In addition, we also showed that inhibition of H3R by siRNA or CPX inactivated the PI3K/Akt and MEK/ERK signaling pathways, while inhibition of Akt or ERK activity with antagonists or siRNAs suppressed H3R agonist (R)-(α)-(−)- methylhistamine dihydrobromide (RAMH) mediated invasion and reorganization of cadherin-household. In conclusion, overexpression of H3R is associated with glioma progression. Inhibition of H3R leads to suppressed invasion and EMT of GBM by inactivating the PI3K/Akt and MEK/ERK pathways in gliomas.Histamine receptor 3 (H3R) is expressed in various tumors and correlated with malignancy and tumor proliferation. However, the role of H3R in tumor invasion and epithelial to mesenchymal transition (EMT) remains unknown. Here, we explored the H3R in the highly invasive glioblastoma (GBM) and U87MG cells. We found that H3R mRNA and protein levels were up-regulated in the GBM and glioma cell lines compared to normal brain tissue and astrocytes. In U87MG cell line, inhibition of H3R by siRNA or the antagonist ciproxifan (CPX) suppressed proliferation, invasiveness, and the expression of EMT activators . In addition, expression of epithelial markers (E-cadherin and ZO-1) was up-regulated and expression of mesenchymal markers (vimentin and N-cadherin) was down-regulated  Diffuse infiltration of tumor cells in the brain is a central hallmark of high-grade astrocytomas, especially glioblastoma (GBM) , 2. TumoCompared with the other three histamine receptors subtypes, histamine receptor 3 (H3R) is mostly expressed in neurons, in which regulates the synthesis and release of histamine and neurotransmitters as presynaptic autoreceptors and heteroreceptors –8. AlthoH3R functions as a presynaptic autoreceptor and heteroreceptor at numerous neurotransmission crossroads, and it regulates the release and synthesis of neurotransmitters, such as histamine, acetylcholine, dopamine, norepinephrine and 5-hydroxytryptamine . It has In order to verify our hypothesis, the present studies were designed to (1) explore the H3R expression in the human astrocytoma tissues and cell lines by immunohistochemistry assay, real-time qPCR and western blot; (2) inhibit the overexpressed H3R by small-interfering RNA (siRNA) or antagonist in the U87MG cells to evaluate the role of H3R in the metastasis nature, EMT activators and markers in the U87MG cells; (3) explore the signaling pathways downstream of H3R by siRNAs or the selective agonist/antagonist in the U87MG cells; (4) explore the effects of knockdown or blockade of H3R on a xenografted model.To date, evidence for the expression of H3R in glioma was lacking and there was only one study reporting that H3R is not expressed in U373MG cells . Here, 4P < 0.01). In addition, the PEA and MD of H3R protein expression in the GBM tissue were significantly higher than in the AA tissues (P < 0.01). These results were confirmed using real-time PCR and western blot techniques . In addition, the relative level of H3R mRNA and protein expression in HGA was significantly higher than that in the LGA (P < 0.01). Both mRNA and protein expression of H3R in the GBM were also significantly higher than in the AA (P < 0.01).Figure  Figures . We founin vitro experiments. In order to explore the role of H3R in these cells, a siRNA specific for H3R was employed. Western blot analysis of lysates of cells incubated with the H3R siRNAs .H3R expression levels in the normal human astrocytes (hAstrocytes) and glioma cell lines  were measured using real-time qPCR and western blot techniques. The results in Figure s Figure  showed t4 cells/well) were incubated with the H3R selective agonist (R)-(α)-(−)-methylhistamine dihydrobromide (RAMH) or/and its antagonist ciproxifan (CPX). We found that pretreatment with the H3R selective agonist RAMH (10−3-10 μmol/L) promoted proliferation of U87MG cells in a dose-dependent manner. In contrast, blocking H3R activity with an antagonist CPX (10−3-10 μmol/L) suppressed proliferation of U87MG cells in a dose-dependent manner . Down-regulation of H3R expression also resulted in fewer cells infiltrating the membranes in the transwell assay compared to the control , which was inhibited by CPX. In addition, blockade of H3R by CPX led to a sharp decline in migration (P < 0.01). As expected, RAMH increased the number of infiltrating U87MG cells in the transwell assay .A selective H3R agonist and antagonist were used in parallel in the wound healing and transwell assays to confirm our results as shown in Figure P < 0.01, Figure P < 0.01, Figure The mRNA levels of the EMT-activators snail, slug and twist were measured in U87MG cells depleted of H3R. We found that all markers of EMT were significantly down-regulated compared with the control group . In both cases, the total protein levels of PI3K, Akt, MEK and ERK . These observations indicated that the H3R mediated activation of PI3K/Akt and MEK/ERK signaling pathways in U87MG cells depended on PTX sensitive Gi/o-proteins.The Akt and ERK signaling pathways have been reported to play a role in modulating cell invasion and the progression of EMT. Here, we examined the effect of H3R on these two signaling pathways. We found that H3R down-regulation by siRNA significantly decreased the phosphorylation of PI3K, Akt, MEK and ERK, but not their total protein levels  siNC group: nude rats were inoculated with the U87MG cells with mock-transfection for 4 w; (2) siH3R group: nude rats were inoculated with the U87MG cells transfected with H3R siRNAs for 4 w; (3) Vehicle group: nude rats were inoculated with the U87MG cells. When the subcutaneous tumors reached 50 mm3 (about 2 w), the nude rats received I.V. injections of vehicle (0.9% NaCl) once a day for 4 w; (4) CPX group: nude rats were inoculated with the U87MG cells. When the subcutaneous tumors reached 50 mm3 (about 2 w), the nude rats received I.V. injections of CPX (3 mg/kg) once a day for 4 w. After 4 w, the tumor volume in the siNC group is higher than that in the siH3R group  inhibited hyperplastic biliary growth by inhibition of cAMP [o) decreased the growth of cholangiocarcinoma Mz-ChA-1 cells by inositol triphosphate (IP3)/Ca2+, which is independent of cAMP activation [The up-regulated expression of H3R in the GBM compared to other grades of astrocytomas suggested a possible role in tumor progression of the GBM. In order to investigate the role of H3R in tumor progression, we used the H3R antagonist CPX and a specific siRNA for H3R isorders –29. We frocytoma , 30. Introcytoma , while arocytoma , 32. Therocytoma , while Hrocytoma . Second,rocytoma , 33. Spe of cAMP , while Rtivation –16. In dtivation , while Hin vitro, while activation of H3R by an agonist had the opposite effect. Studies in GBMs showed that EMT (−like) processes are of clinical relevance in malignant brain tumors [in vitro and in vivo as shown in animal studies and in patient-derived specimens [Even though the role of H3R in tumor proliferation has been extensively documented, it remained to be determined whether H3R is involved in the regulation of invasion. Here we showed for the first time that inhibition of H3R suppressed migration and invasion of glioma cells n tumors –37, whicn tumors , 39. Mempecimens –42. In oi/o protein manner. In order to further demonstrate the role of above signaling pathways in the H3R related cell invasion and EMT progress, we applied the selective antagonist or we down-regulated the expression of signaling components of the PI3K/Akt and MEK/ERK pathways. Either knockdown or blockade of the PI3K/Akt or MEK/ERK signaling pathways inhibited RAMH-mediated increase in cell invasion and reorganization of the cadherin-household, confirming that PI3K/Akt and MEK/ERK signaling pathways play a role in the H3R-mediated tumor invasion and EMT progression.A possible explanation for the different tumor behaviors of GBM is the deregulation of signal transduction pathways such as the PI3K/Akt and MEK/ERK pathways caused by a large number of genetic abnormalities . A largeSince histamine and histamine receptors were discovered, the evidence of their role in oncology, including astrocytoma. It has been suggested that there are multiple and context-dependent effects of H3R on proliferation in tumors. However, direct evidence of the expression of H3R in glioma or an understanding of its involvement in tumor invasion and EMT progress was lacking. Here we found that H3R was over-expressed in glioma cells and tissues and inhibition of H3R suppressed invasion and EMT in glioblastoma through the PI3K/Akt and MEK/ERK signaling pathways. The biological functions of H3R found in this study provided a basis for additional pathological and clinical investigations, suggesting that H3R may be a novel target for therapeutic intervention in GBM.The protocol for this study was approved by the ethics committee of the Fourth Military Medical University. Forty patients from the Tangdu Hospital diagnosed with astrocytoma between 2008 and 2012 were selected for the study. All patients involved in the study provided consent. Patients with other medical conditions or those who received treatment prior to surgery were also excluded. The pathological specimens were reviewed, and histological classifications of astrocytoma were made based on the World Health Organization (WHO) Classification of brain tumors . We idenHuman glioma cell lines, C6MG, IMR-32, U87MG and U251MG cells were purchased from the American Type Culture Collection (Rockville) and the Cell Bank of the Chinese Academy of Sciences. Cells were cultured using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. Normal human astrocytes (hAstrocytes) were obtained from ScienCell and propagated in astrocyte medium (ScienCell).U87MG cells growing in 6-well plates were incubated with human siRNAs (100 nmol/L) for H3R/Akt2/ERK1. Mock-transfection was performed using a negative control siRNA (Santa Cruz Biotechnology) as control. Cells were harvested at 48 h post transfection, washed and stored for future experiments. The knockdown efficiency was assessed using the western blot assay.4 cells/well) for 24 h. At this time, 10 μL CCK-8 reagent (Dojindo) was added to each well and then the plates were incubated at 37°C for 90 min. Absorbance was measured using a Microplate Reader (Bio-Rad).Cells were plated in 96-well plates  fitted with polyethylene terephthalate (8.0 mm pore size). The agonist/antagonists for H3R, PI3K, MEK or the Gi/o protein  were then added to the upper chamber as follows  and 900 μl DMEM media with 20% FBS was added to lower chamber of each well. After 48 h, cells were fixed using 4% paraformaldehyde and dyed with crystal violet. Cells on the top surface of the insert were removed with a cotton swab. Cells adhering to the lower surface were fixed with methanol, stained with Giemsa solution and counted under a microscope in five predetermined fields (200×).The upper chamber of a 24-well hanging cell culture insert was pre-coated with 24 μg/μl Matrigel (BD Biosciences). 