IFNB1, IFNL1 and IFNL2/3) and proteins (IFN-λ1 and IFN-λ2/3). The lower levels of IFN expression correlated with weaker induction of tyrosine-phosphorylated STAT1 and reduced RIG-I protein levels. Our findings demonstrate that influenza viruses can develop increased resistance to the antiviral activity of type III interferons.Influenza A viruses pose a constant potential threat to human health. In view of the innate antiviral activity of interferons (IFNs) and their potential use as anti-influenza agents, it is important to know whether viral resistance to these antiviral proteins can arise. To examine the likelihood of emergence of IFN-λ1-resistant H1N1 variants, we serially passaged the A/California/04/09 (H1N1) strain in a human lung epithelial cell line  in the presence of increasing concentrations of recombinant IFN-λ1 protein. To monitor changes associated with adaptation of this virus to growth in Calu-3 cells, we also passaged the wild-type virus in the absence of IFN-λ1. Under IFN-λ1 selective pressure, the parental virus developed two neuraminidase (NA) mutations, S79L and K331N, which significantly reduced NA enzyme activity (↓1.4-fold) and sensitivity to IFN-λ1 (↓˃20-fold), respectively. These changes were not associated with a reduction in viral replication levels. Mutants carrying either K331N alone or S79L and K331N together induced weaker phosphorylation of IFN regulatory factor 3 (IRF3), and, as a consequence, much lower expression of the IFN genes ( Influenza A viruses are respiratory tract pathogens that can cause widespread annual epidemics and occasional pandemics with a huge impact on public health . Recent in vivo.Virus-induced interferons (IFNs) are a complex group of biologically active cytokines that include type I and type III IFNs. These cytokines represent a major component of the innate immune response to influenza virus infection –24. TypeInfluenza virus infection induces co-expression of type I and type III IFNs by target cells such as airway epithelial cells . The subin vitro  open reading frame in pHW72-EGFP replaced with the firefly luciferase gene) [hRluc/CMV), which expresses Renilla luciferase , was used as an internal control for a dual-luciferase assay. As a negative control, 293T cells were transfected with the same plasmids, with the exception of the NP expression plasmid. After 24 h of incubation at 37°C cell extracts were harvested and lysed, and luciferase levels were assayed with a dual-luciferase assay system (Promega) and a Synergy 2 multimode microplate reader . Experiments were performed in triplicate.Subconfluent monolayers of 293T cells (7.5 × 10se gene)  and a mii.e., IFNB1, IFNL1, and IFNL2/3). Total cellular RNA was isolated from virus-infected Calu-3 cells (MOI = 1) using RNeasy Minikit . The RNA samples were then treated with DNase, and 1 μg of each purified RNA sample was then reverse-transcribed to cDNA with Quantiscript reverse transcriptase (Qiagen). The cDNAs were mixed with RT2 SYBR® green qPCR Mastermix (Qiagen), and qPCR analyses were performed using the ViiA™ 7 instrument . IFNB1, IFNL1, and IFNL2/3 gene copy numbers were assayed using Taqman gene expression assay primer/probe sets and master mix  and ViiA™ 7 software v.1.2.2 (Applied Biosystems). The values were determined by comparison to standard curves for each gene. Graphing and statistical analysis of the qPCR results were performed using Prism 6.0 (GraphPad Software). Values are the means of three independent determinations.Quantification of changes in IFN gene expression was carried out by quantitative real-time polymerase-chain reaction (qPCR) measurement of individual IFN genes . The levels of viral PB1 and M1 proteins in H1N1 viruses carrying silent PB1 and M1 mutations were analyzed by western blot with rabbit anti-PB1  and mouse monoclonal anti-M1 Abs , respectively.The levels of tyrosine-phosphorylated STAT1, RIG-I, IFN regulatory factor 3 (IRF3), and phospho-IRF3 were measured by Western blot analysis as described previously ,46. Whol50 values, virus yield, plaque size and number, binding to sialyl receptors, NA enzyme kinetic parameters (Km and Vmax), polymerase activities of ribonucleoprotein (RNP) complexes, and IFN gene/protein expression values induced by the wild-type and mutant H1N1 influenza viruses were compared by unpaired t-test or analysis of variance (ANOVA). Probability values ≤ 0.05 indicate statistically significant differences.The EC50 = 45.0 ± 8.4 ng/ml. However, IFN-λ1 did not fully protect Calu-3 cells from influenza H1N1 virus infection as it was observed previously [50 value was selected as the initial passage concentration of IFN-λ1 to obtain drug-resistant H1N1 mutants .We first measured the sensitivity of influenza A/California/04/09 (H1N1) virus to treatment with recombinant IFN-λ1. We evaluated the antiviral activity of this agent by cell ELISA, an assay that measures the level of inhibition of cell-associated virus yield in Calu-3 cells. A/California/04/09 virus was sensitive to inhibition by IFN-λ1: mean ECeviously . This EC mutants . We then10PFU/ml), and a mixed viral population was detected by plaque morphology analysis of culture supernatants at passage 15 (data not shown). Notably, the parental virus remained sensitive to inhibition by IFN-λ1 by cell ELISA after 15 passages (data not shown). Passage of the A/California/04/09 virus in the absence of IFN-λ1 caused the greatest reduction in plaque number at passage 23 .Analysis of the A/California/04/09 virus following culture in the presence of increasing concentrations of IFN-λ1 showed the largest decrease in plaque number at passage 21 . In contrast, the CA/04+IFN-λ1 variant exhibited significantly reduced sensitivity to IFN-λ1 compared to the wild-type A/California/04/09 virus (EC50 ˃ 1000 ng/ml). The EC50 values determined by cell ELISA and by virus reduction assay (data not shown) were comparable. These findings indicated that the CA/04+IFN-λ1 variant may have acquired one or more mutation(s) that decreased its sensitivity to the antiviral activity of IFN-λ1.After 30 serial passages in the presence or absence of IFN-λ1, we prepared stocks of two selected H1N1 variants: CA/04+IFN-λ1 and CA/04−IFN-λ1 viruses in MDCK cells. Both of the passaged variants grew to significantly higher titers than the parental strain, and they formed larger and more homogeneous plaques than the wild-type virus . The yields of the CA/04−IFN-λ1 variant were also significantly higher than the parental virus at all time points . These findings indicate that sequential passage of the parental H1N1 influenza virus in Calu-3 cells in the presence or absence of IFN-λ1 promoted selection of variants with enhanced growth properties.We next examined growth of the wild-type, CA/04+IFN-λ1 and CA/04−IFN-λ1) at the end of the passaging protocol  or A183G in PB1 or M1, respectively. Sequence analysis of the genome of the CA/04−IFN-λ1 virus revealed three mutations in HA1 , one mutation in M2 (E70K), two nucleotide mutations in PB1 (C975T and T2136C), and one nucleotide mutation (C1149T) and one amino acid substitution (V14I) in the PA protein  database: www.fludb.org). Three of the amino acid substitutions that we identified in our study, V14I in PA and G155E and S183P in HA1, were also present in 3.3% of the related H1N1 strains in the Influenza Research Database. The remaining eight mutations  were found in <0.6% of contemporary H1N1 isolates.To determine if the amino acid changes identified in our CA/04+IFN-λ1 and CA/04−IFN-λ1, in comparison to the wild-type virus. The binding level to the “avian-type” receptor (3′SL/N) was negligible (data not shown). Based on the Ka values .To determine if the HA1 mutations that we identified affect the HA affinity for sialyl receptors, we examined the receptor specificity of two selected variants, CA/04a values , both of−IFN-λ1 variant altered its viral transcription activity, we analyzed reconstituted RNP complexes in 293T cells using a luciferase mini-genome reporter assay (P < 0.01).To determine if the observed amino acid substitution V14I in the PA protein of the CA/04er assay . We founA183G nucleotide change and the E70K amino acid mutation into the background of the wild-type CA/04 virus to generate the recombinant viral constructs, CA/04-M1A183G and CA/04-M2E70K. Our data showed that A183G change slightly increased M1 protein expression level (E70K virus were significantly lower than the CA/04 virus only at 72 h post-infection (P < 0.05). We next measured sensitivity of our H1N1 recombinant viruses to the antiviral activity of IFN-λ1 by cell ELISA. Both of the mutant viruses, CA/04-M1A183G and CA/04-M2E70K, exhibited sensitivity to IFN-λ1 that was equivalent to the wild-type CA/04 strain in Calu-3 cells . We first measured the sensitivity of these three recombinant viruses  to the antiviral activity of IFN-λ1 by cell ELISA using Calu-3 cells as target cells . We also observed that the EC50 values determined by cell ELISA and by virus reduction assay . We also noted that the plaque sizes of all three mutant viruses were significantly increased (↑~4.1-fold) compared to those of the parental CA/04 virus (P < 0.05). To further evaluate the replication ability of our recombinant H1N1 viruses carrying the different NA mutations, we assayed their viral yields in comparison to the wild-type CA/04 strain after several replication cycles in Calu-3 cells. As shown in S79L,K331N virus grew to significantly higher titers than the wild-type CA/04 virus at 24 h post infection . Furthermore, the yields of the CA/04-NAS79L and CA/04-NAK331N mutants were also higher than that of the wild-type virus at 24 and 96 h post-infection time points in Calu-3 cells .We next examined replication of the CA/04-NAm and Vmax values for the CA/04, CA/04-NAS79L, CA/04-NAK331N, and CA/04-NAS79L,K331N viruses  than the wild-type virus NA (P < 0.05). Furthermore, the S79L mutation correlated with significantly decreased NA enzyme activity  in Calu-3 cells. As shown in S79L virus induced similar levels of IFN gene expression as those induced by the parental virus. However, both the CA/04-NAK331N and CA/04-NAS79L,K331N viruses induced significantly lower levels of IFNB1, IFNL1, and IFNL2/3 gene expression at 48 and 72 h post-infection (P < 0.05). We next measured IFN-λ1 and IFN-λ2/3 protein levels in Calu-3 cells after infection with our mutant viruses by ELISA .We compared the ability of our recombinant H1N1 viruses containing the NA mutations to induce IFN gene expression  and IFN proteins (IFN-λ1 and IFN-λ2/3) by target respiratory epithelial cells. The reduced IFN gene/protein expression levels also correlated with decreased activation of STAT1, suppressed expression of RIG-I, and reduced sensitivity to the antiviral activity of IFN-λ1.In the present study, we generated an IFN-λ1-resistant mutant of the A/California/04/09 (H1N1) virus by serially passaging the parental virus in the presence of increasing concentrations of IFN-λ1 for a total of 30 passages. The selective pressure mediated by IFN-λ1 induced acquisition of several viral genomic mutations. One of these mutations, K331N in the NA protein, correlated with weaker phosphorylation of IRF3 and decreased ability of the virus to induce expression of the IFN genes ; however, it did not compromise viral growth. Importantly, our results showed that the K331N but not the S79L NA mutation was associated with markedly increased resistance to IFN-λ1 (˃ 20-fold increase in the mean EC50) and correlated with reduced anti-viral responsiveness to IFN-λ1. We have not studied the impact of the K331N substitution on sensitivity to IFN-λ2 and -λ3 because of the initial resistance of the parental virus to these type III IFNs (data not shown).We found that two amino acid changes in the NA protein, S79L and K331N, were selected in the A/California/04/09 virus after extended passaging in Calu-3 cells in the presence of IFN-λ1. The S79L mutation is located in the stem domain, and K331N is located near the active site of the NA protein. We observed that both of these NA mutations were viable and genetically stable on the H1N1 virus background. The S79L mutation significantly reduced the activity of the NA enzyme  was associated with the IFN-λ1-resistant phenotype, the decreased NA activity mediated by the S79L mutation may be important for H1N1 virus viability in human epithelial cells. This finding coincides with previously reported data showing that decreased NA activity is necessary for the adaptation of highly pathogenic H5N1 influenza viruses for growth in human airway epithelium [+IFN-λ1 variant that was selected in the presence of IFN-λ1. In this study we examined the phosphorylation status of IRF3 and the kinetic profile of IFN gene/protein expression by Calu-3 cells after infection with recombinant H1N1 viruses carrying the S79L and/or K331N mutations. We found that mutants carrying either the single amino acid change (K331N) or the double mutation (S79L and K331N) induced weaker antiviral responses than the parental CA/04 virus—as evidenced by reduced IRF3 phosphorylation, weak induction of the IFNB1, IFNL1, and IFNL2/3 genes, and low expression of IFN-λ1 and IFN-λ2/3 proteins in Calu-3 cells. Moreover, infection with the CA/04-NAK331N or CA/04-NAS79L,K331N virus resulted in decreased activation of STAT1 and reduced expression of RIG-I compared to those induced by infection with the parental CA/04 virus or the CA/04-NAS79L virus. It is unclear how influenza infection of Calu-3 cells pretreated with IFN-λ1 can select for a virus that induces less IRF3 phosphorylation and a consequent weaker production of IFN-λ1. Interestingly, previous study by Grimm et al. [Our results showed that the CA/04-NAithelium . Notablyithelium . Additioithelium , the decm et al.  demonstr+IFN-λ1 and CA/04−IFN-λ1 variants revealed that M257I HA1 substitution was associated with a significant decrease in binding to α2,6 sialic acid-linked receptor . It is likely that the observed HA1 changes located next to the HA receptor-binding pocket contribute to the increased ability of the parental virus to infect and spread in mammalian respiratory epithelium.To monitor the potential emergence of amino acid changes associated with adaptation of the A/California/04/09 virus to growth in Calu-3 cells, we also passaged the parental virus in parallel in the absence of IFN-λ1. We identified three mutations in the HA1 protein , one amino acid change in the PA protein (V14I), and one mutation in the M2 protein (E70K), that partially, and more likely independently, controlled adaptation of the pandemic 2009 isolate to growth in respiratory epithelial cells. Notably, two of the HA1 mutations that we identified in this study, G155E and S183P, were shown previously to be necessary for the enhanced virulence of the A/California/04/09 (H1N1) isolate in mice . FurtherC975T, PB1 T2136C, PA V14I, and PA C1149T, were involved in the adaptation of the pandemic H1N1 isolate to growth in Calu-3 cells. A single amino acid substitution (V14I) in the PA protein was sufficient to significantly enhance the transcription activity of the polymerase complex of the A/California/04/09 virus (P < 0.01). The V14I mutation occurred only in the CA/04−IFN-λ1 virus most likely due to the lack of additional IFN-λ1 selective pressure during sequential passaging. This mutation was likely necessary for optimal interaction of the viral polymerase with human host proteins and enabled the CA/04−IFN-λ1 virus to replicate efficiently in Calu-3 cells.We found that mutations at four points in the RNP complex, namely PB1 +IFN-λ1 and CA/04−IFN-λ1, this indicates that this change is not a random mutation. We believe that this change reflects a specific character of variation, most likely linked to adaptation of this virus for growth in the human lower respiratory tract. Our results add to the growing body of mutational analysis data defining the structural and functional changes that govern adaptation of H1N1 viruses to human respiratory epithelial cells. Our findings demonstrate that multiple adaptive changes occurred when the A/California/04/09 (H1N1) virus was passaged for an extended time period in Calu-3 cells. These viral adaptations included optimizing receptor specificity, NA enzyme catalytic activity, and interaction of viral polymerase components with host cell factors. These changes provide a means by which the pandemic H1N1 virus can achieve an optimal competitive advantage in the human lower respiratory tract.Passage of H1N1 virus in the presence or absence of IFN-λ1 resulted in acquisition of an M2 mutation, E70K, which was associated with decreased replication ability in Calu-3 cells at 72 h post-infection. However, since an amino acid change of the same residue (residue 70) occurred independently in both variants, CA/04To our knowledge, this is the first study to evaluate the emergence of resistance of pandemic A/California/04/09 (H1N1) influenza virus to the anti-viral activity of a type III interferon. Future characterization of the genetic stability, infectivity, and pathogenicity of our IFN-λ1-resistant H1N1 virus in human primary lung epithelial cells or in an appropriate animal model should help to define its pathogenicity. Finally, the findings described in this report may aid in the development of more effective therapeutic strategies to prevent and treat influenza virus infection.S1 FigThe levels of viral PB1 and M1 proteins in concentrated and purified H1N1 viruses carrying silent PB1 and M1 mutations were analyzed by western blot with rabbit anti-PB1  and mouse monoclonal anti-M1 Abs , respectively.(PPTX)Click here for additional data file.


JMJD2B gene expression by binding to a canonical p53-consensus motif in the JMJD2B promoter. JMJD2B induction attenuates the transcription of key p53 transcriptional targets including p21, PIG3 and PUMA, and this modulation is dependent on the catalytic capacity of JMJD2B. Conversely, JMJD2B silencing led to an enhancement of the DNA-damage driven induction of p21 and PIG3. These findings indicate that JMJD2B acts in an auto-regulatory loop by which p53, through JMJD2B activation, is able to influence its own transcriptional program. Functionally, exogenous expression of JMJD2B enhanced subcutaneous tumor growth of colon cancer cells in a p53-dependent manner, and genetic inhibition of JMJD2B impaired tumor growth in vivo. These studies provide new insights into the regulatory effect exerted by JMJD2B on tumor growth through the modulation of p53 target genes.The p53 tumor suppressor protein plays a critical role in orchestrating the genomic response to various stress signals by acting as a master transcriptional regulator. Differential gene activity is controlled by transcription factors but also dependent on the underlying chromatin structure, especially on covalent histone modifications. After screening different histone lysine methyltransferases and demethylases, we identified JMJD2B/KDM4B as a p53-inducible gene in response to DNA damage. p53 directly regulates  The p53 protein responds to a variety of cellular stresses, including genotoxic damage, hypoxia, nutrient depletion and aberrant proliferative signals through oncogene activation. Following stress, p53 protein translation and half-life increase, and p53 binds to DNA as a tetramer in a sequence-specific manner. The increase in p53 protein results in the transcriptional regulation of genes involved in mediating key cellular processes, such as DNA repair, cell-cycle arrest, senescence, apoptosis, autophagy and metabolism –3.Modulation of chromatin through covalent histone modification is a fundamental way of regulating DNA accessibility during processes such as gene transcription, DNA replication and DNA damage repair . A majorN-methyltransferase (PRMT) family have been shown to methylate arginine residues. These histone methyltransferases have the capability to methylate histones as well as non-histone proteins (A plethora of histone lysine methyltransferases (HMTs) and demethylases (HDMs), which affect the dynamic regulation of lysine methylation and demethylation respectively, have been discovered in the past decade. Three families of enzymes have been identified that catalyze the addition of methyl groups to histones. The SET-domain-containing proteins and DOT1-like proteins have been shown to methylate lysines, and members of the protein arginine proteins . In contproteins .JMJD2B is induced by p53 activation, supporting the notion that it is a bona fide p53 target. By chromatin immunoprecipitation (ChIP) assay and functional promoter analysis, we also demonstrate that p53 can bind directly to a distal region on JMJD2B promoter, which has strong homology to canonical p53-binding motifs. Further, we identify a list of key p53 target genes that are affected by the overexpression of JMJD2B in the context of DNA damage response. In particular, we show that p53 was less efficient in inducing its target genes p21, PIG3 and PUMA upon DNA damage under conditions in which JMJD2B expression is elevated, and most importantly JMJD2B catalytic activity is required to modulate this response. The attenuated response of key p53 targets is reflected by a decrease in the H3K4me3 permissive mark, concomitantly with an increase in the H3K9me3 repressive mark at those specific promoters. In agreement with these findings, JMJD2B silencing leads to an enhancement in the DNA-damage driven induction of the p53 targets p21 and PIG3. Functionally, we show that exogenous expression of JMJD2B significantly enhances the ability of human colon cancer cells to grow in vivo in a p53-dependent manner, whereas genetic inhibition of JMJD2B significantly delays in vivo