasian and North American lineage H7 viruses, which are suitable for development against other zoonotic influenza viruses. H7 subtype avian influenza virus has caused outbreaks in poultry, and even human infection, for decades. Both North American and Eurasian lineages of H7 viruses have been associated with human infection , and theTo date, H7 influenza viruses have not caused a pandemic, but have the potential. Vaccination is an effective strategy to prevent and control enzootic influenza infection. However, antigenically distinct Eurasian and North American lineage subtype H7 viruses require the development of appropriate vaccine candidates. Currently available influenza vaccines mainly provide hemagglutinin strain-specific protection, but rarely provide cross-protection against divergent strains. It is therefore particularly important to develop an intra- and inter-subtype universal vaccine. In recent years, a methodology called computationally optimized broadly reactive antigen (COBRA) was used to design intra-subtype universal influenza vaccines . COBRA eDNA vaccines constitute a powerful alternative to conventional vaccines because they can induce both humoral and cellular immune responses. With DNA vaccinations, because the encoded protein is synthesized in its native form inside the host cell, the antibody responses induced in mice by consensus H7 DNA vaccination in our study reflected the real immunogenicity of the antigen in mice. In this study, four consensus H7 immunogens were generated based on the COBRA approach. Immunization with DNA vaccines encoding the four antigens were then evaluated in a mouse model. The mice elicited broadly reactive antibodies against H7 viruses from Eurasian and North American lineages. Further infection with Eurasian H7N9 and North American H7N3 virus strains confirmed that two consensus antigens (CH7-22 and CH7-24) conferred the most effective protection against hetero-lethal challenge.This study was reviewed and permitted by the Ethics Committee of the Wuhan Institute of Virology, Chinese Academy of Sciences. Animal experiments were carried out in an animal biosafety level 2 facility. The experiments were conducted under the Chinese national guidelines of ethics and policies for the care of laboratory animals and were certified by the Welfare and Ethical Review Board of Wuhan Institute of Virology’s Institutional Animal Care and Use Committee.50) of the viruses was determined with eight replicates in fresh medium containing 1 μg/mL TPCK-trypsin .) [Madin Darby Canine Kidney (MDCK) and BHK-21 cells were obtained from the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The H7N9 virus used in the animal experiments was NIBRG-267 (H7N9), a vaccine strain from the National Institute for Biological Standards and Control (NIBSC) and adapted to mice . H7N7 A. H7N3 [A, USA).) . n = 1502) were downloaded from the NCBI influenza virus resource and GISAID  database until early 2017. A multiple alignment analysis was conducted and a maximum-likelihood phylogenic tree was generated using MEGA7 software. Consensus generation of each round was performed as described previously [Human H7 HA protein sequences . Sequencing was performed to ensure no mutations were in the plasmid. The HA genes of A/Shanghai/02/2013 (H7N9) and A/Phalacrocorax carbo/Hubei/HH179/2013 (H7N7) were amplified and cloned into pVAX1 to generate pH7N9 and pH7N7, following the same method for COBRA HAs.After designing the consensus immunogens, the four consensus sequences were subjected to codon/RNA optimization to enhance gene expression in mammal cells as described previously ,25. The 6) were transfected with 3 μg pCH7-22, pCH7-24, pCH7-26, pCH7-28, and pVAX1 (empty vector) using Lipofectamine 3000 Transfection Reagent (Invitrogen). After incubation for 5 h, the medium was replaced with fresh medium containing 10% fetal bovine serum. At 24 h post-transfection, the cells were washed with PBS, fixed with 4% paraformaldehyde (pH 7.4) for 30 min, permeabilized with 0.2% Triton X-100 in PBS for 30 min, and then stained with Hoechst 33258 for 30 min. Indirect immunofluorescence staining was performed with monoclonal antibodies CR9114  [BHK-21 cells  of H7N9, H7N7, H7N3, and H7N1 was added to each well. After incubation of the plate at room temperature for 1 h, 50 μL of 0.5% (v/v) chicken red blood cells were added to each well, and the plate was incubated at room temperature for 30 minutes. The HAI titers were determined as the highest serum dilution that completely inhibited hemagglutination.50 H7N9, H7N7, H7N3, and H7N1 at room temperature for 1 h. The virus-serum mix was then transferred to MDCK cells. Culture medium was added, and the plates were incubated for 72 h. Endpoints were determined by the hemagglutination titer.Titers of neutralizing antibodies were determined as described previously . Serum tAn ELISA was performed using a 96-well plate  that was first coated with H7N9, H7N7, H7N3, and H7N1 (4 HAU/well) and then incubated with serial dilutions of RDE-treated serum, followed by goat anti-mouse IgG (γ-chain specific)  conjugated with HRP. The amount of chromogen produced was measured based on the absorbance at 420 nm using an ELISA reader .50) of each stock was determined using the Reed–Muench method.The mouse-adapted virus was prepared as described previously . To enhan = 10) were anesthetized and challenged with the mouse-adapted H7N9 and H7N3 viruses at 10 × LD50 by intranasal administration in 20 μL viral suspension. This infection caused rapid and widespread viral replication in the lungs and death of the unimmunized mice within 7 days. At 5 days post-infection, the trachea and lungs in each group (n = 4–5) were removed and washed twice by injecting 2 mL PBS containing 0.1% BSA. The bronchoalveolar wash was used for virus titration after removing cellular debris by centrifugation. The remaining mice were observed for 14 days to record survival rates and weight loss. Three weeks after a boost, mice in