5 × 107 in 100 mL of saline) were suspended in a volume of 0.2 ml serum-free medium and injected subcutaneously into the hind flanks of the animals. At the end of the treatment, the nude rats were sacrificed and tumor volumes were calculated (0.5 × length (mm) × width2 (mm2). Tumors were harvested, fixed in formalin and embedded in paraffin.All animal procedures were performed following the National Animal Experimentation guidelines and were approved by the ethics committee of the Fourth Military Medical University. All mice were bred in aseptic conditions at a constant humidity and temperature with standard 12 h light-dark cycles and free access to drinking water and standard chow. U87MG cells (1 × 102O2 and 60% methanol PBS (pH 7.4) for 30 min and then treated with 0.01 mol/L sodium citrate buffer at 95°C in a microwave oven for 13 min . Specimens were blocked using a 5% normal goat serum and 5% bovine serum albumin in PBS. Before each step, sections were rinsed three times in PBS buffer. Incubation with H3R primary antibodies  was performed in a PBS-based solution of 1% bovine serum albumin for 12 h at 4°C in the recommended dilutions. After rinsing with PBS, sections were incubated with the corresponding secondary biotinylated antibodies for 1 h at room temperature. A streptavidin/horseradish peroxidase complex was then applied as a detection system for 1 h . Finally, staining was developed using 3, 3′diaminobenzidine tetra-hydrochloride in 0.05 mol/L Tris-HCl buffer and 0.1% H2O2. Negative control sections were incubated without the primary antibody.The pathology specimens were quickly removed, post fixed overnight at 4°C, dehydrated in graded sucrose and processed for embedding in paraffin wax with the anatomical orientation preserved. Sections 3–4 μm in thickness were mounted on silane-coated slides and air-dried. Before immunohistochemistry staining, sections were placed in a bathing solution of 3% HImage analysis was performed using a DMR-X microscope coupled to a DC500 digital camera (Leica) and the image analysis system Quantimet Q550 (Leica). Nine randomly selected discontinuous fields (20×) per samples were evaluated. The positive area, total area and the integrated optical density of H3R were stained and quantified using the Image-Pro Plus software (Media Cybernetics). The PEA was measured as positive area/total area × 100%. The MD of H3R expression in a selected discontinuous fields was measured as the integrated optical density/total area × 1000. The average of PEA and MD form the above nine fields were calculated as the behalf of a sample for the further data analysis.Tissue samples and cells were put into liquid nitrogen for 10 min and then into an ultra freezer at 80°C. Total RNA was extracted from glioma cell lines and sample tissues using the Trizol reagent according to the manufacturer's instructions (Takara). RNA was transcribed to cDNA using the PrimeScript RT reagent Kit (Takara). For H3R expression analysis, the products were separated by electrophoresis on 1.5% (v/v) agarose gels, stained with ethidium bromide, and visualized under UV light. The qRT-PCR for other investigated genes was performed using the CFX96TM real-time system (Bio-Rad), and the relative gene expression was normalized to the internal control β-actin. The primers used were as follows: H3R: Forward: 5′-TCATCGTGAGCATCTTTGGG-3′, Reverse: 5′-ACAGAGGGTAGAGG ACAGGGTT-3′; Snail: Forward: 5′-TTTACCTTCCAGCAGCCCTA-3′, Reverse: 5′-GACAGAGTCCCAGATGAGCA-3′; Slug: Forward: 5′-ACAGCGAACTGGAC ACACAT-3′, Reverse: 5′-GGAATGGAGCAGCGGTAGT-3′; Twist: Forward: 5′-CACCATCCTCACACCTCTGC-3′, Reverse: 5′-GCTGATTGGCACGACCTCT-3′.Proteins were extracted from tissue samples or cells. Total protein concentration was determined using a bicinchoninic acid (BCA) kit for protein determination (Takara). The samples were resolved in 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Immunoblots were probed with primary antibodies overnight at 4°C. The following antibodies used were from Abcam: H3R (1:1000), E-cadherin (1:10000), N-cadherin (1:5000), ZO-1 (1:50), vimentin (1:1000), phospho-PI3K (Tyr607)(1:500). The following antibodies were from Cell Signaling: phospho-Akt (Ser473) (1:1000), Akt (1:1000), phospho-ERK1/2 (Thr202/Tyr204)(1:1000), ERK1/2 (1:1000), phospho-MEK1/2 (Ser217/Ser221)(1:1000), MEK1/2 (1:1000), PI3K (1:1000) and β-actin (1:1000). All primary antibodies were used according to the instructions provided by the supplier. After that, the membrane was incubated with the corresponding HRP-linked anti-rabbit IgG antibody  at room temperature for 1 h. The blots were visualized using the ECL-Plus reagent (Millipore).U test was used for data with heterogeneity of variance. A value of P < 0.05 was considered significant.SPSS 16.0 (SPSS Inc) and Graph Pad Prism 5.0 software (Graph Pad Software) were used to analyze the data and all values were expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA  followed by Bonferroni's multiple comparison tests where appropriate. The Mann-Whitney


Our findings suggest that adaptive changes that enhance viral receptor binding, thermostability, and replicative capacity in mammalian cells can collectively enhance the transmissibility of H9N2 AIVs by direct contact in the guinea pig model.H9N2 avian influenza viruses circulate worldwide in poultry and have sporadically infected humans, raising concern whether H9N2 viruses have pandemic potential. Here, we use a guinea pig model to examine whether serial passage results in adaptive viral changes that confer a transmissible phenotype to a wild-type H9N2 virus. After nine serial passages of an H9N2 virus through guinea pigs, productive transmission by direct contact occurred in 2/3 guinea pig pairs. The efficiency of transmission by direct contact increased following the fifteenth passage and occurred in 3/3 guinea pig pairs. In contrast, airborne transmission of the passaged virus was less efficient and occurred in 1/6 guinea pig pairs and 0/6 ferret pairs after the fifteenth passage. Three amino acid substitutions, HA1-Q227P, HA2-D46E, and NP-E434K, were sufficient for contact transmission in guinea pigs (2/3 pairs). The two HA amino acid substitutions enhanced receptor binding to α2,3-linked sialic acid receptors. Additionally, the HA2-D46E substitution increased virus thermostability whereas the NP-E434K mutation enhanced viral RNA polymerase activity  Influenza viruses pose a continual threat to public health in the form of seasonal epidemics and occasional pandemics. To date, influenza viruses bearing H1, H2, H3, H5, H6, H7, H9 and H10 hemagglutinin (HA) subtypes have infected humans12342The ongoing evolution of H9N2 influenza viruses in nature poses a significant public health risk. Many avian H9N2 influenza viruses can replicate in mammals without prior adaption101113Several experimental approaches have been used to explore the capacity of avian influenza subtypes to acquire a transmissible phenotype. In general, these approaches rely upon the genetic reassortment of an avian subtype virus with a human influenza virus, the adaptation of an avian subtype virus in mammals through serial passage, or a combination of both of the above. These approaches have been used to demonstrate that reassortant H5N1 viruses containing 2009/H1N1 genes were capable of respiratory droplet-mediated transmission in guinea pigs and ferrets161719http://blast.ncbi.nlm.nih.gov/Blast.cgi), the limited number of reported human H9N2 infections suggests that α2,6-linked SA receptor binding is not sufficient for infection or human-to-human transmission.The ability of avian influenza viruses to transmit between mammalian hosts appears to be require multiple viral features, including α2,6-linked sialic acid (SA) receptor binding (the human-type receptor) as well as high levels of heat stability and increased polymerase activity in human cells161718212223242526272930Serial passage of AIVs in mammalian hosts can result in adaptive viral changes that confer an increased replication capacity in mammalian cells and may enhance transmission between hosts. We sought to explore the impact of mammalian adaptation on transmission dynamics of an H9N2 AIV using a guinea pig model. A wild-type H9N2 AIV isolate with affinity for both α2,6-linked SA (the human-type receptor) and α2,3 -linked SA (the avian-type receptor) moieties was serially passaged through guinea pigs. After every third passage, we evaluated the ability of the adapted virus to transmit by direct contact in guinea pigs. Guinea pig-adapted H9N2 viruses acquired the ability to transmit by direct contact, but were less efficiently transmitted via respiratory droplets in guinea pigs. These findings suggest that mammalian adaptation of H9N2 viruses may generate viruses capable of transmission by direct contact in mammals, although additional amino acid substitutions are likely required for efficient airborne transmission.50 values >6.5  (abbreviated as SD2/H9N2), A/Chicken/Shandong/Li-3/2010 (H9N2) (abbreviated as SD3/H9N2), and A/Chicken/Jilin/Hu-3/2006 (H9N2) (abbreviated as JL3/H9N2), isolated from chickens for properties of virulence in mice and receptor binding specificity. Each of the three H9N2 viruses displayed MLDA solid-phase binding assay was used to further assess the receptor binding specificities of each virus. The wild-type SD2/H9N2 virus, possessing a phenylalanine residue at HA position 226, preferentially recognized the human-type receptor and only weakly recognized the avian-type receptor . The SD3296 EID50 of SD2/H9N2, SD3/H9N2, or JL3/H9N2. The XD/H1N1 virus was used as a positive control for viral transmission between guinea pigs. Each inoculated guinea pig was housed in a separate cage and isolator. Twenty-four hours after inoculation, a naïve guinea pig was placed into each cage with an inoculated animal. To assess productive transmission, we evaluated nasal washes collected from the naïve contact guinea pigs at various time points post-contact for the presence of virus and assessed serum for HI antibody responses after 21 days. While virus was detected in the nasal washes of all inoculated guinea pigs following i.n. inoculation with each H9N2 virus from days 2 to 4 post-infection, none of the naive contact guinea pigs seroconverted or had detectable virus in nasal washes , passage 12 (P12) and passage 15 (P15) acquired the ability to transmit to naive guinea pigs in a direct contact model . The P9 th passage, five amino acid substitutions were identified, including PB2-D195N, HA1-Q227P, HA2-D46E, NP-E434K, and M1-Q211R  was productively transmitted from inoculated guinea pigs to 3/3 paired naive contact animals . NotablyWe next generated a panel of recombinant viruses in which one or more of the five adaptive substitutions identified in the P15 virus was omitted