tumor growth. Taken together, these studies lead us to propose that the development of JMJD2B specific inhibitors might represent a valuable approach for cancer therapy.Although several epigenetic enzymes are able to methylate and demethylate p53 ,10, littHCT116 p53+/+ and p53−/− colon carcinoma cells, RKO p53 wt and isogenic RKO-E6 cells transfected with a stably integrated human papillomavirus (HPV) E6 oncogene under control of the cytomegalovirus promoter, mouse embryonic fibroblasts (MEFs) and 293FT cells were maintained in Dulbecco's modified Eagle medium; HeyA8 ovarian cancer cells were grown in RPMI medium. Media were supplemented with 10% (v/v) fetal bovine serum (Omega Scientific), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Doxorubicin, Nutlin-3, Etoposide and 5-Fluorouracil (5-FU) were purchased from Sigma-Aldrich. γ-irradiation was performed with a Mark I irradiator containing a Cesium 137 source (J.L. Sheperd and Associates).https://lifescience.roche.com/en_us/brands/universal-probe-library.html) and are listed in the RNA was isolated using Trizol (Invitrogen) and subsequently treated with DNase I (Fermentas). First-strand cDNA synthesis was performed with SuperScript II Reverse Transcriptase and random primers (Invitrogen) according to the manufacturer's instructions. qPCR was carried out using Power SYBR Green Master Mix (Life Technologies), detection and data analysis were executed with the 7900HT Fast Real-Time PCR System (Applied Biosystems) by computing the results relative to a standard curve made with cDNA pooled from all samples, normalized to 18S. Primer sequences used to amplify specific target genes were obtained from the Universal Probe Library Assay Design Center  were purchased from Open Biosystems. The target sequences in JMJD2B mRNA are 5΄- GCCCATCATCCTGAAGAAGTA-3΄ for shRNA-2 and 5΄-GTGGAAGCTGAAATGCGTGTA-3΄ for shRNA-4. Control hairpin against GFP in the pLKO.1 backbone (pLKO.1-shGFP) was a kind gift from Silvestre Vicent (Stanford University). For lentiviral stock preparation, 293FT cells were transfected with pLKO.1-based vectors and two helper plasmids (pCMV-ΔR8.2 and pCMV-VSV-G) using Lipofectamine and Plus Reagent according to the manufacturer's protocol (Invitrogen). About 24–48 h after transfection, media containing packaged lentivirus were collected, passed through 0.45 μ filters and added to target cells along with 5 μg/ml of Polybrene (Sigma-Aldrich). Infected cells were selected in puromycin-containing media (1 μg/mL) for 1 week.Short hairpin RNA (shRNA) lentiviral constructs against human TP53 and JMJD2B, and siGENOME Non-Targeting siRNA Pool #2. Cells were transfected at a final concentration of 100 nM using DharmaFECT 1 transfection reagent, according to the manufacturer's instructions (Dharmacon).Small interfering RNA (siRNA) was carried out using Dharmacon siGENOME SMART pool siRNAs for JMJD2B/KDM4B full-length human cDNA was obtained from Open Biosystems  and subcloned by HindIII and XhoI (New England Biolabs) into the retroviral expression vector pLPC  by PCR, using forward primer 5΄- AGAGAGAAGCTTAGCCATGGGGTCTGAGGACC-3΄ and reverse primer 5΄- ATATATCTCGAGGGCCAGCTGTCCTAGAAGGG-3΄. JMJD2B catalytic mutant (JMJD2BH189A) was generated with the QuikChange II XL site-directed mutagenesis kit (Agilent) by using forward primer 5΄-ACCACCTTCGCCTGGGCGACCGAGGACATGGAC-3΄ and reverse primer 5΄- GTCCATGTCCTCGGTCGCCCAGGCGAAGGTGGT-3΄, as per manufacturer's instructions. The JMJD2BH189A construct was subcloned by HindIII and XhoI (New England Biolabs) into pLPC by PCR. All constructs were verified by sequencing. Retrovirus production and infections were done as described previously . The JMJD2B mutant p53-binding site #3 construct was generated by altering the CATG and CTGG p53-consensus half-sites in TCCC and TCCC, respectively. DNA regions containing BS3, mut BS3 or BS6/7 sites were subsequently inserted by NheI and XhoI (New England Biolabs) into the pGL3-Promoter vector upstream of the firefly luciferase gene (Promega), while the insert containing BS9 and BS10 sites was subcloned by NheI and HindIII into the pGL3-Basic vector. All constructs were verified by sequencing. Transient transfection of luciferase-reporter plasmids was mediated by Lipofectamine and PLUS Reagent according to the manufacturer's instructions (Invitrogen). Twenty-four hours after transfection, HCT116 p53+/+ and p53−/− cells were exposed to doxorubicin for 18 h or left untreated. Alternatively, HCT116 p53−/− cells were co-transfected with either pLPC-empty vector (pLPC(EV)) or pLPC vector carrying human N-FLAG-p53 (pLPC-p53) along with the luciferase-reporter plasmids BS3 or mut BS3, following doxorubicin treatment. Cells were lysed and assayed for firefly luciferase activity using a Bright-Glo Luciferase Assay System (Promega) on a Monolight 2010 luminometer . Assays were performed in duplicate and repeated three times.For protein analysis, cells were harvested in lysis buffer  supplemented with protease and phosphatase inhibitor cocktails (Roche), then incubated on ice for 15 min, vortexed and centrifuged at 13 000 rpm. Protein lysates were quantified using BCA Protein Assay kit (Pierce) and 25–100 μg of protein samples were resolved by SDS-PAGE according to standard methods, then transferred onto 0.2 μm Supported Nitrocellulose membranes (Bio-Rad Laboratories). The following primary antibodies were used to detect specific proteins: JMJD2B , p53 , phospho-p53 S15 , p21 (Santa Cruz Biotechnology), PIG3 (Oncogene Research Products), MDM2 (Santa Cruz Biotechnology), histone H3K9me3 (Active Motif), Hsp70 (Sigma-Aldrich), α-tubulin , β-actin (Sigma-Aldrich). Secondary antibodies used in this study were HRP-conjugated goat anti-mouse and anti-rabbit (Vector Laboratories). Immunoblots were developed with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and visualized with ChemiDoc XRS+ imaging system equipped with Image Lab Software (Bio-Rad Laboratories). Protein bands were quantified by densitometry using ImageJ software .Protein lysates were prepared and quantified as described under Western blotting section. Approximately 250 µg of proteins were pre-incubated with Protein A/G Dynabeads (Thermo Fisher Scientific), followed by overnight incubation with 2 μg of p53 DO-1 antibody (Santa Cruz Biotechnology) at 4°C with gentle agitation. Normal mouse IgG (Santa Cruz Biothechnology) was used as a non-specific IgG control, and 10% of the sample from each immunoprecipitation was reserved for input control. Subsequently, lysates were incubated with 25 μl of Protein A/G Dynabeads for 3 h at 4°C and immunoprecipitated proteins were collected using magnetic stand, washed three times, boiled in 1× sample loading buffer for 10 min and analyzed by WB.For xenograft studies, tumor specimens were fixed in 10% (v/v) neutral buffered formalin, permeabilized with 95% (v/v) ethanol and embedded in paraffin. Tumor sections were subsequently deparaffinized with xylene, rehydrated in ethanol solutions and subjected to antigen retrieval using 10 mM citric acid buffer (pH 6.0) in microwave for 10 min. Slides were probed with primary anti-Ki67 antibody (Thermo Fisher Scientific) overnight at 4°C, followed by secondary detection using biotinylated anti-rabbit antibody (Vector Laboratories) and streptavidin-HRP conjugated antibody (EMD Millipore), both for 30 min at 37°C. Negative controls for all samples were tissue sections treated with secondary antibodies alone. Proteins were visualized with DAB Chromogen System (DAKO), counterstained with Hematoxylin (VWR) and mounted on slides with Fluoromont-G (Southern Biotech). Pictures were captured using a Leica DM6000B microscope (Leica Microsystems) equipped with Image-Pro Premiere 9.0 software (Media Cybernetics).2 × length) × 0.5.All procedures involving animals and their care were performed in accordance with Institutional and National guidelines, and approved by Stanford University's Administrative Panel on Laboratory Care (APLAC). HCT116 p53+/+ and p53−/− cells stably expressing pLPC(EV) and pLPC-JMJD2B, or carrying shGFP, shJMJD2B-2 and shJMJD2B-4 lentiviral constructs were implanted subcutaneously into the lower flanks of SCID Hairless 6–8 week-old female mice (Charles River Laboratories). Tumors were measured with calipers at regular intervals, and tumor volume was calculated according to the following formula: volume = . Significance was determined by two-tailed Student's TP53 gene (HCT116 p53+/+ cells) and in the isogenic cell line lacking TP53 (HCT116 p53−/−), following treatment with the chemotherapic agent doxorubicin. The screening of 25 proteins comprising both families of amine oxidase and jumonji C–domain containing iron-dependent dioxygenases, several of which have been found to possess histone demethylase activity, revealed different patterns of regulation following DNA damage , was found to be regulated by p53. These genes were induced about 2.5-fold selectively in HCT116 p53+/+ cells, but not in HCT116 p53−/− cells in response to doxorubicin treatment. A second group of genes, including JARID2 , JMJD6 (jumonji domain containing 6 demethylase) and the lysine-specific demethylase 1A , was selectively repressed by p53. Indeed, their transcript levels were increased about 1.5–2-fold in both control and doxorubicin-treated HCT116 p53−/− cells. Finally, a third subset of genes, including the hair growth associated gene HR , the lysine-specific demethylase 2B  and the lysine-specific demethylase 4D , was induced in response to the DNA damaging agent, but in a p53-independent manner. In fact, only HCT116 cells lacking p53 protein showed a 2–2.5-fold increase in the expression of these epigenetic enzymes in response to doxorubicin treatment, and their mRNA levels were unchanged in unstressed conditions.In order to identify novel p53-transcriptionally regulated genes among the different families of epigenetic enzymes, we performed a qPCR screen by analyzing changes in the expression of HDMs and HMTs in cells harboring a WT e Figure . In partJMJD2B was of particular interest given the apparent paradox of being activated by p53, yet being recently reported to be overexpressed in numerous cancers , KMT2B , SETD1B (SET domain containing 1B), SETD1A (SET domain containing 1A), SETD7 (SET domain containing-lysine methyltransferase 7), SETD2 (SET domain containing-lysine methyltransferase 2), EHMT2 , SMYD2 (SET and MYND domain containing 2) and SUV39H1 (suppressor of variegation 3–9 homolog 1 (Drosophila)) displayed a marked repression exerted by p53. Indeed, their transcript levels were increased about 1.5-fold in both control and doxorubicin-treated HCT116 p53−/− cells. This result is in line with the recent findings of Mungamuri et al. and Zheng et al., showing the ability of p53 to influence its own transcriptional program by downregulating the expression of the histone