WNT signaling pathway, which plays a key role in T2D metabolism. Our results highlighted the potential functional importance of T2D super enhancer SNPs, which may yield novel insights into the pathogenesis of T2D.Clinical studies in type 2 diabetes (T2D) primarily focused on the single nucleotide polymorphisms (SNPs) located in protein-coding regions. Recently, the SNPs located in noncoding regions have also been recognized to play an important role in disease susceptibility. The super enhancer is a cluster of transcriptional enhancers located in noncoding regions. It plays a critical role in cell-type specific gene expression. However, the exact mechanism of the super enhancer SNPs for T2D remains unclear. In this study, we integrated genome-wide association studies (GWASs) and T2D cell/tissue-specific histone modification ChIP-seq data to identify T2D-associated SNPs in super enhancer, followed by comprehensive bioinformatics analyses to further explore the functional importance of these SNPs. We identified several interesting T2D super enhancer SNPs. Interesting, most of them were clustered within the same or neighboring super enhancers. A number of SNPs are involved in chromatin interactive regulation and/or potentially influence the binding affinity of transcription factors. Gene Ontology (GO) analysis showed a significant enrichment in several well-known signaling pathways and regulatory process,  Type 2 diabetes (T2D) is a long-term metabolic disorder which is characterized by hyperglycemia and the aberrant metabolism of fat and protein due to the deficient action of insulin . Over the.g. DNA methylation valleys and locus control regions) which plays an important role in cell-type specific gene expression [Previous studies have shown that SNPs in protein-coding region are associated with hundreds of diseases by affecting the expression of their target genes . Howeverpression . A recenpression . For exapression . HoweverIn this study, we integrated T2D GWAS meta-analysis dataset with T2D cell/tissue-specific histone modification ChIP-seq data to identify T2D super enhancer SNPs, followed by comprehensive bioinformatics analyses to further elucidate the potential functional importance of these SNPs in the pathogenesis of T2D. Our results may yield novel insights into the genetic basis of T2D.http://diagram-consortium.org/downloads.html). To our knowledge, it is the standard and the largest T2D meta-analysis study which conducted in 34,840 cases and 114,981 controls of European descent. Next, we used proxy SNAP (https://www.broadinstitute.org/mpg/snap/ldsearch.php) to identify SNPs in LD with retrieved T2D SNPs. The search was based on genotype data from the 1000 Genomes Project with the Utah residents with European ancestry (CEU) population panel. The inclusion criteria for LD SNPs were set as a distance limit of 500 KB from the query SNP with r2 > 0.8 from pairwise LD calculations. A total of 1,086 potential functional T2D SNPs was identified.We retrieved 792 potential T2D SNPs at GWAS significance threshold  from Diabetes Genetics Replication and Meta-analysis (DIAGRAM) Consortium (https://www.ncbi.nlm.nih.gov/geo/). A detailed list of all T2D-relevant cell/tissue types and sample information are included in S1 Table.We selected T2D-relevant cell/tissues based on their crucial role in pathogenesis of T2D through comprehensive, systematic literature searches in PubMed. In total, we identified 27 T2D-relevant cell/tissue types, such as adipose tissue –13, aorthttp://bowtie-bio.sourceforge.net/index.shtml). MACS v1.4.1 was used to identify enhancer regions [local, to calculate the p-value of each candidate peak and removes potential false positives due to local biases. The significant threshold was defined as Bonferroni adjust p-value < 1×E-9. Super-enhancers were separated from enhancers using the ROSE algorithm [S2 Table) and mapped 1,086 potential functional T2D SNPs to these super enhancers. Finally, we achieved 286 T2D super enhancer SNPs (S3 Table).Histone modification WIG/BIGWIG files were mapped to the human reference genome hg19) using Bowtie v1.1.2  and regulatory motif alterations within sets of genetically linked T2D super enhancer SNPs. Furthermore, we performed transcription factor enrichment analysis using SNP2TFBS  tool which selects and visualizes user defined variants that affect single or multiple transcription factors.Characterization of the regulatory features of T2D super enhancer SNPs was carried out through the bioinformatics tool rVarBase . rVarBashttp://jjwanglab.org/gwas3d) systematically calculates the probability of GWAS-associated