and evaluated transmission efficiency in the guinea pig contact model. Recombinant viruses lacking either HA1-Q227P, HA2-D46E or PB2-D195N replicated well in inoculated guinea pigs, but productively transmitted to only 2/3 naive contacts by direct contact in each case, suggesting that these substitutions are not strictly required for transmission, but may enhance transmission efficiency . A recomThe above results suggest that NP-E434K and either HA1-Q227P or HA2-D46E substitutions are required for contact transmission of the adapted virus in guinea pigs. To test the impact of these amino acid substitutions in combination, we generated recombinant viruses harboring NP-E434K, HA1-Q227P, and HA2-D46E by reverse genetics. The HA1-Q227P-, HA2-D46E- and NP-E434K-containing recombinant virus was transmitted in 2/3 guinea pig pairs in the direct contact model . In cont1P/D462E) displayed enhanced binding to both avian- and human-type receptors , whereas HA2-D46E is located in the α-helix of the HA2 subunit. Solid-phase binding assays were performed to determine whether these substitutions affected virus receptor preference. The HA1-Q227P and HA2-D46E substitutions independently increased the ability of each virus to bind an avian-type α2,3 SA receptor when compared to the parental r-SD2/H9N2 virus . The comeceptors . These rWe evaluated the thermostability of viruses with wild-type HA or variant HA proteins harboring the HA1-Q227P and/or HA2-D46E substitutions as previously described22P < 0.001; one-way ANOVA test). Since the P15 virus possessed two amino acid substitutions in the viral replication complex, PB2-D195N and NP-E434K, we respectively assessed the impact of each single substitution on viral polymerase activity. The PB2-D195N substitution increased polymerase activity by about 3.5-fold , while the NP-E434K substitution resulted in 14-fold higher polymerase activity when compared to wild-type polymerase activity . These results show that the PB2-D195N and NP-E434K substitutions, which were required for direct contact transmission, increased viral polymerase activity.We next assessed whether adaptation in guinea pigs led to enhanced viral RNA polymerase activity. The polymerase activity of the parental SD2/H9N2 virus was compared to that of the P15 adapted virus using a mini-replicon assay in 293T cells at 37 °C . The pol6 EID50 of the r-P15 virus, or the wild-type SD2 virus or XD/H1N1 virus as transmission controls. Twenty-four hours later, naïve contact animals were placed in cages next to the inoculated animals. In the guinea pig model, all inoculated animals had detectable virus in nasal washes on days 2 and 4 post-infection . These results suggest that the adaptive changes acquired during serial passage of the H9N2 virus in guinea pigs did not substantially enhance viral virulence in mice.To explore whether the pathogenicity of the adapted H9N2 virus changed, groups of three 5 to 7-week old BALB/c mice were inoculated i.n. with 10We evaluated changes in viral antigenicity that may have occurred during serial passage of the H9N2 virus in guinea pigs as previously describedPrevious studies have demonstrated that amino acid substitutions acquired during mammalian adaptation of AIVs can enhance transmission efficiencies in mammals17We explored the mechanistic basis by which these three amino acid substitutions may contribute to the ability of the adapted H9N2 virus to transmit by direct contact. Receptor binding preference is widely recognized to be an important factor for influenza virus transmission in different animal model systems1819232436in vitro assay to characterize viral polymerase activity, we showed that the PB2-D195N and NP-E434K substitution independently enhanced polymerase activity in mammalian cells. However, the combination of these two substitutions did not further increase the viral polymerase activity compared to the effect of single NP-E434K substitution, implying the role of PB2-D195N substitution should be further studied . The protocol of the study was conducted in accordance with guidelines of animal welfare of World Organization for Animal Health.Studies with H9N2 virus and adapted variants were performed in a biosecurity level 3 laboratory approved by the Military Veterinary Research Institute of the Academy of Military Medical Sciences. The research program, procedures, occupational health plan, security and facilities are reviewed by the Academy of Military Medical Sciences.All H9N2 viruses used in the study were isolated from chickens and include: A/Chicken/Shandong/Li-2/2010(H9N2), A/Chicken/Shandong/Li-3/2010 (H9N2), and A/Chicken/Jilin/Hu-3/2006(H9N2). The 2009 pandemic H1N1 influenza virus, A/Changchun/01/2009(H1N1), was acquired from the Academy of Military Medical Sciences. Viruses were stored at −80 °C.Hartley strain female guinea pigs weighing 300–350 g  and 4-month-old male ferrets  serologically negative for prior influenza virus infection were used in these studies. Ketamine (20 mg/kg) and xylazine (1 mg/kg) were used to anesthetize animals by intramuscular injection.6 EID50 of SD2/H9N2, SD3/H9N2, JL3/H9N2 or XD/H1N1 in 300 μl. Each inoculated guinea pig was housed in a separate cage in an independent isolator. Twenty-four hours later, one naive guinea pig was introduced into each cage with an experimentally inoculated animal. Nasal washes were collected from each guinea pig at two day intervals, beginning two days post-inoculation . Virus titers were determined by titration in eggs. To prevent inadvertent physical transmission of virus by investigators, the contact guinea pigs were handled first, and all materials used to handle and manipulate the guinea pigs during nasal wash collection were changed between animals. The ambient conditions for these studies were set at 20–22 °C with 30–40% relative humidity. Horizontal airflow in the isolator was set a speed of 0.1 m/s. Sera were collected from all animals after 21 days for hemagglutinin inhibition (HI) antibody detection.To evaluate the efficiency of each virus to transmit by direct contact, groups of three guinea pigs were inoculated intranasally (i.n.) with 106 EID50 of SD2/H9N2 virus or a guinea pig-adapted virus. As controls, groups of three guinea pigs or ferrets were inoculated i.n. with 106 EID50 of SD2/H9N2 virus or XD/H1N1 virus. Each inoculated animal was housed in a separate cage in an independent isolator. After 24 hours, a naïve animal was placed in a chamber adjacent to the chamber housing the inoculated animal (separated by 2cm). Nasal washes were collected at 2 day intervals, beginning on day 2 post-inoculation (1 day post-contact). Viral titers were determined by titration in eggs. Sera were collected from all animals on day 21 for HI antibody detection.To measure the capacity of each virus to transmit by respiratory droplets, groups of six guinea pigs or ferrets were inoculated i.n. with 106 EID50 of SD2/H9N2, SD3/H9N2, JL3/H9N2, or guinea pigs adapted virus. Mice were monitored daily for weight loss and mortality for 14 days. Mice that lost more than 25% of the initial body weight were recognized as experimental end point and humanely euthanized. Serum was collected from each mouse on day 21 for HI antibody detection.To evaluate the pathogenicity of H9N2 AIVs in mice, groups of three 5 to 7 week-old female BALB/c mice  were lightly anesthetized with dry ice and inoculated i.n. with 106 EID50 of SD2/H9N2 virus. Three days later, a naïve guinea pig was inoculated i.n. with 300 μl of nasal wash from the previously inoculated guinea pig. Fifteen successive passages were performed. Successful infection of each guinea pig in the series was confirmed by titrating virus present in the nasal washes from 2 to 5 days p.i. in eggs. As the constellation of adaptive amino acid substitutions in viruses generated each independent line of guinea pigs was found to be identical after 15 passages, one lineage of adapted H9N2 was used for subsequent analyses. For the selected adapted lineage, the viral genome was sequenced after every third passage and the capacity for transmission assessed in the guinea pig contact model as described above.SD2/H9N2 was serially passaged in two independent lines of guinea pigs in order to identify amino acid substitutions associated with mammalian adaptation of an avian-origin H9N2 virus. In brief, the first guinea pig in each independent line was inoculated i.n. with 300 μl containing 10Viral RNA was extracted from the nasal wash of guinea pigs on day 3 post-inoculation after every third passage. Viral genes were amplified by reverse transcription-PCR using QIAGEN One-Step RT-PCR Kit  according to the manufacturers’ instructions. Amplified cDNAs were sequenced by Comate Bioscience Corporation.Each of the eight SD2/H9N2 viral gene segments was amplified and rescued by using the pBD plasmid as previously describedSialidase-treated Chicken red blood cells (CRBCs) were prepared as previously describedVirus receptor specificity was evaluated using a direct solid phase assay as described previously25The thermostability of wild-type virus and viruses harboring amino acid substitutions in the HA protein was evaluated as follows. Sixty-four HA units of each virus were incubated at 50 °C–58 °C in 2 degree increments for 60 minutes, or were incubated at 54 °C for 1–5 hours. Heat-treated viruses were evaluated by using a haemagglutination assay.A dual-luciferase reporter assay system  was used to compare the polymerase activities of viral RNP complexes. The PB2, PB1, PA and NP gene segments of wild-type and the adapted virus generated following 15 passages in guinea pigs were cloned into the pCDNA3.1 expression plasmid and transfected to 293T cells with the p-Luci luciferase reporter plasmid and the Renilla internal control plasmid. Cell lysates were analyzed 36 hours after transfection to measure firefly and Renilla luciferase activities. Values shown are the mean ± standard deviation (SD) of three independent experiments and are normalized to the values obtained for wild-type virus at 37 °C (set as 100%).Vibrio cholerae  receptor-destroying enzyme before hemagglutination inhibition (HI) tests. For antigenicity analysis, the sera were collected from BALB/c mice inoculated i.n. with 106 EID50 of wild virus, P9 virus and P15 virus on day 21 p.i. The antigenic relatedness between the different viruses was expressed as R-value based on the Archetti and Horsfall formula35Sera were treated with P-values less than 0.05 were considered statistically significant.Statistically significant differences between experimental groups were evaluated using analysis of variance  with the GraphPad Prism 5 software package . How to cite this article: Sang, X. et al. Adaptation of H9N2 AIV in guinea pigs enables efficient transmission by direct contact and inefficient transmission by respiratory droplets. Sci. Rep.5, 15928; doi: 10.1038/srep15928 (2015).