methyltransferase SUV39H1  or a non-targeting control siRNA (siCON), and treated with doxorubicin. Consistent patterns of JMJD2B induction upon DNA damage were observed in the two cell lines treated with a non-targeting control siRNA. Specifically, HCT116 cells exhibited about 9-fold induction  and ionizing radiation (IR). We found that JMJD2B expression, at both mRNA  were identified, according to the consensus sequence RRRC(A/TA/T)GYYY (N)0-13 RRRC(A/TA/T)GYYY  from the TSS (over 9-fold enrichment upon DNA damage treatment compared to control). The occupancy of p53 on the PCNA promoter, an established p53 target gene  (Since our data indicate that A/T)GYYY , within e Figure . p53 ChIe Figure . Endogenget gene , was useget gene . Interest Figure . Notablyn Figure , indicatand BS5) .JMJD2B and to pinpoint the functional p53-consensus, we generated several reporter constructs by subcloning promoter fragments containing BS3, BS6/7 or BS9+10 into the pGL3-Promoter luciferase reporter vector  in response to doxorubicin relative to the vector control (pGL3prom), but not in cells transfected with BS6/7 or BS9+10. Furthermore, mutation of the BS3 site completely abolished this response, demonstrating that the observed p53-dependent activity is mediated through this consensus motif. In order to confirm that the JMJD2B regulatory region BS3 responds specifically to p53, the same reporter assay was conducted using HCT116 p53−/− cells -transfected cells, and about 1.5-fold versus the control samples for pLPC-JMJD2B overexpressing cells. These effects were even more profound in cells challenged with 5-FU for 48 h. We found that p21 transcripts were induced following DNA damage in cells treated with either 5 or 10 μg/ml of 5-FU at both 24 and 48 h time points, and this induction was significantly diminished by the overexpression of JMJD2B. Similarly, the mRNA levels of PIG3  were increased more than 20-fold versus the untreated samples for pLPC(EV)-transfected cells and about 15-fold versus the unstressed samples for pLPC-JMJD2B overexpressing cells, but overall PIG3 mRNA induction was robustly attenuated by JMJD2B following DNA damage , another key modulator of p53-mediated apoptotic activity, in the context of JMJD2B overexpression. In fact, PUMA transcript levels were reduced in cells expressing pLPC-JMJD2B versus pLPC(EV) transfected cells, with a rather modest effect when cells were treated with a lower dose of 5-FU, but with a more profound effect in cells challenged with 10 μg/ml of 5-FU. Whereas the expression of p21, PIG3 and PUMA was significantly impaired after JMJD2B overexpression at both time points, NOXA transcript levels were not affected at 24 h but modestly, although significantly, reduced at 48 h post stimulation with 5-FU.The p53 transcription factor regulates the expression of an array of different genes, which mediate the p53 response to different forms of stress . Activate Figure . We alsoIn line with these results, we observed a significant reduction of p21, PIG3 and PUMA proteins accumulation in cells expressing pLPC-JMJD2B compared to control cells following DNA damage, at both 24 and 48 h time points, with a more robust effect after a 48 h treatment Figure . Also, Np21 and PIG3 in cells treated with either doxorubicin  . These findings clearly demonstrate that the JMJD2B impingement on p53-dependent transcriptional activity is dependent on the catalytic capacity of JMJD2B.To unravel the functional requirement of JMJD2B for the attenuation of p53-mediated transcriptional activity, we utilized a catalytically dead variant of JMJD2B (pLPC-JMJD2B) Figure . QPCR anp21, PIG3 and PUMA gene expression , in response to 5 or 10 μg/ml of 5-FU treatment for 24 h, respectively. Efficacy of siRNA delivery was tested, and JMJD2B gene silencing ranged from 70 to 75% in treated and untreated samples. Similarly, DNA-damage driven induction of PIG3 mRNA was also enhanced in a statistically significant manner by knocking down JMJD2B.Because JMJD2B counteracts the p53-dependent induction of key p53 targets in response to DNA damage, we wanted to assess the impact of JMJD2B silencing on n Figure . QPCR anp21, PIG3 and PUMA seems paradoxical. In fact, JMJD2B would be expected to further activate the activity of these p53 targets rather then repressing it, by removing the inactive H3K9me3 mark. Thus, we wanted to document changes in histone methylation marks at those promoters by analysing the effect of JMJD2B overexpression on the levels of H3K4 and H3K9 trimethylation, hallmarks of active and inactive chromatin respectively. We performed ChIP followed by qPCR analysis using nuclear extracts from HCT116 WT cells, stably expressing pLPC-JMJD2B construct or pLPC-empty vector control. The ratio of H3K4me3  on histone H3, reducing both modifications to the monomethylated state ,12, the 3 Figure  and H3K93 Figure  to bulk 3 Figure . Overexpn Figure , in accon Figure . JMJD2B p21, PIG3 and PUMA promoters  or hairpin targeting a control protein (shGFP) into the dorsal flanks of SCID hairless mice , a second group selectively repressed by p53  and a third group induced in response to DNA damage but in a p53-independent manner . In contrast, the screening of HMTs revealed a unique pattern of regulation, as most of these genes are repressed by p53, both in stressed and unstressed conditions . The identification of SUV39H1 as a p53-repressed methyltransferase in our screen is in agreement with recent reports showing that induction of p53 by various methods led to decreased RNA and protein levels of SUV39H1  domain. JMJD2B specifically targets the trimethylated lysine 9 of histone H3 (H3K9me3) for demethylation at pericentric heterochromatin and euchromatin . JMJD2B rouracil . Recent rouracil .p21 promoter upon exposure to adriamycin . However, it is possible that this consensus site might represent an alternative and/or additional binding site for p53 to use under certain conditions.Recently, it has been reported that a 1-kb fragment of the onstruct , althougp21, PIG3 and PUMA, without affecting the overall cellular levels of the H3K9me3 mark, thus highlighting a repressive regulatory loop by which the p53 tumor suppressor is able to influence its own transcriptional program. In contrast, we detected no changes on NOXA transcript levels upon ectopic expression of JMJD2B, suggesting that JMJD2B exerts selective regulation of p53 transcriptional activity by inhibiting only a subset of p53 target genes. Because H3K9 trimethylation is a hallmark of inactive chromatin and is normally mutually exclusive with H3K4 trimethylation, which is a mark for active promoters, the recruitment of JMJD2B to active promoters would guarantee that the H3K9me3 epigenetic marks become demethylated, which will then amplify gene transcription at specific sites. Our findings of an attenuated response of p21, PIG3 and PUMA expression following JMJD2B overexpression, seems paradoxical as JMJD2B would be predicted to remove the inactive epigenetic mark and facilitate gene transcription of these p53-target genes, instead of repressing their activity. Different models can be conceived to explain this regulatory feedback repressive loop exerted by JMJD2B on p53 function. One model invokes the involvement of MDM2 protein, a well know p53 gatekeeper, which can inhibit p53 activity by a variety of means. First, by binding to the transactivation domain of p53, MDM2 sterically blocks the function of that domain. Moreover, by acting as a p53-specific E3 ubiquitin ligase, MDM2 promotes the ubiquitylation and subsequent proteasomal degradation of p53 (BHLHE40 (ZNF420 (ID1 (BHLHE40, ZNF420 and ID1 transcriptional repressors after JMJD2B ectopic expression in HCT116. Nevertheless, consistent with a role of a transcriptional repressor, we have provided data documenting changes in histone methylation marks at the p21, PIG3 and PUMA promoters. Specifically, we have shown a decrease of the H3K9me3 permissive mark along with an increase of the H3K9me3 repressive mark at those promoters, which recapitulates our findings of an attenuated response of these p53-targets after JMJD2B overexpression. Most importantly, we have presented data demonstrating that the JMJD2B catalytic activity is required to attenuate the p53-mediated transcription activity of p21 and PIG3 targets, following DNA damage. These findings, together with evidences showing that JMJD2B is not recruited on the promoters of those p53 target genes, allowed us to strengthen our model implying the requirement of a transcriptional repressor.In an effort to identify a list of key p53 target genes that were affected by JMJD2B in response to DNA damage, we show that JMJD2B is able to attenuate the response of p53 established target genes, such as n of p53 –59. Actin of p53 . JMJD2B n of p53 . Our dat(BHLHE40 , ZNF420  (ZNF420  and ID1 420 (ID1 , followi420 (ID1 . Howeverp21, PIG3 and PUMA. Indeed, lysine methylation has been shown to occur on histones as well as on non-histone proteins (Alternatively, a third model might imply the ability of JMJD2B to form a complex with p53, and such physical interaction, via a demethylation process, would cause the repression of p53-dependent transcriptional activation of its targets proteins , and sevproteins . Conversproteins . HoweverA final model can evoke the ability of JMJD2B to potentially demethylate H3K36me3, which would lead to decreased expression of the p53 transcriptional targets, since the removal of the trimethylated mark on H3K36 would repress transcription. In fact, comprehensive studies have determined the histone demethylase specificity of JMJD2 proteins, as well as shown that JMJD2B, similarly to JMJD2A and JMJD2C, can act on the tri- and di-methylated forms of both H3K9 and, less efficiently, H3K36 substrates ,12,65,66JMJD2B-/- mice are viable and do not display gross abnormalities might also argue against a proposed cancer predisposition (Several recent studies have supported the evidence that alteration in the functioning of histone demethylases might have a profound role in cancer, given the potential contribution of an imbalance of histone methylation to oncogenic transformation . In lineposition .JMJD2B/KDM4B as a bona fide p53-responsive gene, which acts as a rheostat for the p53-dependent transcriptional program. Our findings reveal a novel auto-regulatory repressive loop by which p53, through JMJD2B activation, is able to influence its own transcriptional activity by inhibiting the expression of a subset of its target genes. More importantly, ectopic expression of JMJD2B enhances tumor growth in vivo, suggesting that JMJD2B may represents an attractive epigenetic target for therapeutic intervention of cancers harboring WT p53.In summary, the data presented here identified the histone demethylase Supplementary DataClick here for additional data file.