SNPs which affect regulatory pathways and underlie disease associations by integrating functional genomics, chromatin state, sequence motif, and conservation information [2 = 0.8, CEU population panel (HapMap phase I+II+III), binding site p-value = 0.01, size of 30 variants, and 3 interaction size.GWAS3D  of T2D super enhancer SNPs, we assigned each T2D super enhancer SNPs to their nearest genes based on the human genome assembly GRCH37 (hg19) from Genome Reference Consortium. We carried out GO analyses using these T2D super enhancer SNPs target genes through GOEAST toolkit (http://omicslab.genetics.ac.cn/GOEAST/). The p-values were calculated by hypergeometric tests and adjusted for multiple comparisons using stringent Yekutieli (FDR under dependency) method [To find out the functional enrichment  method . The stahttp://string-db.org/) is a database of known and predicted protein interaction, including physical and function association. It quantitatively integrates interaction data derived from genomic context, high-throughput experiments, co-expression and previous knowledge [S3 Table), we construct gene (protein) interaction networks via the STRING v10.0 database with default settings .STRING v10.0 (nowledge . The datS2 Table). Then 1,086 potential functional T2D SNPs were mapped to the identified T2D cell/tissue-specific super enhancer. Finally, we obtained a total of 286 potential functional T2D super enhancer SNPs (S3 Table).In this study, we first retrieved 792 potential T2D SNPs at GWAS significance threshold  from the largest T2D meta-analysis study DIAGRAM Consortium. By using LD information from the 1000 Genomes Project with CEU population panel, we inferred 1,086 potential functional T2D SNPs. Then we took advantage of the public available human histone modification ChIP-seq datasets from GEO database to construct a comprehensive list of super enhancer in T2D-relevant cell/tissue types. In total, we examined and confirmed 18,422 super enhancer regions in 27 human cell/tissue types (S4 Table). Notably, 160 SNPs were mapped to super enhancer in a wide variety of cell/tissue types (S4 Table). Interestingly, some of these SNPs enriched in the same or neighboring super enhancers, the strong enrichment of T2D super enhancer SNPs on the chromosome 2p21 (THADA), 3q21.1 (ADCY5), 4p16.1 (WFS1) and 10q25.2 (TCF7L2) are rather striking , e.g. the SNP pair rs11196182 and rs12245680 with low LD (r2 = 0.016) were mapped to the same super enhancer in adipose tissue (10q25.2). The full list of SNPs that map to a specific super enhancer and LD between those SNPs is included in S5 Table.To identify whether T2D super enhancer SNPs enriched in specific cell/tissue types, we characterized the distribution of the T2D super enhancer SNPs in different cell/tissue types and identified a significant SNP enrichment in adipose tissue (161/286), aorta (115/286), pancreas (113/286) and brain (44/286)  by using data from ENCODE and GTEx projects through HaploReg v4.1. Finally, we observed that 242 T2D super enhancer SNPs changed transcription factor binding motif, this result highlighted the strong regulatory potential of these T2D super enhancer SNPs (S7 Table). Moreover, we identified 186 T2D super enhancer SNPs which were reported eQTL evidences, including 120 T2D super enhancer SNPs showed eQTL evidences in a wide variety of tissues and cell types (S7 Table). Importantly, we found that all 57 regulatory SNPs identified by rVarBase were replicated in HaploReg regulatory prediction results (S7 Table).To investigate the potential functional impact of the identified T2D super enhancer SNPs, we annotated 286 T2D super enhancer SNPs to various regulatory elements through rVarBase . rVarBasFig 2). Transcription factors Tcf12 is an important component member of WNT signaling and ERK signaling pathway. Diabetogenic factors have been found to influence insulin signaling through activation of the ERK signaling pathway [The effect of T2D super enhancer SNPs on the transcription factor binding affinity was further analyzed by SNP2TFBS. The top five enrichment transcription factors are Tcf12, Sox6, JUNB, Myog, and Gata4 . For example, SNP rs13411629, located in the intronic region of THADA on chromosome 2, has a long-range interaction signal with locus 11p15.4. SNP rs2124500, located in the intronic region of ADCY5 on chromosome 3, has two long-range interaction signals with locus 7q36.1 and 18q12.2. SNP rs1552224, located in the intronic region of ARAP1 on chromosome 11q13.4, showed two long-range interaction signals with locus 10p23.2 and locus near POTEG respectively. Furthermore, GWAS3D also