The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory. + T cells, in particular T follicular helper (Tfh) cells, have a major role in mediating antiviral immunity by providing help to B cells, which is key to a strong and efficient anti-HIV antibody response. The unique RV254 cohort provided the best setting to analyze immune responses during very early acute HIV, as the study was able to enroll individuals that were infected for less than 2 weeks and initiated treatment approximately 1–2 days after recruitment. We aimed to study the capacity of memory circulating Tfh (cTfh) cells to promote B cell help in acute HIV infection, and found the interaction to be dysfunctional in the later stage compared to the very early stages, accompanied by increased levels of proinflammatory cytokines and a reduction in regulatory cytokines. High levels of plasma viremia correlated with low cTfh-mediated B cell antibody production in later stage acute individuals; and memory B cells were significantly decreased but could be restored with ART, compared to chronically infected individuals, who could not normalize this compartment compared to healthy individuals. Overall, we show that the cTfh- B cell interaction and B cell memory compartment is altered in late stage acute infection, mainly attributed to an increase in inflammation and skewing of the response away from helper to proinflammatory. Identifying individuals for treatment in the earliest stages of acute infection, prior to immune damage, could preserve cTfh function and the anti-HIV B cell response, therefore reducing the chances of viral rebound upon the cessation of ART.The HIV-specific T cell memory response diminishes rapidly even after the initiation of anti-retroviral treatment (ART), and there is no control of viral rebound if treatment is interrupted. Restoration or preservation of memory T cells or B cells with treatment, to allow for control of virus replication after ART is stopped, is therefore very important. CD4 The progressive nature of immune dysfunction in HIV infected individuals has implied that early treatment could play a critical role in reducing immune defects and in preserving T and B cell memory responses against HIV infection –3. Despi+ memory cells [Even with ART, low level HIV replication in lymphoid tissues has been shown to maintain a state of chronic immune activation . B cell ry cells –19.+CXCR5+ T cells in the blood known as memory circulating Tfh (cTfh), that are also very efficient at inducing B cell differentiation and providing B cell help; and much progress has been made to characterize and understand their biology [+ Tfh function, and that phenotypic perturbations of T and B cell populations that are characteristic of acute HIV infection are better resolved in very early treated individuals. As the HIV-associated microenvironment likely affects the Tfh program [CD4 T follicular helper cells (Tfh) are specialized in providing help to B cells and support B cell maturation and differentiation to long-lived plasma cells in the germinal center (GC) . It ther biology , 21–23.  biology . We have biology . It is t program , it is athG stages 1 and 2 grouped)  and stage 3 (4thG stage 3)  [The unique RV254/SEARCH010 Thai cohort is strategic in that HIV infected participants are recruited during the earliest stage of acute infection and receive ART almost immediately . This poxposure) ; and inv+CXCR3-) in peripheral blood from stage 1/2 are similar to those from stage 3 at week 0. Similar frequencies of cTfh cells were observed in all stages  were performed. Cocultures of cTfh cells from stage 3 elicited significantly less help to B cells compared to cocultures of cTfh cells from stage 1/2 individuals, as evidenced by reduced levels of total IgG  suggesting that the defect in stage 3 is specific for cTfh/B cell interaction. Of note, although CXCR5+CXCR3- cTfh cells are mostly enriched in a subset that provides efficient B cell help, the CXCR5+CXCR3+ T cell subset can also provide help, but to a lesser degree , activated memory B cells (CD21-CD27+) or exhausted tissue-like B cells (CD21-CD27-) between stage 1/2 and stage 3   with cTfh from healthy individuals (effective) in coculture with memory B cells (obtained from stage 3). We found that replacing cTfh showed signs of increased total and HIV-specific antibody levels , as wellIn order to further discern the contribution of memory B cells and determine the extent of B cell impairment prior to coculture with cTfh, we measured memory B cell proliferation and antibody production following polyclonal stimulation with CpG oligonucleotide. Our results indicated that similar proliferation profiles between the two stages and apparent non-significant increase in total IgG production in stage 1/2 when compared to stage 3 . OverallAdditionally, we found that cocultures of cTfh cells obtained from patients at stage 3 are polarized towards a proinflammatory phenotype. Our results imply that this skewing of cTfh cells towards a non-cTfh program is associated with elevated levels of viral load observed at stage 3 when compared to stage 1/2. This has been confirmed by the significant correlation between viral load at week 0 and the impaired cTfh function as measured by the production of total and HIV-specific antibodies in culture supernatants. It is not clear from our study, however, whether this impairment is a direct effect of the virus or indirectly through viral-induced microenvironmental changes. Previously published data has suggested elevated proinflammatory cytokine levels including RANTES and TNF-α during chronic HIV infection , 29. Thi+ T helper cells in X-linked lymphoproliferative disease (XLP) patients impaired the development of B cell responses [The regulatory cytokine IL-10 has been shown to be important in B cell proliferation and maintenance , 39–41, esponses . The incRapid initiation of ART for maintaining low viremia and preserving immunity is a key objective of the RV254 study. We determined that stage 1/2 individuals had significantly lower viral load than stage 3 and consequently less plasma HIV-specific IgG and terminally differentiated B cells compared to stage 3 individuals. We know one of the characteristic effects of increased viremia on the B cell response is hypergammaglobulinemia , 10. In Resting memory B cells play a critical role in eliciting strong humoral immunity and were significantly lower in stage 3 than in stage 1/2. Interestingly, the frequencies of these resting memory B cells in both stage 1/2 and stage 3, but not chronic individuals were restored to levels similar to what is observed in healthy subjects. These results strongly suggest that intervention at the earliest stages of infection can maintain the integrity and survival of resting memory B cells, which are significantly capable of strengthening humoral defenses and very important in preventing HIV disease progression. This is similar to other studies comparing the effects of early ART versus initiation at the chronic phase, where the resting memory B cell subset was better restored with early treatment , 45.As we investigate what occurs in the periphery, we are cognizant that this environment may not necessarily represent what occurs in GCs in secondary lymphoid tissue. Using cTfh and memory B cells as surrogates allows us to interpret what may be going on generally and these results will help is to refine our future investigations. However, based on our previous work , 24 and Of note, there has been pioneering but not extensive work done on the timing of ART for controlling viral rebound after treatment interruption. This study as well as work done by others, has shown that the timing of ART is important to preserve T cell-dependent B cell responses , 45 and The RV254/SEARCH 010 study is an ongoing study in Bangkok, Thailand , recruiting participants at the earliest stages of acute HIV infection. Peripheral blood mononuclear cells (PBMCs) from participants in the study cohort were used for these experiments  Tables. The RV254/SEARCH010 study (NCT00796146) was approved by the Institutional Review Boards (IRBs) of Chulalongkorn University, Walter Reed Army Institute of Research (WRAIR), University of California at San Francisco (UCSF), Yale University, University of Hawaii (UH), University of Texas Medical Branch at Galveston, University of Sydney, and Centre Hospitalier de l’Université de Montréal Le Comité d’Ethique de la Recherche. The RV304/SEARCH013 study (NCT01397669) was approved by the IRBs of Chulalongkorn University, Walter Reed Army Institute of Research (WRAIR), University of California at San Francisco (UCSF), and Yale University. The SEARCH011 study (NCT00782808) was approved by the IRBs of Chulalongkorn University, University of California at San Francisco (UCSF), and University of Hawaii (UH). Initiation of ART was voluntary and done as part of enrollment. All participants gave written informed consent for all the studies detailed above.