Thus, we analyzed the capacity of STAT5A and STAT5B to control expansion of human hematopoietic stem/progenitor cells (HS/Pc) and transformation of murine HSC using oncogenic STAT5A/5B variants.Signal transducer and activator of transcription 5A and 5B (STAT5A/5B) are encoded by closely related and chromosomally juxtaposed genes. Both proteins play a major role in normal hematopoiesis.+ cells cultured with SCF. We first verified that STAT5A/5B proteins were endogenously expressed and activated by SCF in CD34+ cells (+ cells in vitro and this was accompanied in long-term culture by the graduate downregulation of endogenous STAT5A/5B expression (+ cells cultured with SCF10 (Y694F) failed to induce expansion of CD34+ cells (+ cells transduced with TAT-STAT5A protein were not able to grow in the absence of SCF (data not shown). These data indicated that sustained expression and activation of STAT5A are sufficient to promote CD34+ cell growth. We then asked whether TAT-STAT5A or TAT-STAT5B recombinant proteins maintain similar impact on CD34+ cells. A schematic overview on the modular design of TAT-STAT5A/5B recombinant proteins used in this study is shown in 10 The purity and identity of both recombinant proteins were confirmed by Coomassie gel staining and western blot using either anti-HA or anti-STAT5-specific antibodies  that does not activate STAT5 in CD34+ cells ) cells were infected with recombinant retrovirus expressing constitutively active STAT5A (cS5a) or STAT5B (cS5b) followed by IRES-GFP or GFP alone as control. GFP+ cells were sorted and cultured with SCF at indicated times. Uninfected LSK cells were cultured in the presence of SCF and IL-3 (SCM medium) as positive control  775 and aa 785 is of critical importance for STAT5A-induced CD34+ cell growth. Importantly, deletion of this sequence also abrogated the leukemogenic potential of the hyperactive cS5a variant  in normal human HS/Pc  failed to induce CD34+ cell proliferation .l region . Recombil region . In orde mutants . TAT-STAl growth . In cont variant . Sequenc variant . Constit4+ cells . The P21an HS/Pc . We addrern blot . Transduperiment . Mutatioferation .779 differentially fine-tunes proliferation and transformation of HS/Pc with higher capacity over STAT5B. The highly related STAT5A/B proteins regulate hematopoiesis or neoplastic cell growth through distinct and specific signaling mechanisms, possibly explaining differences in leukemic disease development associated with gain-of-function STAT5A or STAT5B variants.In conclusion, our findings indicate that specific phosphorylation of STAT5A proteins at Ser


We compared the hyperoxia-induced proliferative retinopathy (HIPR) model in two mouse strains (C57BL/6J and C57BL/6N). Pups were exposed to 75% oxygen, starting at birth and continuing for 14 days (P14). Mice were euthanized at P14, or allowed to recover in room air for one day (P15), seven days (P21), or 14 days (P28). We quantified retinal thickness and the length of residual photoreceptors not affected by rosette formation. In addition we explored differences in retinal immunostaining for NADPH oxidase 4 (NOX4), Rac1, vascular endothelium, and activated Mϋller cells. We analyzed photoreceptor oxidative stress using DCF staining in cross sections and quantified NOX4 protein levels using western blotting. C57BL/6N mice in HIPR showed increased oxidative stress, NOX4, and Rac1 in the photoreceptors at P14 and P15 compared to C57BL/6J. In addition, we observed significant progression of photoreceptor degeneration, with significantly accelerated rosette formation in C57BL/6N under HIPR, compared to their room air counterparts. Furthermore, C57BL/6N under HIPR had significantly thinner central retinas than C57BL/6J in HIPR. We did not find a difference in vascular disruption or Mϋller cell activation comparing the two strains in hyperoxia. In HIPR, the C57BL/6N strain carrying the rd8 mutation showed significantly accelerated photoreceptor degeneration, mediated via exacerbated photoreceptor oxidative stress, which we believe relates to Rac1-NOX dysregulation in the setting of Crb1 loss-of-function.To investigate the impact of photoreceptor oxidative stress on photoreceptor degeneration in mice carrying the  On funduscopic examination, the rd8 mice show large white spots in the nasal quadrant, progressively spreading along with a slow process of photoreceptor degeneration [rd8 mutation is characterized by retinal folding, photoreceptor rosettes, outer nuclear layer loss, and retinal degeneration [rd8 at 28 days of age, progressing to disorganized outer segments, with shortened inner segments by 10 weeks of age [The mutation known as retinal degeneration 8 , which is comprised of highly specialized junctions that maintain retinal integrity and polarity. One of these junctions is the subapical region (SAR), where the photoreceptor inner segments and Mϋller glial cells connect. Crb1 localizes at the SAR on the microvilli of Mϋller glial cells [The protein product of the al cells –5. Loss al cells –5.Drosophila, crb has been shown to control epithelial polarity by counteracting phosphoinositol 3-kinase (PI3K) and Rac1, a Rho GTPase[Drosophila model, loss of crb was shown to lead to dysregulation of the Rac1-NOX pathway, causing superoxide overproduction, and destruction of the photoreceptors [rd8 mutation [In ho GTPase–10. Rac1ho GTPase. Crb repho GTPase. In a Dreceptors . Similareceptors . Histolomutation , 13.rd8 mutation. We used the hyperoxia-induced proliferative (HIPR) model [rd8 mutation, while the C57BL/6J carried the wild-type Crb1 [rd8 phenotype characterized by rosette formation in B6N mice via Rac1 dysregulation. We found that C57BL/6N develop an accelerated retinal degeneration, with loss of about 80% of photoreceptors within the first 4 weeks, compared to their room air raised littermates. These findings demonstrate that Crb1 is important in regulating photoreceptor oxidative stress and may have important translational implications to human disease, particularly infants carrying CRB1 mutations.Given the importance of Crb1 in regulating Rac1 and limiting the production of photoreceptor superoxide, we sought to explore the effect of neonatal hyperoxia and neonatal proliferative retinopathy on mice carrying the R) model , where nype Crb1 , 16. We This study was approved by the Institutional Animal Care and Use Committee at Northwestern University and all animal procedures were performed in compliance with the Institutional Animal Care and Use Committee at Northwestern University and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were sacrificed with isoflurane overdose and cervical dislocation.rd8 mutation while the C57BL/6J mice carry the wild-type Crb1 gene [Two strains of mice, C57BL/6J  and C57BL/6N , were used. The C57BL/6N mice carry the rb1 gene , 16. We rb1 gene , 17, 18.rd8 alleles as previously described, using the mCrb1-mF1, mCrb1-mF2, and mCrb1-mR primers [rd8 allele produces a 244 bp fragment.DNA was isolated from tail biopsies and DNA samples were amplified separately for the wildtype and mutant Enucleated eyes were fixed in 10% neutral buffered formalin for 24 hours or fixed in 4% paraformaldehyde for four hours. Paraffin embedded eyes were sectioned (7 μm), deparaffinized, and hematoxylin and eosin stained. Images were taken with Nikon 80i Eclipse microscope  using a Photometrics CoolSnap CF camera .distance_between_polylines.java ImageJ plug-in [Retinal thickness analysis was performed using ImageJ software , as previously reported . Thickne plug-in . This toThe percentage of residual healthy photoreceptors was determined by dividing the length of visible photoreceptors by the total retinal pigment epithelium length, per retinal cross-section. Two independent masked observers analyzed four to five separate cross-sections (40 μm apart) per eye. The measurements obtained by the two observers were compared to calculate the interclass correlation coefficient (ICC).Retinas were processed and stained as previously reported . Eyes weEnucleated eyes were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (7 μm), deparaffinized, and underwent antigen retrieval in sodium citrate buffer  at 80°C for 20 minutes NADPH oxidase 4 (NOX4). For Rac1, RhoA, and Cdc42, enucleated eyes were fresh frozen, sectioned (7 μm), and fixed for 15 minutes with 4% paraformaldehyde. Sections were blocked  for one hours and then incubated with primary antibody for 18 hours at 4°C. Primary antibodies used were rabbit anti-glial fibrillary acidic protein , rabbit anti-NOX4 , mouse anti-Rac1 , rabbit anti-RhoA , and rabbit anti-Cdc42 . After washes, the sections were incubated with donkey anti-rabbit FITC , IB4 , donkey anti-mouse Alexa Fluor 647 , or donkey anti-rabbit Rhodamine Red  for 1 hour. Rinsed sections were stained with 0.1% or 0.5% Sudan black diluted in 70% ethanol to quench autofluorescence and counterstained with 4’, 6-diamidino-2-phenylindole , rinsed, mounted, and sealed. Sections were imaged with Nikon A1R+ confocal laser microscope system.2DCFDA, Thermo Fisher Scientific), is converted to the highly fluorescent 2’, 7’-dichlorofluorescein (DCF) when acetate groups were cleaved by intracellular esterases and oxidation. DCF staining is a direct measure of ROS. Unfixed retinal cryosections (10 μm) were incubated with 10 μM H2DCFDA at 37°C in a dark, humidified chamber for one hour. Sections were imaged immediately with a Zeiss LSM-510 Meta confocal microscope .Reactive oxygen species (ROS) generation was examined by dichlorofluorescein staining. The non-The sections were then stained for an adhesion receptor restricted to the Mϋller glial cell end-feet, CD44 , 23. ImmWe determined fluorescence intensity using three different cross-sections per eye (N = 3 eyes per group) using ImageJ (NIH) , 25. TheWestern blotting was performed as previously described . Immediapost hoc comparisons. Results were considered statistically significant for p-value < 0.05.All data were analyzed using SPSS . Results are expressed as mean ± SEM. Mice from different litters (4–5 per group) were analyzed. Group differences were evaluated using ANOVA with Bonferroni’s rd8 eyes (B6N) at P28  and C57BL/6N (B6N) strains, histologic cross-sections were analyzed. In room air controls of both strains, we did not see any evidence of rosette formation at any time point (N = 3–4 per time point). The B6J in HIPR did not display the rosette formation and had continuous intact photoreceptors throughout the retina , while t) at P28 .The total retinal thickness was measured near the optic nerve and in the peripheral retina. In B6N mice, retinas were significantly thinner in HIPR than room air controls at P15, P21, and P28 near the optic nerve  and onlyComparing the two strains near the optic nerve, B6N HIPR had significantly thinner retinas than B6J HIPR at P14  and P28  staining, which fluoresces upon contact with reactive oxygen species (ROS). In a diabetic mouse model study, DCF staining was increased in the photoreceptor inner and outer segments compared to controls . SimilarQuantification of averaged DCF corrected total cell fluorescence intensity (CTCF) showed different time-course for ROS generation in the two strains. In HIPR, B6J only showed higher DCF staining at P21 compared to controls  . In contNOX are a major source of ROS in the retina , 30. TheNOX2 and, more prominently, NOX4 are present in photoreceptors . We haveSince the photoreceptors were a major source of ROS in HIPR , we deciDrosophila in the setting of crb knock-down [rd8 mutation. At all time points, Rac1 was present in the photoreceptors  and Leber congenital amaurosis (LCA) [CRB1 mutations are detected in 6% of patients with RP [CRB1 mutations manifest either severe progressive pericentral, visual field loss in adult onset RP or profound loss of vision from birth in LCA [rd8 are not significantly different from wildtype mice, even at the most advanced stages of retinopathy (> 9 months) [CRB1 phenotypes are significantly more severe than in mice. In contrast to rd8 (Crb1 mutants), mice deficient in Crb2 showed more severe, early disorganization throughout the entire retina [CRB2 mutations have rarely been reported with a retinal phenotype [CRB2 mutation has been reported, presenting with reduced visual acuity, nystagmus, and irregular retinal pigmentation [CRB1 mutations alone can cause severe photoreceptor degeneration, while defects in multiple components of the crumbs complex may be required to generate the severe phenotype in mice. In addition, we show that enhanced photoreceptor oxidative stress, in HIPR, leads to significant acceleration of the rd8 phenotype, which may present an opportunity to explore the severe phenotype of LCA seen in human CRB1 mutations.In humans, mutations in the is (LCA) –59. CRB1 with RP , 61 and  with RP , 58, 62.h in LCA . In cont months) . It is ne retina . Crb1 ane retina . Howeverhenotype . Recentlentation . This dard8  mutations [rd8 phenotype suggests that higher vigilance may be indicated to avoid photoreceptor oxidative stress in infants and children carrying these mutations [rd8 and, ultimately human CRB1-related retinal degenerations.In summary, our results show that the HIPR model in the B6N mouse strain exposes the photoreceptors to significant oxidative damage, with acceleration of rosette formation and significant degeneration of the photoreceptors by P28, unlike the otherwise mild natural history of this mutation. We found significant differences in ROS, NOX4, and Rac1 between the B6J and B6N substrains in HIPR. The two strains did not show significant differences in angiogenesis or Mϋller glial cell activation. These findings highlight the importance of understanding strain differences, and alerts researchers to rule out the utations  as potenutations . Based oS1 FigRetinal cross sections were hematoxylin and eosin stained. Rosettes involving photoreceptors were seen in C57BL/6N HIPR mice at P21 and P28 (blue arrowheads). Scale bar, 500 μm (N = 3–5).(TIF)Click here for additional data file.S2 FigWestern blots of NOX1 (A) and NOX2 (B) retinal lysates in HIPR mice were quantified with densitometry and normalized with a β-actin loading control. The values shown were fold change over respective room air controls. (A) There were no statistical differences for NOX1 protein expression. (B) NOX2 was significantly increased at P15 in C57BL/6N HIPR mice while in C57BL/6J HIPR NOX2 was significantly increased at P21. Values were mean ± SEM (N ≥ 4). ‡ p<0.05 P15 HIPR compared to P14 HIPR of the same strain. § p<0.05 P21 HIPR compared to P15 HIPR of the same strain. * p<0.05 HIPR C57BL/6J compared to HIPR C57BL/6N.(TIF)Click here for additional data file.S3 FigRetinal central cross sections were stained with NOX4 (green) and nuclei were stained with DAPI (blue). Minimal staining of NOX4 was present in room air control (RA) retinas in both strains, localizing to the retinal ganglion cell layer and blood vessels. In HIPR, both strains showed NOX4 staining in the retinal ganglion cell, inner plexiform, and inner nuclear layers. Representative images from the central retina are shown (N = 3). Scale bar, 50 μm. INL, inner nuclear layer. GC, ganglion cell layer. IPL, inner plexiform layer.(TIF)Click here for additional data file.S4 FigRetinal central cross sections stained with Rac1 (red) and counterstained with DAPI (blue) showed minimal staining in room air controls (RA). Both strains had Rac1 staining in the inner nuclear layer, inner plexiform layer, and retinal ganglion cell layer. C57BL/6N had increased Rac1 staining compared to C57BL/6J. Representative images from the central retina are shown (N = 3). Scale bar, 50 μm. INL, inner nuclear layer. GC, ganglion cell layer. IPL, inner plexiform layer.(TIF)Click here for additional data file.S5 FigRetinal central cross sections stained with RhoA  or Cdc42  and counterstained with DAPI (blue). Both strains showed minimal RhoA and Cdc42 staining in the photoreceptors and inner nuclear layer in room air controls (RA) and HIPR. There were no distinct differences detected at any time point at the level of the photoreceptors. At P21 in HIPR in both strains, RhoA and Cdc42 were increased in the Mϋller cells, compared to respective room controls. Representative images from the central retina are shown (N = 3). Scale bar, 50 μm. ONL, outer nuclear layer. RPE, retinal pigment epithelium.(TIF)Click here for additional data file.