revealed 105 SNPs which may affect promoter activity by changing transcription factor binding site affinity (S8 Table), including 76 SNPs have direct effect by GWAS leading SNPs and 29 variants have indirect effect by high LD of GWAS leading SNPs. Interestingly, among the 105 functional SNPs identified by GWAS3D, 96 SNPs were also confirmed by HaploReg regulatory prediction results (S8 Table).GWAS3D was used to identify T2D super enhancer SNPs which have a long-range interaction signal with their distal regulatory elements. Finally, we detected 82 SNPs  affecting the long range interaction based on the CEU population and all cell types listed in GWAS3D (S3 Table) through GOEAST. We identified 151 significant GO terms with Yekutieli adjust p-value < 0.05 (S9 Table). Interestingly, we observed a significant enrichment in signaling pathways and regulation process (S9 Table), e.g. WNT signaling pathway and G-protein coupled receptor signaling pathway, which play a key role in the pathogenesis of T2D [To systematically investigate whether the identified T2D super enhancer SNPs were specifically associated with T2D, we conduct GO analysis using 26 T2D super enhancer SNPs target genes  into STRING v10.0 database with default settings . Fig 4 revealed a strong association between the topological properties and biological function of T2D target genes. The hub genes with the strong connections were KCNJ11, PPARG, ABCC8, THADA, WFS1, TCF7L2, and ADCY5. Interestingly, these genes are involved in the energy reserve metabolic process, glucose homeostasis, and negative regulation of pancreatic β cell apoptotic process, which revealed their potential functional impacts on the pathogenesis of T2D.To partially characterize the functional partnership and their interaction networks among the identified T2D super enhancer SNPs target genes, we uploaded the 26 T2D super enhancer SNPs target genes ,  process .e.g. we only focus on SNPs from the 1000 genomes project with MAF > 0.001. We admit that a number of known variants satisfying the MAF criterion were missed in the functional prediction analysis. In additional, the results of functional annotation exclusively depend on computationally predicted regulation features, therefore the model and algorithm selection is critical for this kind of analysis. For example, the reliability of the transcription factor binding site predicted by SNP2TFBS is a function of the accuracy of the PWM model which is less accurate than recently proposed approaches [In the current study, we performed comprehensive bioinformatics analyses to demonstrate the functional importance of the T2D super enhancer SNPs. However, the databases used in these bioinformatics analyses are specialized, proaches . Furtherproaches . TherefoIn conclusion, by integrating T2D GWAS and histone modification ChIP-seq data, we identified hundreds of T2D super enhancer SNPs. Comprehensive bioinformatics analyses of T2D super enhancer SNPs highlighted their potential functional importance in the regulation of T2D pathogenesis, which may yield novel insights into the genetic basis of T2D.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.


Alchornea floribunda leaves are widely used in ethnomedicine for the management of immuno-inflammatory disorders. We investigated the in vivo and in vitro antioxidant activity of the leaf extract, fractions and isolated compounds of A. floribunda. 4). Further analysis on the isolated phenolic compounds were done using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and hydrogen peroxide scavenging activity tests. The ethyl acetate fraction of the methanol leaf extract was subjected to several chromatographic separations to isolate compounds 1-4. The structures of the isolated compounds were elucidated by a combination of 1D and 2D NMR and mass spectrometry. Oxidative stress was induced with carbon tetrachloride . (-) epicathechin showed scavenging of DPPH radical with EC50 value of 19 μg/ml while in the FRAP assay, it had EC50 value of 46 μg/ml which was lower than that of the standard, ascobic acid (EC50 = 66 μg/ml). The ethyl acetate fraction at 200 mg/kg produced significant (p<0.05) elevations of catalase enzyme activity and a significant (p<0.05) reduction in serum malondialdehyde. The chemical investigation of the ethyl acetate fraction led to the isolation of three flavans, (-) cathechin (1), (-) epicathechin (2), (+) epicathechin (3) and a flavanone, 2R, 3R dihydroquercitin (4). In hydrogen peroxide scavenging assay, (-) epicathechin exhibited an ECA. floribunda may be due to the antioxidant activities of its phenolic compounds.The