+CD4+CD45RA-CXCR5+CXCR3-, less efficient helper T cells denoted CXCR5+CXCR3+ and defined as CD3+CD4+CD45RA-CXCR5+CXCR3+; and resting memory B cells defined as CD3-CD19+CD10-CD27+. Sorted cTfh or CXCR5+CXCR3+ T cells were placed in coculture with sorted memory B cells at an equal ratio with 100ng/mL of staphylococcal enterotoxin B (SEB) (Toxin Technology) in RPMI 1640 (Corning) with 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco). SEB was required to induce T cell activation and promote T/B crosstalk [PBMCs from HIV-infected patients were incubated with fluorochrome-conjugated antibodies for at least 15–20 minutes at 4°C or on ice, protected from light. Samples were sorted on a BD™ FACSAria II for: cTfh cells defined as CD3rosstalk . Of notePBMCs from HIV-infected or HIV-negative patients were incubated with fluorochrome-conjugated antibodies for at least 15–20 minutes at 4°C or on ice, protected from light. The following fluorochrome-conjugated anti-human antibodies were used: CD3 (HIT3α), CD4 (RPA-T4), CD10 (HI10a), CD20 (2H7), CD25 (BC96), CD27 (O323), CD38 (HIT2), PD-1 (EH12.2H7), and CXCR3 (G025H7) were all from BioLegend. CD19 (HIB19), CXCR5 (RF8 B2), ICOS (DX29) and CD21 (B-ly4) were from BD Biosciences and CD45RA (2H4LDH11LDB9) from Beckman Coulter. LIVE/DEAD Fixable Dead Cell Stain (Life Technologies) was used to gate on live cells; and in some cases both LIVE/DEAD stain and Annexin V (BD Biosciences) were used. Samples were acquired on a BD LSR II.3PO4. The OD values were read at 450nm using a spectrophotometer .Total and HIV-specific IgG was measured by ELISA on culture supernatant and plasma as previously described . Total IIL-1β, IL-6, IL-10, IFN-γ, MIP-1α, RANTES and TNF-α were measured in 7 day culture supernatant from T and B cell coculture experiments using the Bio-Plex Pro™ Human Cytokine Luminex Kit (Bio-Rad) exactly according the manufacturer’s instructions. Samples were added neat to the plate and cytokine standards were supplied by the manufacturer. The samples were acquired using a Bioplex-200 system and the data analyzed on the BioPlex Manager Software (Bio-Rad).All data were analyzed using Graphpad Prism v10. Unpaired Student’s t test (Mann Whitney) was used when comparing two groups. The unpaired multiple t test and non-parametric one-way ANOVA (Dunn’s) test was used when comparing more than two groups to each other. Comparisons by correlations were analyzed using nonparametric spearman correlation. P values of less than 0.05 were considered statistically significant.S1 Fig+CD4+CD45RA-CXCR5+CXCR3-, the less efficient helper T cells identified as CD3+CD4+CD45RA-CXCR5+CXCR3+; and non- helper T cells (denoted CXCR5+CXCR3+) identified as CD3+CD4+CD45RA-CXCR5- cells. Frequencies of (B) cTfh and the frequency of (C) PD-1, (D) ICOS, (E) CD25 and (F) CD38 expression on cTfh was determined. (G) The mean fluorescence intensity (MFI) of CD38 was determined for cTfh after stimulation with SEB for 3 days. For graphs bars represent mean ±SD and symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares). Statistics were carried out using the Mann-Whitney non-parametric test. * P< 0.05.PBMCs from Stage 1 and 2 and Stage 3 patients at week 0 (W0) (n = 17 and n = 16 respectively) were analyzed by flow cytometry. (A) Representative plots showing cTfh identified as CD3(TIF)Click here for additional data file.S2 Fig+CXCR3-), (CXCR5+CXCR3+) were placed in culture with autologous CD10-CD21+CD27+ resting memory B cells in the presence of or without SEB. Quantification of cTfh-mediated B cell help was carried out by measuring total IgG ELISA in 7 day culture supernatant.PBMCs from week 0 stage 1/2 (n = 9) and stage 3 (n = 4–7) individuals were sorted. cTfh cells (CXCR5(TIF)Click here for additional data file.S3 Figα. Bars represent mean ±SD. Symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and statistics were carried out using the Mann-Whitney non-parametric test.Cocultures of cTfh and autologous resting memory B cells from week 0 stage 1/2 (n = 9) and stage 3 (n = 7) individuals were analyzed for the presence of cytokines (A) IL-1β, (B) IL-6, (C) IFN-γ and (D) MIP-1(TIF)Click here for additional data file.S4 Fig+CD10-CD20-CD21-CD38hiCD27hi cells. (B) Representative plots from a healthy individual showing total B cells identified as CD19+; activated memory (AM) B cells CD19+CD10-CD21-CD27+ cells, Tissue-like (TL) B cells CD19+CD10-CD21-CD27-, resting memory (RM) B cells CD19+CD10-CD21+CD27+ and resting naïve (RN) B cells CD19+CD10-CD21+CD27-IgD+.(A) Terminally differentiated plasmablasts in stage 1/2 and stage 3 individuals from week 0 were identified as CD19(TIF)Click here for additional data file.S5 FigcTfh cells from a sorted pool of healthy controls (HC) were used to substitute cTfh cells from stage 3 HIV-infected individuals placed in coculture with memory B cells from stage 3 subjects. (A) Total IgG, (B) HIV-specific IgG, (C) IL-10 and (D) RANTES levels were assessed in coculture supernatant. Symbols on the graphs represent cTfh from Stage 3 individuals (black squares) and cTfh from HCs (open diamonds). Statistics carried out using the Mann-Whitney non-parametric test.(TIF)Click here for additional data file.S6 Fig+CD27+ resting memory B cells from stage 1/2 and stage 3 (n = 4–5) were stimulated in vitro with CpG ODN for 5 days. (A) CFSE expression on cells was analyzed by flow cytometry and (B) total IgG was quantified in the supernatant by ELISA. (C) The expression of BCL2 on CD21+CD27+ memory B cells from stage 1/2 and stage 3 individuals from day 0 was measured ex vivo by flow cytometry. Symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares). Statistics were carried out using the Mann-Whitney non-parametric test. * P< 0.05.CFSE labeled sorted CD21(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants. Flaviviridae family. The HCV genome encodes a polyprotein of ~3000 amino acids. The polyprotein is proteolytically cleaved by host and viral proteases to yield 10 proteins  that are responsible for viral replication and assembly /(Kd + [L]), where [L] is the concentration of 3H-LDV.3H-LDV (30 nM) and varying concentrations of unlabeled LDV, DCV, or BMS-Biotin (in DMSO). Each reaction, in a final volume of 200 μl of buffer C, was incubated at room temperature for 4 hr and processed as described above. Data were fit to a single site competitive binding model to obtain the concentration of half-maximal binding (IC50): %Bound = Bmin + (Bmax-Bmin)/(1 + [S]/IC50), where %Bound is the fraction of 3H-LDV bound relative to control with no competitor, Bmin and Bmax are parameters related to %Bound when fully inhibited and uninhibited respectively, and [S] is the concentration of competitor. The inhibitor dissociation constant (Ki) was determined using the Cheng-Prusoff equation [d determined from the saturation binding experiment: Ki = IC50/(1 + [3H-LDV]/Kd).For competition reactions, 50 nM NS5A-6HIS was mixed with a fixed concentration of 3H-LDV  nM and v6 cells per T175 flask and cultured at 37°C. On Day 2, cells were switched to medium A containing 1 μM DCV or 25 μM BMS-Biotin. After incubation for 24 hr, cells were washed with PBS, trypsinized, and harvested by centrifugation.On day 0, Huh7-lunet cells stably expressing the genotype 1b, Con 1 replicon or the genotype 1b, Con1 replicon containing mutant NS5A-Y93H  were pla6 cells per T75 flask and cultured at 37°C. On day 1, cells were transfected with the indicated plasmid using Lipofectamine LTX (Invitrogen). On day 2, cells were switched to medium containing BMS-Biotin and varying amounts of LDV or DCV. After incubation for 42 hr, cells were washed with PBS and harvested by centrifugation.On day 0, Huh7-lunet cells were plated in Medium A without antibiotics at a density of 1x10Cells were lysed by passage through a 27-gauge needle, and membranes were isolated by centrifugation . Membranes were solubilized with PBS containing 0.1% C12E8 and solubilized protein was isolated by centrifugation . BMS-Biotin was precipitated from the soluble fraction by incubation with streptavidin-agarose beads (Pierce). The pelleted beads were washed and co-precipitated material was eluted with SDS loading buffer. Samples were subjected to SDS-PAGE and immunoblot analysis using an anti-NS5A monoclonal antibody (Apath LLC).