Surveillance of wild aquatic birds and free-range domestic ducks in the Tanguar Haor wetlands in Bangladesh has identified influenza virus subtypes H3N6, H7N1, H7N5, H7N9, and H15N9. Molecular characterization of these viruses indicates their contribution to the genesis of new genotypes of H5N1 influenza viruses from clade 2.3.2.1a that are dominant in poultry markets in Bangladesh as well as to the genesis of the highly pathogenic H5N8 virus currently causing disease outbreaks in domestic poultry in Europe and the Middle East. Therefore, we studied the antigenicity, replication, and pathogenicity of influenza viruses isolated from Tanguar Haor in the DBA/2J mouse model. All viruses replicated in the lung without prior mammalian adaptation, and H7N1 and H7N9 viruses caused 100% and 60% mortality, respectively. H7N5 viruses replicated only in the lungs, whereas H7N1 and H7N9 viruses also replicated in the heart, liver, and brain. Replication and transmission studies in mallard ducks showed that H7N1 and H7N9 viruses replicated in ducks without clinical signs of disease and shed at high titers from the cloaca of infected and contact ducks, which could facilitate virus transmission and spread. Our results indicate that H7 avian influenza viruses from free-range ducks can replicate in mammals, cause severe disease, and be efficiently transmitted to contact ducks. Our study highlights the role of free-range ducks in the spread of influenza viruses to other species in live poultry markets and the potential for these viruses to infect and cause disease in mammals. They play a major role in the global distribution of IAVs and the emergence of novel IAVs, which can affect human and animal health worldwide3. IAVs are classified on the basis of surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Currently, IAVs are classified into 18 HA subtypes (H1–H18) and 11 NA subtypes (N1–N11). Only subtypes H1, H2, and H3 have been associated with human pandemics. However, subtypes H5, H6, H7, H9, and H10 can cross the species barrier from birds to mammals, including humans, and cause sporadic infections, but have not yet acquired the ability to transmit between humans7. The reasons for this remain unclear.Wild aquatic birds are the primary natural reservoir for influenza A viruses (IAVs)8. This was evident in the emergence of an LPAI H7N9 IAV in China in 2013 that spread to humans in live poultry markets. Epidemiological data indicated that this virus originated in domestic ducks and spread to chickens11. The recent cases of human infections with H10N8 IAVs in China highlight that domestic ducks at sentinel duck farms act as intermediate hosts12. Free-range ducks have also been associated with H5N1 outbreaks13. Thus, free-range ducks are considered to be the first line of connection between wild and domestic birds and may be key to the emergence and spread of zoonotic AIVs.Domestic ducks can perpetuate most avian influenza viruses (AIVs), asymptomatically shed and spread influenza viruses, and play an important role in epidemiology and reassortment of low-pathogenic avian influenza (LPAI) viruses14. In Bangladesh, H5-HA genes of the first H5N1 viruses identified in 2007 belonged to clade 2.2.2. Subsequently, 2 subclades of H5N1 viruses, 2.3.2.1a and 2.3.4.2, were detected. Subclade 2.3.2.1a viruses have been dominant in Bangladeshi poultry since 201116. As of 30 September 2017, 8 laboratory-confirmed human cases of H5N1 in Bangladesh have been reported to the World Health Organization (WHO), of which 1 case was fatal. Circulation of HPAI H5N1 viruses may increase the chances of emergence of pandemic strains through reassortment. H5N1 reassortant viruses containing the polymerase basic 1 (PB1) gene from H9N2 viruses have been reported18. Recently, we described the emergence of a novel genotype of H5N1 virus that contained the HA, matrix (M), and NA genes of circulating Bangladeshi H5N1 viruses and five genes from Eurasian-lineage LPAI viruses19.Active surveillance of poultry has been conducted in Bangladesh since November 2008, after the first highly pathogenic avian influenza (HPAI) H5N1 outbreak in 2007. Several influenza virus subtypes, including H5 and H9, have been isolated in live poultry markets20. Therefore, we sequenced the full genomes of H3, H7, and H15 AIVs and analyzed their genetic and antigenic characteristics to better understand the evolution of these influenza viruses that have played important roles in the emergence of the new H5N1 genotype currently circulating in Bangladesh and in the genesis of H5N8 circulating in Europe and the Middle East. We also evaluated the replication and pathogenicity of these isolates in mice and mallard ducks.Bangladesh is located in the central Asian flyway and is near the Eastern Asian–Australian and Black Sea–Mediterranean flyways. Tanguar haor, a vast wetland in northeastern Bangladesh, also serves as the major wintering ground for birds migrating in both the Central Asian and Eastern Asian–Australian flyways. As this location facilitates contact between wild birds and free-range ducks, surveillance for influenza viruses was conducted in wild birds and free-range ducks in Tanguar haor in Bangladesh in February 2015 and February 2016. Various LPAIV subtypes, including H3, H7, and H15, have been isolated in the Tanguar haor area. Our previous study showed genetic similarities between internal gene segments of these viruses with some genes of HPAI H5N8 clade 2.3.4.4 origin, suggesting their potential role in the genesis of the HPAI A(H5N8) virus that emerged in 2016Thirteen AIVs were isolated from wild birds and free-range flocks of domestic ducks in the Tanguar haor region in February 2015 and February 2016. Four influenza A (H3N6), four influenza A (H7N1), one influenza A (H7N5), three influenza A (H7N9), and two influenza A (H15N9) viruses were isolated from free-range flocks of domestic ducks. Also, an influenza A (H7N5) virus was isolated from a migratory black-tailed godwit. The intravenous pathogenicity index (IVPI) of the seven viruses  ranged from 0.0 to 0.05 in chickens  against Eurasian and North American viruses. Antigenic analysis showed that H3 viruses were homogenous and similar to the Eurasian lineage. Three H3 viruses reacted with the antiserum against A/duck/Shantou/1283/2001 (H3N8) at higher titers than for other antisera against viruses from the Eurasian lineage (Table 6 egg infectious dose (EID50) of each virus in 30 μL of phosphate-buffered saline (PBS) (n = 5 mice per virus isolate), and morbidity and mortality were monitored daily for 14 day post inoculation (dpi)   was detected at 8 dpi in mice infected with A/black-tailed godwit/Bangladesh/24734/2015 (H7N5) (PI-0). There was no replication detected in the intestines of mice infected with any of the viruses. A/duck/Bangladesh/24692/2015 H7N1 (PI-4), and A/duck/Bangladesh/26980/2015 (H7N9) (PI-2) replicated in the heart, liver, and brain. Viral titers were detected in the heart, liver, and brain of mice infected with A/duck/Bangladesh/24692/2015 (H7N1) (PI-4) at all time points (Table 0.75/mL–104.5/mL EID50); in the liver at 2 and 6 dpi (101.75/mL–103.5/mL EID50); and in the brain at 2, 4, 6, and 8 dpi (100.75/mL–102.75/mL EID50) of mice infected with A/duck/Bangladesh/26980/2015 (H7N9) (PI-2)  (PI-4), A/duck/Bangladesh/26980/2015 (H7N9) (PI-2), and A/black-tailed godwit/Bangladesh/24734/2015 (H7N5) (PI-0)—and viral replication in lungs, liver, heart, brain, and intestines was determined at 2, 4, 6, and 8 dpi, respectively. All three viruses replicated in the lungs. A/duck/Bangladesh/24692/2015 (H7N1) (PI-4) and A/duck/Bangladesh/26980/2015 (H7N9) (PI-2) had higher shedding than did A/black-tailed godwit/Bangladesh/24734/2015 (H7N5) (PI-0), with average viral titers in the lungs for each group ranging from 10The extent and severity of histopathologic lesions in the lungs of mice infected with H7N1, H7N9, and H7N5 virus isolates correlated with the viruses’ differing levels of pathogenicity. The more rapid and extensive spread of infection in H7N1-infected lungs was evident at 2 dpi. At this early time point, there was no notable pulmonary inflammation Fig.  but viruAt all time points, the severity and extent of virus infection was less in H7N9-infected lungs than in H7N1-infected lungs. Although H7N9 caused extensive infection of the tracheal epithelium by 2 dpi, there was no notable inflammation in the lung parenchyma Fig.  and only6 EID50/mL. None of the ducks infected with H7N1 or H7N9 viruses by inoculation or after contact with infected birds died or exhibited clinical symptoms  (PI 4) and A/duck/Bangladesh/26980/2015 (H7N9) (PI 2) at a dose of 10s Table S than did0.75/mL–107.75/mL EID50) and from oropharyngeal swabs at 3 and 5 dpi (100.75/mL–103.5/mL EID50). The H7N9 virus was detected in cloacal swabs at 3, 5, and 7 dpi (101.5/mL–107.25/mL EID50) and from oropharyngeal swabs at 3 and 5 dpi (100.75/mL–102.5/mL EID50)  and oropharyngeal swabs at 3 and 5 dpi (101.5/mL–103/mL EID50). Also, contact ducks infected with the H7N9 virus had extended shedding from cloacal swabs until 7 dpi (101.25/mL–105.75/mL EID50), whereas virus was detected only at 3 dpi in oropharyngeal swabs (100.75/mL–103.37/mL EID50).Replication and transmissibility of these viruses in ducks were evaluated. Cloacal shedding was higher than oropharyngeal shedding in all ducks infected with H7N1 or H7N9 viruses by inoculation or after contact with infected birds. The H7N1 virus was detected in cloacal swabs from inoculated ducks at 3, 5, and 7 dpi  at the cleavage site between HA1 and HA2, which have been associated with low-pathogenic effects in poultry. All H7 viruses isolated in our study belonged to the Eurasian-lineage H7 viruses, which have two motifs with one arginine residue at the HA cleavage site PETPKGR↓GLF and PELPKGR↓GLF .The HA protein was examined for the presence of N-linked glycosylation motifs, using the N-(P-[S/T]-P) motif to indicate a potential N-linked glycosylation site. The H3N2 viruses had six potential glycosylation sites at positions 22, 38, 165, 285, and 483, whereas H7N1 and H7N5 viruses had five potential glycosylation sites at positions 22, 38, 240, 411, and 483. There were seven potential glycosylation sites for H15N9 viruses at positions 22, 38, 92, 165, 411, 483, and 487. There was a specific insertion in the HA1 subunit at positions 253–262 (SNALSGVEYN) in two isolate H15N9 viruses that characterized the 22, and in our study all isolates had an arginine residue at this location. The PB2 amino acid substitutions E249G, G309D, and T339M increase replication in human lung epithelial cells23, all isolates in this study had substitution 309D, except A/duck/Bangladesh/24704/2015 (H15N9) virus. All our isolates expressed PB1-F2 of 90 amino acids. Further, two H7N1 