medicinal uses of  Oxidative stress has been implicated in the development and progression of many diseases such as inflammation, cancer, coronary heart disease, atherosclerosis and Alzheimer’s disease  is a branched monoecious shrub widely distributed in Africa. It has been used widely as a traditional treatment for a wide range of health conditions including hepatitis, wounds, ringworm, eczema, pains in the heart, and a variety of infections and inflammatory disorders, as an antidote to poison and for general body healing  and the extract was concentrated in vacuo with a rotary evaporator to obtain a dark green semisolid. The dried extract was reconstituted in 20 ml of methanol and the dispersion made up to 200 ml with water, sonicated for about 10 min and subsequently, partitioned successively against hexane (750 ml × 3), ethyl acetate (750 ml × 3) and n-butanol (500 ml × 2). All fractions were taken to dryness using rotary evaporator. About 5 g of ethyl acetate fraction was subjected to VLC  eluting with 500 ml each of hexane:ethyl acetate  and dichloromethane:MeOH  resulting in 12 pooled fractions EF1 to EF12. About 200 mg of the sub-fraction EF4 (eluted with dichloromethane:MeOH 3:7) was subjected to Sephadex LH-20 (3X60) separation using dichloromethane:MeOH 1:1 and finally, purified by semi-preparative reverse phase HPLC to obtain compounds 1 (10 mg), 2 (10.0 mg), 3 (11 mg), 4 (12.0 mg).About 500 g of pulverized dried leaves of Phytochemical testQualitative determination of secondary metabolites in the extract and fractions were carried out using the methods described by Odebiyi and Sofowora, (1978)Estimation of total phenolic content2CO3 solution was added to the mixture followed by the addition of 2 ml of deionised distilled water. The mixtures (in triplicate) were incubated at 40oC for 30 min and the absorbance was read at 760 nm. The total phenolic content was determined from extrapolation of calibration curve which was made using gallic acid solution and expressed as milligrams of gallic acid equivalents (GAE) per gram of the extract/fractions.The total phenolic content of the extract and fractions were determined using the method described by Kim et al. (2003)Structural elucidation of isolated compounds1H, 13C, DEPT, HMQC and HMBC) were recorded with Bruker ARX 500 NMR. MS (ESI) was obtained with Finnigan LCQ Deca mass spectrometer. Analytical HPLC was carried out with a Dionex P580 HPLC system coupled with a photodiode array detector (UVD340S). Routine detection was at 235, 254, 280 and 354 nm. The separation column  was prefilled with Eurospher-10 C18 , and a linear gradient of nanopure water (adjusted to pH 2 by addition of formic acid) and methanol was used as eluent. Silica gel 60 F254  was used for analytical TLC. Compounds were detected under UV absorbance at 254 nm (fluorescence absorption) and 366 nm (fluorescence). Vacuum Liquid Chromatography (VLC) was carried out with silica gel , Sephadex LH-20  was used for CC while TLC was performed on silica gel G254 coated plates (Merk) using the following solvent systems: DCM-MeOH (9:1 and 4:1).NMR spectra  and mice (25 - 30 g) were obtained from the animal house of the Department of Pharmacology and Toxicology, Nnamdi Azikiwe University, Awka. The animals were housed under standard laboratory conditions and fed with rodent feed (Guinea Feeds Nigeria Ltd). They had free access to food and water. All animal experiments were conducted in compliance with NIH guide for care and use of laboratory animals (Pub No: 85-23 Revised 1985) as well as with our Institutional ethical guidelines for use of laboratory animals.Acute toxicity studyAcute toxicity test was conducted according to OECD guidelines  served as the standard while the control groups  received 10 ml/kg tween 20. Oxidative stress was induced using single dose of CCl4 6 hr after the last treatment. On the 11th day, blood samples were collected from the animals through retro-orbital plexus for determination of antioxidant enzymes  and lipid peroxidation. The effect of the extract and fractions on CClDetermination of superoxide dismutase (SOD) activity -1cm-1 was used in calculating activity.Superoxide dismutase activity was determined by its ability to inhibit the auto-oxidation of epinephrine determined by the increase in absorbance at 480 nm as described by Sun and Zigma (1978)Determination of catalase (CAT) activityCatalase activity was determined according to the method of Beers and Sizer as described by Usoh et al., (2005). An extinction coefficient for H2O2 at 240 nm of 40.0 M-1cm-1 as described by Aebi (1984)Lipid peroxidation (LPO) assayoC for 15 