Human infection with avian influenza A(H7N9) virus is associated mainly with the exposure to infected poultry. The factors that allow interspecies transmission but limit human-to-human transmission are unknown. Here we show that A/Anhui/1/2013(H7N9) influenza virus infection of chickens  is asymptomatic and that it generates a high genetic diversity. In contrast, diversity is tightly restricted in infected ferrets, limiting further adaptation to a fully transmissible form. Airborne transmission in ferrets is accompanied by the mutations in PB1, NP and NA genes that reduce viral polymerase and neuraminidase activity. Therefore, while A(H7N9) virus can infect mammals, further adaptation appears to incur a fitness cost. Our results reveal that a tight genetic bottleneck during avian-to-mammalian transmission is a limiting factor in A(H7N9) influenza virus adaptation to mammals. This previously unrecognized biological mechanism limiting species jumps provides a measure of adaptive potential and may serve as a risk assessment tool for pandemic preparedness. H7N9 bird flu viruses cause mild disease in poultry but can occasionally infect humans with fatal consequences. Here, the authors show that viral genetic diversification is low in ferrets and high in chickens, suggesting that a genetic bottleneck limits H7N9 adaptation to mammals In February 2013, avian influenza AH7N9) virus crossed the species barrier in China and for the first time caused human infections34N9 virus Sporadic clusters of H7N9 virus transmission among family members have been reported8911121317202123To elucidate the transmission dynamics and transmission barriers of the emerging H7N9 influenza viruses, we evaluated the replication efficiency, genomic diversification and host adaptation of A/Anhui/1/2013 (H7N9) influenza virus after the inoculation of chickens and ferrets and during ferret-to-ferret transmission. Here we provide evidence that H7N9 virus infection of chickens, while asymptomatic, promotes genetic diversity in the virus population, increasing the likelihood of substitutions that favour mammalian infection and transmission. Conversely, the virus shows poor airborne transmissibility and no evidence of further adaptation upon replication in ferrets, likely due to a narrow bottleneck constraining genetic diversification.The H7N9 virus was isolated mainly from chickens in live-bird markets suspected to be the source of human infections. Thus, we investigated the virulence and replication of A/Anhui/1/2013 (H7N9), one of the first human H7N9 viruses isolated, in chickens. A/Anhui/1/2013 shares at least 99% identity at the nucleotide and protein levels with H7N9 influenza viruses isolated from birds and live-bird markets .10EID50 (50% egg infectious doses) per ml of virus from the upper respiratory tract at 3 days post inoculation (dpi), and shedding lasted for as long as 7 dpi . Inoculated chickens showed no signs of illness but shed on average 3.75 logas 7 dpi , suggestas 7 dpi . All theas 7 dpi , althoug10 TCID50 (50% tissue culture infectious doses) per ml of virus on 2 dpi) and protracted  virus shedding from the upper respiratory tract, as determined in nasal washes  virus except at 1 dpi, when the titre was much lower in ferrets inoculated with AC virus despite equivalent inoculum . Virus wP<0.05, two-tailed Mann–Whitney U-test) than those isolated from chickens  A/Anhui/1/2013 influenza viruses with single or multiple amino-acid substitutions in the gene/s of interest and compared their replication kinetics with the WT rg-virus in Madin Darby canine kidney (MDCK) cells. In line with the drop in NA and polymerase enzymatic activities, rg-A/Anhui/1/2013 (NAE73K+NAI300V), rg-A/Anhui/1/2013 (PB1D76N+NPI365V) and rg-A/Anhui/1/2013 (NAE73K+NAI300V +PB1D76N+NPI365V) influenza viruses replicated to significantly lower titres than the respective WT strain (0–24), was significantly lower when compared with the WT virus  and rg-A/Anhui/1/2013 (NAI300V) viruses were significantly attenuated in vitro  and rg-A/Anhui/1/2013 (NPI365V) were comparable to those of the WT virus at 37 °C  viruses to a wide array of glycans . Both thAcid stability has been described as one of the markers for regulating host range and virulence of influenza viruses212331Our study revealed a previously unknown population-level factor restricting the further adaptation of avian A(H7N9) influenza virus to mammals. Although the H7N9 virus replicates to high titres in infected ferrets, replication in this host did not select for viruses with enhanced growth, virulence or transmissibility. In contrast, our data suggest that replication in ferrets lead to an attenuated phenotype. Because adaptation depends on the extent of the influenza virus population's diversity in the host, we propose the existence of a ‘genetic bottleneck' that limits the ability of avian A(H7N9) influenza virus to adapt to a mammalian host by limiting its genetic diversity in that host. By using a population-level approach and comparing the genetic diversity of the virus population in the natural avian host and in infected mammals, we found that the genetic diversity of A/Anhui/1/2013 (H7N9) influenza virus was limited after its transmission to mammals. The loss in genetic variation imposed by the narrow genetic bottleneck appears to restrict the capacity of A(H7N9) virus to fully adapt to mammals.Genetic bottlenecks occur frequently during the natural life cycles of RNA viruses1623Several pieces of evidence, including the isolation of the H7N9 virus from chickens at live-bird markets and the exposure history of infected patients, suggest that live-poultry markets are a source of human infection4536Our data show that the H7N9 virus replicates in chicken with moderate efficiency and that the infected chickens shed virus for a protracted period. This shedding appears to be sufficient to allow contact transmission of H7N9 virus among chickens, although with poor efficiencyThe human H7N9 isolates possess, or can readily acquire, several traits that support mammalian adaptation, including the PB2-E627K mutation242743214711252329The presence of a significant subpopulation of variants with the HA mutation Q235L, which confers human receptor-binding preferencein vitro. Importantly, although a human H7N9 virus was passaged in eggs to prepare a sufficient stock for experiments, the effect of egg-selected variants was controlled by using the same parental strain to inoculate chickens and ferrets. Richard et al.et al.In this study, we found that H7N9 viruses that had been transmitted via the airborne route were not better adapted or more transmissible in mammals than the parental strain. Four residues in the three genes were uniquely enriched after airborne transmission, and in the ferrets inoculated with AC virus in round 2 experiments. Two of these substitutions (E73K and I300V) located in the NA gene resulted in a decrease in NA activity, and the PB1-D76N and NP-I365V mutations reduced polymerase activity. These mutations also attenuated virus replication In comparing the genetic diversity of the surface glycoproteins, we observed differences in genetic diversity among species in the HA gene but not in the NA gene. At the amino-acid level, none of the HA variants in the parental virus stock were detectable in the transmitted virus population. This result strongly suggests that a purifying pressure acting mainly on the HA gene (virus entry) shapes the diversity bottleneck observed in ferrets. This purifying pressure did not appear to select variants with novel traits such as enhanced transmissibility of the virus in ferrets. Similar purifying pressure on the HA gene has previously been described in association with the airborne transmission of H5N1 virus. However, in the case of H5N1 the purifying pressure appeared to favour the selection of viruses with enhanced fitness after aerosol transmissionDespite their ability to infect humans, the currently circulating strains of H7N9 viruses did not readily acquire additional adaptive mutations during their limited passage in humans or a mammalian host like ferrets. Our results suggest that viral replication in chickens may enhance the diversity of the H7N9 viruses and increase the potential of a subpopulation of variants to enter the mammalian cells and replicate. Even though these variants can replicate in mammals, the stringency of the genetic bottleneck limits their ability to acquire subsequent mutations that would allow mammal-to-mammal transmission. Whether such fitness enhancing variants get selected in nature is entirely dependent on random events, frequency of infection in the new species and time. Given the continued infection of humans with avian H7N9 viruses, the risk of adaptation and pandemic emergence remains plausible. Therefore, surveillance and risk assessment of H7N9 viruses in poultry along with monitoring infections in humans should remain a focus of pandemic preparedness efforts.L-glutamine. HEK 293T cells were maintained in Opti-MEM  supplemented with 10% heat-inactivated FBS. All the cells were maintained at 37 °C in 5% CO2.MDCK epithelial cells, African green monkey kidney epithelial (Vero) cells and human embryonic kidney (HEK 293T) epithelial cells were obtained from the American Type Culture Collection. MDCK cells were maintained in MEM containing 5% heat-inactivated fetal bovine serum (FBS) and 0.5% penicillin–streptomycin. Vero cells were maintained in DMEM (Gibco) containing 10% heat-inactivated FBS and 2 mM ad libitum to all the animals.All the experiments were performed in a biosafety level 3 enhanced (ABSL3+) containment laboratory in accordance with USDA 9 CFR 121; 7 CFR 331. All the experiments were approved by the Institutional Biosafety Committee and the Dual Use Research of Concern Subcommittee at St Jude Children's Research Hospital. The work in this study was conducted prior to the current U.S. Government gain of function stop work order. The ABSL3+ facility has security and containment features that eliminate risks to humans and the environmentThe human H7N9 viruses A/Anhui/1/2013, A/Shanghai/1/2013, and A/Shanghai/2/2013 were obtained through the World Health Organization network. The virus stock used in the chicken and ferret experiments was grown in 10-day-old embryonated chicken eggs at 35 °C for 48 h.50 assay in 10-day-old embryonated chicken eggs incubated at 35 °C for 48 h. Virus from chicken cloacal and