each group  or Mann-Whitney Test. The survival rate statistical analysis was performed with a Fisher’s exact test.Viral titers were calculated by the Reed-Muench method. All data plotted with error bars are expressed as means with SD. The We applied the COBRA strategy to develop cross-reactive antigens against H7 subtype influenza viruses. Eurasian and North American lineage human H7 viruses were downloaded and aligned. The viruses were divided into four groups  based on the different HA/NA combinations. The H7N9 viruses were further subdivided into five epidemic waves . PrimarySequence alignment of the four consensus antigens showed that the sequence identities ranged from 97% to 98.8%. BLAST analysis of CH7-22, CH7-24, CH7-26, and CH7-28 sequences indicated that each sequence was unique, and the identities between any one of the four sequences and sequences deposited in NCBI range from 97% to 98% (data not shown). Phylogenetic analysis of the four consensus H7 sequences with all H7 HA proteins showed that the four COBRA were located on separate branches without grouping with any viruses of Eurasian and North American H7 lineages A. Using Upon verifying expression of all four consensus H7 constructs, we first examined the capacity of these immunogens to induce immune responses in mice against antigenic and genetically diverse Eurasian and North American lineages H7 viruses. Adult female BALB/c mice were immunized twice with DNA vaccines encoding CH7-22, CH7-24, CH7-26, and CH7-28, their serum was collected and analyzed by ELISA at 2 weeks after the second immunization. As a comparison, plasmids encoding HA proteins of H7N9 and H7N7 were immunized in parallel. The serum was tested against Eurasian H7N9 and H7N7, and North American H7N3 and H7N1 viruses. The results showed that CH-22, CH-24, and CH-26 elicited high antibody titers against all tested H7 viruses, whereas CH-28 elicited relatively low titers against H7N3 . These rWe further investigated whether the elicited antibodies had the potential to cross-neutralize H7 viruses. As shown in 50 A/Shanghai/2/2013/H7N9 virus at 3 weeks after the second immunization. Four mice from each group were sacrificed at day 5 post-infection and their lungs were collected for virus titration. Changes in body weight and survival rates were monitored daily for 2 weeks post-infection. Mice that lost more than 25% of their body weight were euthanized according to guidelines. Mice vaccinated with pCH7-22, pCH7-24, pCH7-26, pCH7-28, or pH7N9 only showed slight weight loss at 3 dpi, recovered quickly, and all survived within 2 weeks . Mice vaccinated with pCH7-22, pCH7-24, and pCH7-26 all survived and showed no symptoms of morbidity or weight loss within 2 weeks  from the North American lineage was used to test the cross-protective efficacy of pCH7-22, pCH7-24, pCH7-26, pCH7-28 pH7N7, or pH7N9. Mice were vaccinated as described above. At week 6, the mice were infected with a lethal dose of mouse-adapted A/Chicken/BC/CN006/2004/H7N3  were designed by COBRA and generated. In vitro experiments confirmed that head-specific P52E03 and stem-specific CR9114 mAbs bound the cell surface-expressed consensus HAs, indicating retention of the antigenicity of the four consensus HAs. It has been shown that COBRA-designed consensus proteins retain highly immunogenic and cross-reactive epitopes . ConsistIn viral infection experiments, protection against lethal H7N9 viral challenge was also provided by DNA immunization with pCH7-22, pCH7-24, pCH7-26, or pCH7-28. The viral titers were undetectable in the lungs of consensus DNA-immunized mice at 5 dpi, suggesting that the consensus H7 vaccines that we designed and tested effectively prevented lethal H7N9 infection in mice. The clinical outcomes of mice immunized with consensus H7 were consistent with the antibody response. The four consensus H7 DNAs induced higher IgG antibody, HAI, and neutralizing antibody titers against H7N9 than pH7N7 and induced a similar high titer compared with pH7N9. Therefore, a lower survival rate, more weight loss, and higher lung viral titers were observed in the pH7N7-immunized group. The H7N9 virus infected humans in 2013 . The 201We next tested the efficacy of the consensus H7 proteins against American lineage H7N3. After challenge with a lethal H7N3 virus, the results showed that all mice vaccinated with pCH7-22, pCH7-24, and pCH7-26 survived and showed no symptoms of morbidity or weight loss within 2 weeks. Consistent with the survival rate and weight loss, no lung viruses were detected in mice vaccinated with pCH7-22 or pCH7-24 at 5 dpi. Mice that received pCH7-28, pH7N7, and pH7N9 had apparent weight loss and only showed partial protection against lethal H7N3 infection with survival rates of 83%, 40%, and 60%, respectively. CH7-28 was derived from CH7-26 sequences by introducing S136N and A143V mutations based on the analysis showing that both mutations emerged in H7N9 HA since the second wave. The introduction of the two mutations may affect the conformational epitopes and subsequently result in less cross-neutralizing, since the antibody titers against American lineage H7N3  in CH-28 immunized group were apparently lower than in the other three consensus H7 protein immunized groups. Based on these results, consensus CH7-22 and CH7-24 proteins showed better efficacy against American lineage H7N3 than the other consensus proteins. Geographical separation of the host species has shaped H7 AIVs into independently evolving Eurasian and American lineages . Among eIn this study, we describe development of a highly effective consensus H7 HA using the COBRA method, which stimulates robust cross-reactivate immune responses. Two (CH7-22 CH7-24) out of four consensus H7 antigens have the potential for development as a H7 universal vaccine candidate. These results also highly suggest that using COBRA to design consensus HAs is an applicable method to develop universal vaccines for not only H7 subtypes, but also other subtypes .