min, allowed to cool and then, centrifuged at 3000 rpm for 10 min. The supernatant was measured using spectrophotometer at 532 nm against a blank. MDA concentration was calculated using the molar extinction coefficient for MDA TBA-complex of 1.56 × 105 M-1 cm-1.Malondialdehyde (MDA), an index of lipid peroxidation, was determined using the method described by Ogbunugafor et al. (2012)Hydrogen peroxide scavenging activity Hydrogen peroxide scavenging activity of the isolated compounds was determined using the method of Kumaran and Karunakaran (2007)Hydrogen peroxide scavenging activity (%) = DPPH free radical scavenging activity testThe DPPH free radical scavenging activity of the isolated compounds was evaluated based on the method described by Patel and Patel (2010)o–A1) / Ao] × 100  % Inhibition of free radical = [ assay3)  and the absorbance of the reaction mixture was measured at 700 nm  in 0.5 ml of methanol were mixed with phosphate buffer  and potassium ferricyanide [K3Fe(CN)6] . The mixture was incubated at 50° C for 20 min. Then, 1.25 ml of 10% tricholoroacetic acid was added to the mixture and centrifuged at 3,000 rpm for 10 min at room temperature. The upper layer of solution (1.25 ml) was mixed with distilled water (1.25 ml) and ferric chloride . 4 were confirmed from significant (p<0.05) elevation of MDA (a product of lipid peroxidation) and reduction in antioxidant defence enzymes in CCl4-induced control  cathechin (1), (-) epicathechin (2), (+) epicathechin (3) and the flavanone, (+) taxifolin  (4)  based on max (PDA) = 280.0 nm. ESIMS (+ve): m/z = 291.1 [M+H]+, 580.9 [2M+1]+, 602.9 [2M+Na]+ ESIMS (-ve): m/z = 289.3 [M-H]-. 1H NMR (MeOH-d4) ((-) Cathechin (1): Light brown solid (10.0 mg); UV: MeOH-d4) . max (PDA) = 280.0 nm. ESIMS (+ve): m/z = 291.1 [M+H]+, 580.9 [2M+1]+, 602.9 [2M+Na]+ ESIMS (-ve): m/z = 289.3 [M-H]-. 1H NMR (MeOH-d4) ((-) Epicathechin (2): Light brown solid (10.0 mg); UV: MeOH-d4) . max (PDA) = 280.0 nm. ESIMS (+ve): m/z = 291.1 [M+H]+, 580.9 [2M+1]+, 602.9 [2M+Na]+ ESIMS (-ve): m/z = 289.3 [M-H]-. 1H NMR (MeOH-d4), ((+) Epicathechin (3): Light brown solid (11.0 mg); UV: eOH-d4), . max (PDA) = 290.0 nm; ESIMS (+ve): m/z = 305.1 [M+H]+, 327.0 [M+Na]+, 630.8 [2M+Na]+; ESIMS (-ve): m/z = 303.2 [M-H]-, 606.8 [2M-H]-; 1H NMR (MeOH-d4) ((+) Taxifolin (4): Light brown solid (12.0 mg); UV: MeOH-d4) .in vitro hydrogen peroxide scavenging activity of the flavans and flavanone isolated from this fraction (2O2 at lower concentrations. (-) epicathechin showed the same EC50 of 8 µg/ml as ascorbic acid while (-) cathechin, (+) cathechin and 2R, 3R dihydroquercitin showed EC50 values of 13, 10 and 18 μg/ml, respectively (4-induced lipid peroxidation while significant (p< 0.05) reduction by the extract and butanol fraction was noticed at 400 mg/kg (The significant (p< 0.05) elevation of CAT enzyme observed with the ethyl acetate fraction was found to be complemented by fraction . It coulectively . The eth00 mg/kg . 50 of 6 µg/ml against 46 μg/ml recorded for 2R, 3R dihydroquercitin  cathechin with an EC50 value of 88 μg/ml.The DPPH scavenging activity of the isolated flavonoid compounds showed a dose-response relationship . 2R, 3R uercitin . Also, (50 of 46 μg/ml compared to ascorbic acid (66 μg/ml)  cathechin was 88 μg/ml. At the tested doses, 2R, 3R dihydroquercitin did not produce up to 50% reduction of ferric ion, showing only 34.7% inhibition at 100 μg/ml.For the ferric reducing antioxidant power (FRAP) assay, (-) epicathechin exhibited higher reducing efficacy with EC6 μg/ml) . The redAlchornea floribunda, which has been widely used in the management of inflammation, arthritis, and wound infection.Oxidative stress has been implicated in many disease states including cancer, inflammation, Azheimer’s and other neurodegenerative disorders  is a known inducer of oxidative stress which acts through its hepatic metabolites . Superoxide dismutase enzyme (SOD) and Catalase (CAT) form important enzymatic defence against oxidative stress. SOD converts superoxide radical to hydrogen peroxide which is inactivated by CAT to water . The abundance of phenolic compounds in the most active fractions may account for their good antioxidant activity. Our results thus corroborated the beneficial roles of phenolic compounds in free radical-mediated processes, which have been previously established  scavenging enzyme but also have direct scavenging effect on circulating H2O2. Biologically, although H2O2 is widespread and poorly reactive, it is capable of direct inactivation of sulfhydryl (–SH) group containing enzymes (Halliwell and Gutteridge, 20072O2 is mainly