oropharyngeal swabs was collected in 1-ml sterile phosphate-buffered saline (PBS) with antibiotics, titrated in 10-day-old embryonated chicken eggs at 35 °C for 48 h and titrated as EID50. Virus from ferret nasal washes and cell culture supernatants from replication kinetic experiments were titrated in MDCK cells at 37 °C for 72 h as TCID50. Virus titres (log10EID50 per ml and log10TCID50 per ml) in nasal washes, swabs, and tissues were estimated by the Reed and Muench method10 EID50 per ml and 1.5 log10 TCID50 per ml.Virus stock was titrated by plaque assay in MDCK cells at 37 °C for 72 h with a 0.4% immunodiffusion grade agarose ; and by EIDGallus gallus domesticus) were inoculated with 106 50% EID50 of A/Anhui/1/2013 (H7N9) influenza virus (0.5 ml) via natural routes . Cloacal and oropharyngeal samples were collected at 3, 5, 7 and 10 dpi with sterile cotton swabs, which were subsequently placed into 1 ml of PBS with antibiotics. Samples were stored at −80 °C for subsequent titration in 10-day-old embryonated chicken eggs as described above. The intravenous virus pathogenicity index was determined as described by Hulse et al.Three 6-week-old, specific-pathogen-free chickens  that were serologically negative by hemagglutination inhibition assay for currently circulating A(H1N1), A(H3N2) and B human influenza viruses were used in the experiments. Six ferrets per group were inoculated intranasally with 106 TCID50 of A/Anhui/1/2013 influenza virus in 0.5 ml PBS under isoflurane anaesthesia. On 3 and 5 dpi, three ferrets per group were humanely killed. Virus in the nasal cavities, tracheas, lungs, brains and the small and large intestines was titrated in MDCK cells as the TCID50. Excised tissue samples preserved in 10% neutral buffered formalin (Thermo Scientific) were paraffin-embedded, sliced into 5-μm-thick sections and stained with hematoxylin and eosin or with immunohistochemical stains for influenza A virus nucleoprotein, pneumocyte type II cells (surfactant protein c), macrophages  and neutrophils (myeloperoxidase). To collect nasal washes, ferrets were anaesthetised by intramuscular administration of ketamine HCl (25 mg kg−1) and sterile PBS with antibiotics (0.5 ml) was instilled into each nostril. The resultant washes were collected into a sterile cup. Samples were titrated by using the TCID50 assay in MDCK cells as described above.Young adult (3–4- month-old) male ferrets  and another four ferrets were placed in AC in an adjacent cage separated from the inoculated ferrets' cage by a perforated divider with controlled air flow.Two rounds of transmission experiments were performed. During round 1, four ferrets were inoculated intranasally with 106 TCID50. At 1 dpi, three naïve ferrets were placed in AC in an adjacent cage as described above. Body weight and clinical signs were monitored for 21 dpi. Virus was titrated in nasal washes collected from inoculated and AC ferrets on 1 dpi and then every other day for 14 dpi or until no virus shedding can be detected for two consecutive sneezes as described above.For the second round of experiments (secondary respiratory droplet transmission), six naïve ferrets were inoculated with a nasal wash sample containing ‘AC' virus obtained at 10 dpi (day 9 post contact) from the ferret infected via AC in the previous round of experiments. The nasal wash was diluted in PBS to a titre of ∼10Seroconversion was tested at 20 days post contact or at 21 dpi. Serum was treated with receptor-destroying enzyme (Seiken) overnight at 37 °C to destroy non-specific inhibitors, heat-inactivated at 56 °C for 30 min, and tested by hemagglutination inhibition assay with A/Anhui/1/2013 (H7N9) virus and 0.5% chicken red blood cells as described previouslyViral RNA was extracted by using an RNeasy kit (Qiagen); reverse-transcriptase PCR was then performed by using a SuperScript III first-strand synthesis kit (Invitrogen) with the Uni12 influenza primer. Multiplex PCR of all the eight gene segments was conducted by using PCR Supermix HiFi (Invitrogen) with the Uni12/13 and influenza A polymerase segment primers under previously described thermal cycler conditionsGenomic sequences were analysed by using a custom work-flow protocol. In brief, sequence reads were filtered for quality scores by using the Nextera adapter trimming protocol, discarding low-quality terminal bases with PHRED scores >20. A sample of 100,000 high-quality read pairs were aligned to the full set of complete NCBI influenza genomes in an exhaustive, competitive fashion to identify potential contaminating samples. Two rounds of alignment were performed: one pairwise alignment and one single-end alignment of unmapped reads from the first round. Genomes with at least one segment with a coverage >0.7 were selected and used to populate a new reference set. The full set of high-quality read pairs was aligned to H7N9 and this reduced reference set, by using the same approach. Genomic variations in H7N9 were detected by using an in-house single nucleotide variants (SNV) detection pipeline that considers base quality of a putative SNV, neighbourhood base quality, strand bias of SNV and several other factors. Heat maps were generated by using the R programming software package . Mean variant frequency for each sample was calculated by using positions that are variable in at least one of the examined samples/reads. If a site had a minor variant frequency >0 in any of the samples, it was included in the numerator; the denominator of the proportion was the total number of variable sites that was variable in all samples.Viral ribonucleoprotein complex genes, including NP, PA, PB1 and PB2 of A/Anhui/1/2013 (H7N9) virus, were cloned into the pHW2000 plasmid to test the viral polymerase activity50 per cell and incubated at 37 °C for 18 h to allow cell surface expression of the HA protein. Cells were then washed with PBS and treated with 5 mg ml−1 of TPCK (L-tosylamido 2-phenylethyl chloromethyl ketone)-treated trypsin (Worthington) for 5 min, followed by neutralization with FBS. Cells were then washed twice with PBS and pulsed with PBS buffers adjusted to 0.1 pH increments by using 0.1 M citric acid. The highest pH at which syncytia were observed was recorded as the pH of HA activation.The pH of HA protein activation was measured by syncytium assay, as previously describedClaI site and subcloning of the product into a pCAGGS expression plasmid, using primers with overhangs that include ClaI and NheI restriction sites  substrate  for 30 min at 37 °C. The reaction was quenched with 100 μl stop solution (150 mM sodium hydroxide in 83% ethanol), and the fluorescence intensity of the released 4-methyl-umbelliferone was measured on a Synergy 2 multi-mode microplate reader (BioTek Instruments) with excitation and emission wavelengths of 360 and 460 nm, respectively. NA activity was quantified as fluorescence above the background fluorescence of mock-transfected cells, expressed in picomoles of 4-methyl-umbelliferone product.To assess the NA activity, Vero cells were transfected with the HA-tagged NA plasmids carrying the mutations of interest (NAI300V and NAE73K), WT plasmid and an empty pCAGGS expression plasmid (mock) by using TransIT-LTI (Mirus Bio) transfection reagents. After incubation of the transfected cells for 20 h at 37 °C, cells were collected and resuspended in assay buffer . Fifty microlitres of the suspension was incubated with an equal volume of 2′-(4-methylumbelliferyl)-α-To confirm the cell surface expression of NA protein, the Vero cells transfected as described above were collected, resuspended in 1X bovine serum albumin blocking buffer in Tris-buffered saline (Thermo Scientific) and stained with monoclonal anti-HA-FITC antibody  for 1 h at 4 °C. HA-tagged NAs was then analysed by flow cytometry on a MACS Quant analyzer (Miltenyi Biotec). The mean fluorescence intensity above that of mock-transfected cells was quantified by FlowJo software.N-linked and O-linked glycans found on mammalian and avian glycoproteins and glycolipids−1. Slides were incubated at room temperature for 1 h, washed with PBS and overlaid with rabbit or goat anti-H7 antibody (1:200) for 1 h, followed by the addition of 10 μg ml−1 anti-rabbit IgG (Alexa Fluor 488) or anti-goat IgG . Slides were washed and scanned on a ProScanArray Express HT (PerkinElmer) confocal slide scanner to quantify the bound virus.Glycan array analysis was performed as previously describedE73K), rg-A/Anhui/1/2013 (NAI300V), rg-A/Anhui/1/2013 (NAE73K+NAI300V), rg-A/Anhui/1/2013 (PB1D76N), rg-A/Anhui/1/2013 (NPI365V), rg-A/Anhui/1/2013 (PB1D76N+NPI365V) and rg-A/Anhui/1/2013 (NAE73K+NAI300V +PB1D76N+NPI365V) were generated by co-transfecting 293T cells  with the appropriate eight plasmids and the virus stocks were prepared in MDCK cells. The presence of the amino-acid substitutions of interest in the recombinant viruses were verified by Sanger sequencing.The plasmids encoding the eight genes of A/Anhui/1/2013 (H7N9) virus cloned into plasmid vector pHW2000 have been described in ref. 50 per cell at 37 °C. After 1 h of incubation, the cells were washed with 0.9% aqueous NaCl solution (pH 2.2) to remove unbound virus particles, washed for three times with PBS to adjust the pH and overlaid with infection medium . Supernatants were collected at 2, 4, 6, 8, 10, 12 and 24 h post inoculation and stored at −80 °C for titration in MDCK cells as described above. To quantify the total amount of virus produced over the course of the experiment, we computed the area under virus titre hour curve from 0 to 24 h (AUC0–24), using peak virus titres at 0, 2, 4, 6, 8, 10, 12 and 24 h post inoculation (log10TCID50 per ml) by GraphPad software.To determine single-step growth curves, confluent monolayers of MDCK cells were infected with reverse-genetic viruses at an identical multiplicity of infection of 1 TCIDU-test was used to compare the genetic diversity. P values <0.05 were considered to indicate a statistically significant difference.GraphPad Prism 5 software (GraphPad Software Inc.) was used for statistical analysis. Two-way analysis of variance was used to compare the groups. Two-tailed Mann–Whitney H.Z. and T.B. designed and performed experiments, and drafted the manuscript; R.C., M.E., G.W. and J.Z. performed computational analysis; B.S.K., J.C.P., R.McB., M.-S.S., J.C.C., J.S., E.K., M.H. and T.F. performed the experiments. J.E.R. analysed the pathology data. J.B., Hu.Z., Y.G., S.S.-C., S.K., R.G.W., E.A.G. and C.J.R. designed the influenza virus infection experiments and contributed to writing the manuscript. R.J.W. designed and supervised the study, critically reviewed and revised the manuscript.How to cite this article: Zaraket, H. et al. Mammalian adaptation of influenza A(H7N9) virus is limited by a narrow genetic bottleneck. Nat. Commun. 6:6553 doi: 10.1038/ncomms7553 (2015).Supplementary Figures 1-5 and Supplementary Tables 1-7Synonymous and non-synonymous mutations detected in chicken isolatesSynonymous and non-synonymous mutations detected in ferret isolates