isolates, three H3N6 isolates, and three H7N9 isolates had an N66S mutation in PB1-F2  have been identified in wild birds, and these birds have recently been implicated in the spread of HPAI H5N1 and H5N8 viruses. Waterfowl are an important host as they are usually asymptomatic and free-range ducks in wetland areas have considerable first-line contact with wild migratory birds, which aids the dissemination of influenza viruses to other birds. In this study, we characterized the genetic and antigenic properties of H3, H7, and H15 viruses isolated from free-range ducks in the Tanguar haor wetland area of Bangladesh; determined the pathogenicity of these viruses in a mouse model; and evaluated the pathogenicity and transmissibility of H7 viruses in a duck model.29. We previously used the DBA/2J mouse model to study the pathogenicity of North American H1N1 and H7 AIVs from migratory waterfowl28. All AIVs used in the current study infect and replicate in mice without prior adaptation. However, H7N1 and H7N9 viruses isolated from free-range ducks caused higher mortality and increased pathogenicity than did H7N5 virus isolated from wild birds and other LPAI subtypes isolated from free-range ducks. In our previous studies, H7 AIVs of duck origin were more pathogenic than those of shorebird origin from the North American lineage21. Our current study confirmed this observation for viruses isolated from Eurasian lineages. Further studies focusing on replication kinetics in various avian and mammalian models are required to understand the mechanisms underlying the differences in replication characteristics.We used DBA/2J mice to evaluate the disease potential of LPAI viruses from free-range ducks and wild birds in mammalian models. Several studies show that DBA/2J mice are more sensitive to influenza A infection than are other strains32. The mallard is the duck model routinely used in our laboratory and is a good model for wild ducks34. We found that 2 avian H7 viruses infected donor ducks and ducks in direct contact with those donors, with no overt clinical signs of disease. Both the donor and direct contact ducks shed virus at higher titers from the cloaca than the trachea, which was expected given that most AIVs preferentially replicate in the gastrointestinal tract of wild ducks and are transmitted primarily via the fecal-oral route32.Pathogenicity and transmissibility of HPAI and LPAI viruses via direct contact in different species of ducks have been previously reported10. Recent studies also show that precursors of H7N3 and H7N8 HPAI viruses in the Americas most likely originated from dabbling ducks and diving ducks, respectively36. It is considered that ducks can perpetuate most AIVs since they generally do not show clinical signs after infection. In this study, we provide evidence that free-range ducks can act as key intermediate hosts by acquiring and maintaining diverse influenza viruses from migratory birds. The H7 viruses evaluated in this study could not only replicate and cause disease in mammals but also be efficiently transmitted to mallard ducks in direct contact groups.H7 AIVs cause sporadic outbreaks in poultry populations worldwide. Epidemiological data on the H7N9 virus circulating in China indicate that it probably transferred from domestic ducks to chickensHA genes of viruses isolated from the Tanguar haor wetland area of Bangladesh showed that they were of Eurasian lineage19. Molecular analysis of viruses revealed no change in four key highly conserved residues E190D, G225D, Q226L, and G228S (H3 numbering) in the receptor-binding site of the HA protein that have been associated with conferring avian-type specificity. Furthermore, these HAs did not contain a cleavage site associated with high pathogenicity, indicating that these viruses are classified as low pathogenic to chickens.Phylogenetic analysis of 37 and more recently in Siberia and Ukraine39. The H15 viruses are members of the subgroup that also includes H7N9 and H10N8 subtypes, which infected humans in 2013. We detected two H15 isolates from free-range ducks in our surveillance in Tanguar haor. Both viruses were genetically different from Australian viruses, but similar to those isolated from Ukraine and Siberia. We also found a specific insertion in the HA1 subunit at positions 253–262, which is characteristic of the HA gene of the H15 subtype37.H15 influenza virus is rarely detected in wild birds, and was previously isolated from Australia from 1979 to 198316. Routine surveillance showed that the H5N1 clade 2.3.2.1a viruses circulating in live poultry markets and poultry farms in Bangladesh have become the most dominant since 2011. The new genotype of HPAI H5N1 viruses contained three segments  from clade 2.3.2.1a, and other segments were more closely related to those from LPAI viruses we isolated from Tanguar haor19, indicating that viruses isolated from Tanguar haor play an important role in the diversity of HPAI H5N1.Our recent study showed the emergence of a new genotype of HPAI H5N1 viruses circulating in live poultry markets in Bangladesh. Multiple clades from HPAI H5N1 were detected in Bangladesh after the first H5N1 HPAI outbreak in 2007Future studies need to determine the effect of new segments that produce new genotypes on the pathogenicity and transmissibility of H5N1. The dynamics and transmission of AIVs through migratory wild birds are complex. Migratory birds infected with avian IAVs are (1) asymptomatic or display subclinical symptoms, (2) maintain the diversity of influenza viruses through various subtype combinations, and (3) allow the virus to spread over long distances. Therefore, monitoring AIVs in migratory wild birds is important for understanding the evolution and emergence of viruses with new biological properties, and surveillance of free-range ducks can be used as an early warning system for AIV outbreaks in poultry and to better understand the potential risk these viruses pose to humans. Our studies highlight the important role of free-range ducks in the spread of influenza viruses from migratory birds to live poultry markets, which is a potential risk to human and veterinary public health.Experiments in mice were conducted in an Animal Biosafety Level 2+ facility at St. Jude Children’s Research Hospital (St. Jude), in compliance with the policies of the National Institutes of Health and Animal Welfare Act and with the approval of the St. Jude Institutional Animal Care and Use Committee. Ducks experiments were approved and performed according to guidelines set by the St. Jude Animal Care and Use Committee in an enhanced Animal Biosafety Level 3+ containment facility.50 according to the method of Reed and Muench40.Thirteen AIVs were isolated from wild birds and free-range flocks of domestic ducks in the Tanguar haor region through collaborative efforts between the Center of Excellence for Influenza Research and Surveillance at St. Jude  and Jahangirnagar University  in February 2015 and February 2016. Four influenza A (H3N6), 4 influenza A (H7N1), one influenza A (H7N5), three influenza A (H7N9), and two influenza A (H15N9) viruses were isolated from free-range flocks of domestic ducks. Also, an influenza A (H7N5) virus was isolated from a migratory black-tailed godwit. All viruses were propagated and titrated for infectious dose in the allantoic cavities of 10-day-old embryonated chicken eggs at 35 °C for 48 h. Viral titers were determined by injecting 100 μL of serial 10-fold dilutions of the virus into allantoic cavities of 10-day-old embryonated chicken eggs and calculating the 50% EID6 EID50/mL of viruses and then boosted after 3 weeks by an intramuscular injection of virus with adjuvant. Blood was collected 1 week later for serum isolation. The HI test was performed according to WHO protocols41.The hemagglutination inhibition (HI) assay was used to antigenically characterize the viruses. The H3N6, H7N1, H7N5, and H7N9 viruses were tested with polyclonal ferret and goat antiserum raised against representative H3, H7, and H15 viruses. Reference antisera against H3 and H7 viruses were selected from North American and Eurasian lineages. The four antisera against A/duck/Bangladesh/26920/2015 (H3N6), A/duck/Bangladesh/26980/2015 (H7N9), A/duck/Bangladesh/24692/2015 (H7N1), and A/duck/Bangladesh/24697/2015 (H15N9) were generated in the current study. Briefly, ferrets were first infected intranasally with 1 mL of 106 EID50/mL of virus. The uninfected (control) group was anesthetized and intranasally inoculated with 30 µL PBS pH 7.2. Upon virus challenge, five mice were monitored for 14 dpi for disease symptoms, weight loss, and mortality. Mortality was recorded on the basis of actual death or a 25% weight loss cutoff, according to our animal protocol. Three mice from each group were killed by CO2 administration and cervical dislocation after 3 days, and lung tissue was collected to determine viral titers. The total pathogenicity score was calculated as the sum of survival and weight loss scores /maximum AUC); weight loss score 0.2 (weight loss AUC/maximum AUC)), as described previously28. The mean weight loss of groups of five mice, each group inoculated with a different virus, was used for these calculations. As mortality was more important than morbidity in our screening, we used 80% weight for survival and 20% weight for weight loss after normalizing AUCs to the maximum AUC that could be obtained with no mortality or morbidity (uninfected group). The AUCs for survival and weight loss were calculated using GraphPad Prism. According to total pathogenicity scores, viruses were divided into five categories and assigned a PI value of 0 through 4, with 0 being nonpathogenic and 4 being the most pathogenic.Seven isolates were selected to investigate pathogenicity in DBA/2J mice. Groups of 8 mice were anesthetized with isoflurane and inoculated intranasally with 30 µL of 106 EID50/mL of A/duck/Bangladesh/24692/2015 (H7N1) (PI-4), A/duck/Bangladesh/26980/2015 (H7N9) (PI-2), and A/black-tailed godwit/Bangladesh/24734/2015 (H7N5) (PI-0). Three mice per group were killed with CO2 at 2, 4, 6, and 8 dpi. Lung, intestine, liver, brain, and heart were collected to determine viral titers. All organs were homogenized in sterile PBS using the TissueLyser II . Organ homogenates were centrifuged at 2000×g for 10 min, and supernatants were transferred to clean tubes. Viral titers were determined by the EID50 assay.Groups of 12 mice were anesthetized with isoflurane and inoculated intranasally with 30 µL of 10Lungs were collected at 2, 4, 6, 8 dpi, and fixed by intratracheal infusion and immersion, using a 10% neutral buffered formalin solution. Tissues were embedded in formalin, sectioned, and stained with hematoxylin and eosin, and immunohistochemistry (IHC) staining of serial histologic