due to its ability to produce highly reactive hydroxyl radical through the Fenton reaction (Thomas et al., 20092O2. The scavenging of H2O2 by the compounds demonstrated their hydrogen atom donating ability, a common feature of most flavonoids compound (Dragan et al., 2007 The observed in vitro DPPH and FRAP tests of the flavans and flavanone isolated from this fraction. Oxidative degradation of unsaturated lipids is another deteriorating effect of free radicals that leads to lipid peroxidation (Barrera, 2012Larix gmelinii has been shown to demonstrate effective antioxidant activity (Kolhir et al., 2006The observed DPPH scavenging activity of the isolated flavonoid compounds is in line with numerous studies on scavenging properties of flavonoids (Okawa et al., 2001The FRAP mechanism is totally single electron transfer unlike DPPH test that combines hydrogen atom transfer with single electron transfer (Huang et al., 2005A. floribunda in our animal models, further study on longer term toxic effects on various organs are needed before the use of the drug in humans is justified.  Although we are much encouraged by the apparent antioxidant properties of


H1N1 swine influenza viruses (SIV) are prevalent in pigs globally, and occasionally emerge in humans, which raises concern about their pandemic threats. To stimulate hemagglutination (HA) of A/Swine/Guangdong/LM/2004 (H1N1) (SW/GD/04) antibody response, eukaryotic expression plasmid pCI-neo-HA was constructed and used as an immunogen to prepare monoclonal antibodies (mAbs). Five mAbs  against HA protein were obtained and characterized. Western blot showed that the 70 kDa HA protein could be detected by all mAbs in MDCK cells infected with SW/GD/04. Three mAbs—8C4, 8C6, and 9D6—have hemagglutination inhibition (HI) and neutralization test (NT) activities, and 8C6 induces the highest HI and NT titers. The protection efficacy of 8C6 was investigated in BALB/c mice challenged with homologous or heterologous strains of the H1 subtype SIV. The results indicate that mAb 8C6 protected the mice from viral infections, especially the homologous strain, which was clearly demonstrated by the body weight changes and reduction of viral load. Thus, our findings document for the first time that mAb 8C6 might be of potential therapeutic value for H1 subtype SIV infection. Eurasian H1N1 swine influenza virus (SIV) was first reported in pigs in 1979 . EurasiaPigs, which serve as ‘mixing vessels’ because of their susceptibility to infection by both human and avian influenza viruses, may be a pandemic threat to public health . SporadiProduction of functional antibodies is highly dependent on the structural integrity of the proteins ,29,30. TAll experiments and procedures involving animals were approved by the Animal Welfare and Ethical Censor Committee at Harbin Veterinary Research Institute (HVRI). All animal experiments in this study were approved by the Animal Ethics Committee of the HVRI of the Chinese Academy of Agricultural Sciences with license SYXK (Heilongjiang) 2011022.v/v) fetal bovine serum  and incubated at 37 °C and in a 5% (v/v) CO2 atmosphere.Two viral strains of the H1N1 were used: A/Swine/Guangdong/LM/2004 (SW/GD/04) (H1N1) and A/Swine/Harbin/2009 (SW/HRB/09) (H1N1). The viruses were propagated in 10-day-old specific-pathogen free (SPF) embryonated chicken eggs or in Madin–Darby canine kidney (MDCK) cells and stored at −70 °C before use. MDCK cells were cultured in Dulbecco’s modified essential medium (DMEM) containing 10% . The cytopathic effect (CPE) was observed daily and the number of wells showing more than 50% pathological changes were recorded. TCID50 titers were calculated as described previously . Each diNhe I/Xho I restriction site. The resulting plasmid pCIneo-HA was purified using a Qiaminiprep kit (Qiagen) as per the manufacturer’s protocols. The extracted plasmid was identified by using a double digest of Nhe I and Xho I . Recombinant plasmids were transformed into TOP10 competent cells. Colonies were screened via PCR to confirm insertion of the gene segments. The plasmid sequencing was conducted by using an ABI 3730 DNA automatic sequencer.Viral RNA was extracted from SW/GD/2004 and SW/HRB/09 allantoic fluids by using a viral RNA extraction kit . Virus-specific cDNAs were obtained by using influenza universal reverse transcription primer uni-12:5′-AGCAAAAGCAGG-3′ with the AMV reverse transcriptase . The HA genes were amplified by using HA gene-specific primers, cloned into the pMD18-T vector , and then sequenced by using an ABI PRISM 3700 DNA Analyzer . Full-length HA was cloned into pCI-neo using specific