Danio rerio), a common model organism for vertebrate development and gene function, has not previously been characterized as a model system for esophageal epithelial development. In this study, we characterized a piece of non-keratinized stratified squamous epithelium similar to human esophageal epithelium in the upper digestive tract of developing zebrafish. Under the microscope, this piece was detectable at 5dpf and became stratified at 7dpf. Expression of esophageal epithelial marker genes  was detected by immunohistochemistry and in situ hybridization. Knockdown of P63, a gene known to be critical for esophageal epithelium, disrupted the development of this epithelium. With this model system, we found that Pax9 knockdown resulted in loss or disorganization of the squamous epithelium, as well as down-regulation of the differentiation markers Krt4 and Krt5. In summary, we characterized a region of stratified squamous epithelium in the zebrafish upper digestive tract which can be used for functional studies of candidate genes involved in esophageal epithelial biology.Mammalian esophagus exhibits a remarkable change in epithelial structure during the transition from embryo to adult. However, the molecular mechanisms of esophageal epithelial development are not well understood. Zebrafish ( The epithelial cells lining the human esophagus transform from simple columnar into ciliated epithelium at an early phase. The ciliated epithelium is then gradually replaced by squamous epithelium until a non-keratinized stratified squamous epithelium forms . AlthougDanio rerio) has attracted much attention as a model system for functional studies. The many favorable characteristics of zebrafish include convenient and economical genetic manipulations, short generation time, optical transparency of embryos, prolific reproduction, external development, and easy maintenance of both the adult and the young [Our previous microarray study globally profiled genes and signaling pathways involved in the development of mouse esophagus, revealing novel players potentially critical for epithelial differentiation and maturation, such as Pax9, Nrf2, PPARβ/δ, and the PI3K/Akt pathway . Neverthhe young .In this study, we aim to establish a zebrafish model for studies on esophageal epithelial biology. Previously we have identified a short piece of non-keratinized stratified squamous epithelium in adult zebrafish . Here, wZebrafish were obtained from Zebrafish International Resource Center . The AB strain was housed in an automatic zebrafish housing system  at 28.5°C. All animal experiments were approved by the Institutional Animal Care and Use Committees at North Carolina Central University (protocol number: XC07-15-2008).http://useast.ensembl.org/Danio_rerio/Gene/Summary?db=core;g=ENSDARG00000044356;r=6:28805219-28923664. Two specific mispair morpholinos were designed according to the sequences of the P63 MO-2 and Pax9 MO-1. These two mispair MOs and a scrambled control MO were synthesized by Gene Tools and then used as controls of MO toxicity and off-target effects. Mispair MOs were designed with the same sequence as the antisense MO, except at the five mispair sites. The sequences of the MOs were as follows:Three antisense MO oligonucleotides each of P63 and Pax9  were designed against exon/intron splice sites. MOs were solubilized in water at a concentration of 0.1–1.0 mM before injection into one to two-cell stage embryos. Preliminary data showed P63 MO-2 and Pax9 MO-1 were the most effective, and they were used for the subsequent knockdown experiments. P63 MO-2 was expected to block expression of all seven transcripts of zebrafish p63 gene. This MO blocked the splicing of intron 2–3 in 5 transcripts . It also blocked the splicing of intron 4–5 in tp63-202, and intron 5–6 in tp63-003. The corresponding DNA sequence can be found at the following link: 5’ TATACTGAGGCTGGAGGAAAAACAA 3’P63 MO-1: 5’ ACATTTGCTGTATGTCTTACCGTCC 3’P63 MO-2: 5’ GAGCATTGGTCTCCAGGTACAACAT 3’P63 MO-3: 5’ AACATGAATATAGATCTTACCCATT 3’Pax9 MO-1: 5’ TGGCTCTAGTTATGCAGATATATAA 3’Pax9 MO-2: 5’ AAACTGAGAGGATGCTTACTTTGCT 3’Pax9 MO-3: 5'-CCTCTTACCTCAGTTACAATTTATA 3'Scrambled control MO: 5’ AGATTTCCTGTATCTCTTAGCGTGC 3’P63 mispair MO: 5’ AAGATCAATATACATGTTAGCCATT 3’Pax9 mispair MO: Zebrafish were sacrificed at 4, 5, 6, 7 and 90 days post fertilization (dpf) and fixed with 4% paraformaldehyde. For adult zebrafish, the upper digestive tract was dissected and fixed with 4% paraformaldehyde. Whole zebrafish or tissue samples were orientated in agarose gel and processed for paraffin embedding. The paraffin blocks underwent serial sectioning (5 μm). Hematoxylin and eosin (H&E) staining was carried out with standard protocols. Alcian blue staining was performed as described previously . BrieflyParaffin-embedded tissue sections were deparaffinized, rehydrated, and pretreated by heating the slides for 5–10 min in 10mM citrate buffer. Staining was performed with the ABC kit (Vector Laboratories) according to the manufacturer’s instructions with a mouse anti-P63 (4A4) antibody diluted at 1: 1000  and a rabbit anti-Sox2 antibody diluted at 1: 500 .Digoxigenin-labeled riboprobes (RNA probes) were synthesized as previously described . DNA tem5' CTGCGTGAACTCCAGTCACAGAT 3'Krt5 forward: 5' TAATACGACTCACTATAGGGAGAAGCAATGCCAAGAAGATACA 3'Krt5 reverse: 5' GATGGACTGGGAAATGAGAAGAT 3'Krt4 forward: 5’ TAATACGACTCACTATAGGATAGCGTTTACTGCTGACGGTGG 3'Krt4 reverse: 5’ AAATGAATCCGACGTACTG 3’Pax9 forward: 5’ TAATACGACTCACTATAGGGAATGGCATGTCCACAGACAC 3’Pax9 reverse: The procedure of ISH on paraffin sections was based on the protocol of a commercial kit . The sections were washed and covered with cover slips for observation under the microscope.CCAGCAGTCCAGCACAGCCAAAT, P63 reverse: CTCTGGTTTCCAATGTGACA; Pax9 forward: GACTCGGAACAGGTCAGAATAGG, Pax9 reverse: GAGTTGGATACGAATACAGGTGGTT). PCR using Advantage RT-for-PCR Kit  was carried out as follows: 95°Cx5’,  X 30 cycles, 72°Cx5’. The control PCR product is 449bp for P63 gene and 562bp for Pax9 gene, and MO injection resulted in un-spliced products of 758 bp for P63 gene and 1,011bp for Pax9 gene.To confirm the effect on splicing induced by P63 and Pax9 MOs, 50 zebrafish embryos injected with P63 MO-2, Pax9 MO-1 and control MO were collected at 3, 5 and 7 dpf. A Total RNA I kit  was used to purify RNA, and the superscript reaction was carried out. Primers specific for P63 or Pax9 splicing variants were designed  at 7dpf as compared with that in control samples . FurtherOur previous microarray studies suggested Pax9 as a critical transcription factor for esophageal development , 16. In A scrambled MO and a Pax9 mispair MO were used to control off-target effects. RT-PCR was performed with zebrafish at 3, 5 and 7dpf, to detect a 562bp spliced transcript and a 1,011bp un-spliced transcript. The spliced transcript was abundant at all the stages injected with control MO, but was reduced by the antisense MO. In the antisense MO injected zebrafish, the un-spliced transcript was abundant at 3dpf and still detectable at 7dpf, suggesting a lasting effect of antisense MO . As expeZebrafish have been a model organism for development and diseases in the intestine, but not in the esophagus, probably due to the fact that its entire digestive system, including the esophagus, is lined with simple columnar epithelium , 19. Onlfl/fl mouse line for conditional knockout of Pax9 in esophageal epithelium. This mouse line survives, and its esophageal phenotypes were similar to those in Pax9 knockdown zebrafish (data not shown).Our study demonstrates there is actually a stratified squamous epithelium located on the dorsal side of zebrafish upper digestive tract between the pharynx and the intestinal bulb. Histologically, it is highly similar to the non-keratinized squamous epithelium in human esophagus, with basal, parabasal, and superficial layers. It is detectable under the microscope at 5dpf and becomes stratified at 7dpf. Expression of esophageal epithelial genes  confirms its squamous features and endodermal origin. We speculate that this piece of squamous epithelium once fully developed may mainly function as a protective layer in adult zebrafish. P63 is exclusively detected in the basal layer of this epithelium in zebrafish esophagus, which is consistent with the fact that P63 is expressed in the basal cells of mammalian esophageal epithelium. In P63 knockout mice, the esophagus is lined with a simple columnar epithelium which fails to develop into stratified squamous epithelium . SimilarThis study opens up a new avenue for future studies on esophageal biology. The explosive growth of next-generation sequencing provides a rapidly increasing database of candidate genes involved in esophageal diseases , 22. Micin vivo, laying a foundation for further studies with mammalian model organisms.In summary, we characterized a stratified squamous epithelium in the zebrafish upper digestive tract as a novel model system for studies on esophageal biology. With the application of MO technology and CRISPR/Cas9 technology , this moS1 FigPercentage of the zebrafish (A) and the number of sections per zebrafish (B) with ESQE in the upper digestive tract at different developmental stages.(JPG)Click here for additional data file.S2 Fig(A) Survival curves (up to 7dpf) after MO injection; (B) Pax9 morphants  and P63 morphants (finless) at 7dpf; (C) Pax9 and P63 morphants with esophageal SQ epithelium at 7dpf; (D) Sections containing ESQE per fish at 7dpf after different MO injection. * P<0.05; ** P<0.01 as compared with UIC (un-injected control) or Control MO.(JPG)Click here for additional data file.S3 FigThe sections show the histology of pharynx (A), esophagus (B-O) and intestine (P). There are 9 sections containing the stratified squamous epithelium (C-K). Scare bar:5μm.(TIF)Click here for additional data file.