sections was performed to determine the distribution of influenza virus antigen. For IHC analysis, a goat primary polyclonal antibody  against anti-influenza A, USSR (H1N1) was used 1:1000 on tissue sections previously subjected to antigen retrieval for 30 min at 98 °C . To quantify the extent of viral infection in the lungs, digital images of whole lung sections stained for viral antigen were first captured using the Aperio ScanScope XT Slide Scanner , and then both uninfected and virus-positive regions were manually outlined and areas were determined using ImageScope software (Leica Biosystems).WHO Manual on Animal Influenza Diagnosis and Surveillance41. The IVPI index was the mean score per chicken per observation over a 10-day period to determine the virulence of influenza virus isolates in chickens.The IVPI was determined in groups of SPF white leghorn chickens as described in the 6 EID50/mL dilution of virus stock of A/duck/Bangladesh/24692/2015 (H7N1) and A/duck/Bangladesh/26980/2015 (H7N9) in PBS via the natural route. Two ducks inoculated with 0.5 mL sterile PBS were used as negative controls. To examine virus transmission, three naive ducks were added to each group at 3 dpi and they shared food and drinking water for 24 h (contact ducks) before being moved to a separate cage. Body weights were determined at days 3, 5, 7, 10, and 14 for donor and contact ducks. Ducks were observed for clinical signs over 14 days. Oropharyngeal and cloacal swabs were collected from all birds at days 3, 5, 7, 10, and 14 to determine virus shedding. Viral titers from oropharyngeal and cloacal swabs were determined by the EID50 assay. Blood samples were taken from donor and contact ducks on day 14 for serology analysis.Three donor ducks were infected with 0.5 mL of 10All statistical analyses were performed by one-way analysis of variance in combination with Bonferroni’s multiple comparisons test by using GraphPad Prism 5.0 (GraphPad Software).Supplemental figure s1Supplementary figure legendsSupplementary tables


Interestingly, while the entire pef operon was necessary to exert toxic activity via the T6SS in P. mirabilis, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two P. mirabilis strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from  Proteus mirabilis requires the lethal action of the T6SS, T6SS-dependent effectors, and immunity proteins. Bacteria that share common immunity proteins are protected against T6SS killing and recognize each other as self. Here we introduce a new group of T6SS immunity effectors that require eight genes for killing and a single immunity gene that differs from the classical effector-immunity pairs. Variation within an effector operon drives specificity and recognition between strains. We propose that subtle sequence variation in the effector operons contribute to self and non-self recognition in these bacteria.Bacterial type VI secretion systems (T6SS) function as contractile nanomachines to puncture target cells and deliver lethal effectors. Little is known about the lifestyle or physiology dictating when bacteria normally express their T6SS. Previously, we have reported that discrimination of self during the multicellular swarming behavior of  Proteus mirabilis is indeed linked to T6S and thus enables discrimination during cell-cell contact to eliminate non-self bacteria from the population /[(CFU of test strain/CFU of HI4320)input].Bacterial killing assays were performed by mixing strains in a 1:1 ratio and spotted onto swarm agar (10 g NaCl/L). Following overnight incubation at 37°C, the entire swarm was collected, serially diluted, and plated onto LB medium containing NaCl (0.5 g/L) and 1.5% agar with rifampicin for HI4320  followed by the nucleotide number where it is joined to the reciprocal gene fragment (either E for pefE or E2 for pefE2) at the 3’ end. Chimeras were transformed into electrocompetent cells of P. mirabilis HI4320 immunity mutant 9C1 and wild-type P. mirabilis BB2000 for further analysis in immunity complementation experiments.Partial gene fragments of genomic DNA sequences, isted in . DigesteE. coli (Life Technologies). Transformants were screened by colony PCR with primers amplifying across the pBAD-MycHisA multiple cloning site containing PefE. Clones with a correct product size of 1 Kb were verified by sequence analysis to confirm each point mutation . Correct site-directed PefE plasmids were transformed into P. mirabilis HI4320 immunity mutant 9C1 and P. mirabilis BB2000 for immunity complementation experiments.Desired point mutations within PefE were constructed using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher). Briefly, target plasmid pBAD-PefE-MycHisA was PCR amplified with two outward facing 5'-phosphorylated primers. Point mutations were introduced by one or more mismatches in the forward binding mutagenic primer. PefE point mutations at aa 137, 138, and 140 were created and substituted with the amino acid residue found in PefE2; PefE_aa137 serine (S) to threonine (T), PefE_aa138 alanine (A) to serine (S), PefE_aa140 lysine (K) to glutamic acid (E). In addition to these three single point mutations, double site-directed mutants of all configurations as well as a triple site-directed mutant was created; PefE_S137T A138S, PefE_S137T K140E, PefE_A138S K140E, and PefE_S137T A138S K140E. Additional single and double site-directed mutants of PefE at residues aa137S and aa140K were constructed to introduce an alanine (A); PefE_S137A, PefE_K140A, and PefE_S137A K140A. Oligonucleotides and respective sequences used in these experiments are listed in . FollowiP. mirabilis HI4320 is the toxic effector of the pef operon, the entire pefD sequence and the tox-only domain of pefD was PCR amplified and ligated into the pETite N-His vector which has a 6xHis affinity tag fused to the amino terminus (Lucigen Expresso-T7 Cloning and Expression system). The resulting constructs were transformed into HI control 10G cells by heat shock and transformants were selected on kanamycin agar plates. HI control 10G cells harbor a single copy BAC plasmid carrying an engineered lacIq1 repressor allele responsible for tightly regulating and minimizing background transcription of the pETite plasmid which is optimal for construction with toxic proteins. Transformants were screened by colony PCR for the correct product size of 1.5 Kb (pefD) or 300 bp (Tox-only domain) and verified by sequence analysis . For protein expression the correct pETite N-His-PefD and pETite N-His-Tox-PefD construct was transformed into HI control BL21 (DE3) cells (Lucigen) and induced in LB medium with 1 mM IPTG. Expression of pETite N-His-PefD and pETite N-His-Tox-PefD was verified by Western blot using THE His Tag HRP antibody (GenScript). Toxicity experiments of pETite N-His-PefD and pETite N-His-Tox-PefD were performed on swarm agar containing 0.1 mM IPTG.To determine whether PefD in The wild-type bacterial isolates used in this study were collected in Baltimore, Maryland in a prior study . All isoS1 Table(XLSX)Click here for additional data file.S1 FigE. coli BL21 in LB medium with kanamycin (closed symbols) and with kanamycin and 1 mM IPTG (open symbols) to induce protein production. Optical density at 600 nm was monitored for 20 hours. All strains induced with IPTG exhibited a growth delay and those expressing PefD or Tox exhibited greatest growth defect and reached a lower final density.Positive control protein (PC), PefD, or the nuclease domain (Tox) were expressed in (TIF)Click here for additional data file.S2 FigE. coli BL21 in LB medium with kanamycin and ampicillin (open symbols) and with kanamycin, ampicillin and 1 mM IPTG to induce PefD or Tox (closed symbols) and with kanamycin, ampicillin, 1 mM IPTG to induce PefD or Tox, and 10 mM L-arabinose to induce PefE (closed symbols). Optical density at 600 nm was monitored for 20 hours. All strains induced with IPTG exhibited a growth delay and those induced with L-arabinose to express PefE did not restore growth.PefE and PefD or PefE and the nuclease domain (Tox) were expressed in (TIF)Click here for additional data file.S3 FigpefE cloned from HI4320 and expressed in BB2000 restored immunity against BB2000 containing the same immunity gene, pefE (black arrow). BB2000 containing pBAD empty vector alone or containing pefE2 cloned from HI4320 were unable to restore immunity against parent strain BB2000 expressing pefE cloned from strain HI4320. Expression of chimera 1, 5, or 6  in BB2000 did not restore immunity against BB2000 containing pefE cloned from HI4320; however, chimera 2, 3, and 4  expressed in BB2000 restored immunity against BB2000 containing pefE cloned from strain HI4320 (white arrows).Following induction on 10 mM L-arabinose (+), (TIF)Click here for additional data file.S4 Figpef operon from BA6163 (PefBA) and BB2000 containing pBAD empty vector and BB2000 expressing pefE. BB2000 expressing PefBA is immune to BB2000 expressing pefE2 cloned from HI4320 (black arrows). Expression of chimera 2, 3, or 4  in BB2000 did not restore immunity against BB2000 expressing PefBA; however, chimera 1, 5, and 6  expressed in BB2000 restored immunity against BB2000 expressing PefBA (white arrows).Following induction on 10 mM L-arabinose, Dienes line formation was observed between BB2000 expressing the (TIF)Click here for additional data file.S5 FigKilling assays of 9C1 against the parental HI4320 are reported as [(CFU of 9C1 strain/CFU of HI4320)output] / [(CFU of 9C1 strain/CFU of HI4320)input]. Strain HI4320 kills mutant 9C1 by 7-logs (pBAD). Mutant 9C1 complemented with PefE is outcompeted by 2-logs. 9C1 containing C2-C4, C7, and C9 all merge with HI4320 and do not form a Dienes line.(TIF)Click here for additional data file.S6 FigpefE mutant 9C1.Alignment of HI4320 PefE and HU1069 PefE predicted amino acid sequences. Amino acids boxed in red indicate the matching residues at VR3. PefE from HU1069 restores kin recognition when introduced into HI4320 (TIF)Click here for additional data file.S7 Fig(A)P. mirabilis HI4320 forms a Dienes line with immunity mutant 9C1 (pBAD) and immunity is restored by expression of PefE (white arrow). Alanine substitution at residues 137 and 140 (S137A K140A) disrupt PefE immunity function and a Dienes line forms (black arrow). Site-directed mutagenesis switching PefE residues to PefE2 residues (S137T A138S K140E) partially disrupts immunity function as PefE and in (B) provides immunity function as PefE2 in BB2000 against BB2000 expressing the pef operon from BA6163. (C) and (D) Chimeric PefE C7 (E475E2) functions as PefE and not as PefE2. Site-directed mutation of C7 containing K140E and any additional change from E to E2 residue is sufficient to change function of C7 from PefE to PefE2 immunity phenotype (white arrows).(TIF)Click here for additional data file.