primers, which introduced a Plasmid DNA, pCIneo-HA was used as the immunogen for development of mAbs in this study. Briefly, five-week-old female BALB/C mice were injected intramuscularly (i.m.) with 50 µg of plasmid DNA in sterile phosphate buffered saline (PBS), pH 7.4. The mice received two boosts of 100 µg of plasmid DNA at a three-week interval. The mice spleens were collected aseptically by using euthanized anesthesia techniques. mAbs were produced using techniques similar to that described previously ,34,35. SThe hemagglutination inhibition (HI) test was performed to evaluate mAbs reactivity against SW/GD/04 as described previously ,37. BrieThe Neutralization Test (NT) was performed using 96-well plates. Mixtures of two-fold serial dilutions of each mAb and virus suspension containing 100 TCID50 of SW/GD/04 were incubated for 1 h at 37 °C and used to inoculate MDCK cells. One hundred microliters (100 µL) of DMEM was added to each well and incubated at 37 °C for three days. The CPE was observed every 24 h. Neutralization titers are presented as reciprocals of the highest antibody dilution, causing a reduction of the virus over 50%. Each dilution was repeated five times.To examine whether anti-HA mAbs recognize the HA protein, Western blot was used to examine the binding ability of mAbs to HA proteins. Approximately 1 μg of purified SW/GD/04 virus was subjected to 10% SDS-PAGE or native PAGE and then transferred to nitrocellulose membranes. The membranes were probed with different mAbs, followed by a secondary HRP-conjugated goat anti-mouse antibody . The ascetic fluids produced from the injection of SP2/0 myeloma cells were used as a negative control.2O2 in PBS for 5 min.MDCK cells were infected with the SW/GD/04 strain (at multiplicity of infection (MOI) 10) and incubated at 37 °C for 24 h. After incubations, the monolayers were washed twice with PBS and fixed in methanol at −20 °C for 30 min. The cells were then incubated separately with different mAbs for 1 h at 37 °C or with negative ascetic fluids. Bound antibodies were processed for immunoperoxidase staining by using alkaline phosphatase-labeled mouse IgG (1:500 dilutions). After extensive washing with PBS, peroxidase activity was revealed by incubation with 0.03% Nitro blue tetrazolium chloride(NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP), 0.006% Hn = 8) were pretreated intramuscularly with mAb 8C6 (two group) or negative ascetic fluids (two group) at a dose of 20 µg per mg of mouse body weight before the viral challenge [n = 8) pretreated intramuscularly with negative ascetic fluids were used as normal controls. After 24 h, mice were intranasally challenged with homologous virus SW/GD/04 or heterologous virus SW/HRB/09, respectively. Mice were monitored for weight changes and clinical symptoms for two weeks at two-day intervals. Three mice per subgroup were euthanized on day 3 post-challenge (p.c.) and samples  were collected for virus titration in eggs. The remaining five mice per group were observed daily for weight changes or clinical signs of infection.mAb 8C6 was selected to evaluate its protective efficacy for H1N1 SIV because of its high HI and NT titers. A total of 40 six-week-old SPF BALB/c mice were used to evaluate the protective efficacy of mAb 8C6. Mice , IgG2a , and IgM (8B1), respectively . ConcentSW/GD/04-infected MDCK cells were used to assess whether the obtained mAbs recognize the native-form HA protein by immunohistochemistry (IH) assays. Five mAbs showed strong specific reactions to SW/GD/04-infected MDCK cells, whereas no immuno-reactivity was observed in normal MDCK cells (as the negative control) . The blaSW/GD/04-infected MDCK cells were used to assess whether the obtained mAbs recognize the native-form or the denatured-form of HA protein by Western blot. mAbs 8C4, 8C6, 9D6, 8A4, and 8B1 reacted strongly with the 70 kDa HA proteins of SW/GD/04-infected MDCK cells in both denatured A and natThe protective efficacy of the mAb 8C6 was evaluated in mice with two different H1N1 strains (the HA gene of SW/GD/04 showed 90% identities to that of SW/HRB/09). Body weight changes were monitored at two-day intervals in both mouse and virus titers  in lung, nasal turbinates, spleen, and kidney were detected on day 3 after the challenge. Compared with the normal control mice, all mAb 8C6 pre-treated mice showed no obvious clinical symptoms after challenge. When challenged with the homologous virus SW/GD/04, no weight changes were observed in the mAb 8C6-treated mice, whereas weight loss in the non-mAb-treated mice was 12.3%. Moreover, compared with mice in the normal control group, there was 15.7% weight loss in mice challenged with the heterologous SW/HRB/09 virus . MoreoveVirus replication in mice was detected on day 3 after the challenge. In the mAb 8C6 pre-treated group, there was no detectable virus in the lung/nasal/spleen/kidney of mice following challenge with SW/GD/04. In the non-mAb-treated group, the challenge SW/GD/04 virus replicated to mean titers of 3.29 log10 EID50 in the nasal turbinates and 4.88 log10 EID50 in the lungs . By contAntibody-mediated passive immunity can provide protection against invading pathogens . Since tIn this study, we developed five mAbs against HA protein of SW/GD/04 with a eukaryotic recombinant plasmid pCI-neo-HA as an immunogen in mice. Since HI and neutralization tests demonstrated that mAbs 8C4, 8C6, and 9D6 both had HI and neutralization activities, we may speculate that this neutralization activity resulted from correct folding of the HA protein. IH assay showed that mAbs reacted with the HA protein of SW/GD/04 (H1N1)-infected MDCK cells, indicating that mAbs were H1-specific. Western blot showed that mAbs recognized the 70 kDa HA protein, 6 kDa larger than the expected 64 kDa HA protein, indicating that HA might be glycosylated.Immunization with anti-HA antibodies is capable of conferring protection against influenza virus infection in both animals and humans. Since mAb 8C6 has the highest HI and NT titers, we examined the protective efficacy in mice by challenging homologous and heterologous strains of H1N1 SIV (SW/GD/04 and SW/HRB/09). Considering H1N1 SIV isolates were not lethal to mice, we investigated the mAb protection efficacy by body weights and detectable virus titers from lungs, spleen, kidney, and nasal turbinates three days after challenge. These data confirm that one-dose pretreatment of mice with mAb 8C6 is sufficient to provide complete protection against homologous SW/GD/04 H1N1 SIV infection.In this study, we also evaluated the ability of mAb 8C6 to protect mice from challenge with the pandemic 2009/H1N1-like virus (the heterologous strain SW/HRB/09). Compared with normal control mice, the replication of the challenge virus in the mAb-treated mice was also inhibited in the lungs and nasal turbinates. These results demonstrate that the treatment of mice with mAb 8C6 also limits the susceptibility of the mice to infection with H1N1 SIV including the HA gene of a recent EA-lineage virus, suggesting that broad cross-protection might be conferred against the pandemic 2009/H1N1 virus. These results suggest that mAb 8C6 could provide a complete protection against the homolog and inhibit disease development caused by heterologous H1N1. The mAb 8C6 is, therefore, a promising treatment for H1 swine influenza virus infection.Sequence alignments indicated that the HA protein of SW/GD/04 showed 90% homology to that of the SW/HRB/09 virus used herein for challenge studies. The significance of this difference for cross-protection remains unknown. Further studies mapping the epitope of this mAb should be conducted to understand the antigenic properties of the H1N1 virus, which might contribute to the prevention and control of the H1N1 virus.


Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh—Shh-brain-enhancer-6 (SBE6)—that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development.The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene ( Shh encodes a secreted signalling protein that imparts patterns of growth and identity to cells during many stages of embryonic development, including neural progenitors throughout ventral regions of the developing central nervous system (CNS) [a).Enhancers orchestrate the regulation of gene expression, which is critical for cell lineage specification and differentiation, and they therefore have a pivotal role during embryonic development . A well-em (CNS) –4 (figurShh is located at one end of a large (approx. 1 Mb) regulatory domain containing a number of known enhancers controlling various Shh expression domains [b). Precise Shh expression is critical for proper spinal cord and brain development, and this is governed by a subset of floor-plate and brain enhancers, many of which were identified by reporter assays. Shh floor-plate enhancer SFPE1, located 8 kb upstream of the Shh transcription start site (TSS), drives expression in the ventral spinal cord, and SFPE2 and Shh-brain-enhancer 1 (SBE1), positioned in the second intron of Shh, show activity in the floor plate of the spinal cord, as well as the ventral midbrain , ventroposterior region of the diencephalon and the zona limitans intrathalamica (zli) [Shh regulatory region—identified SBE2, SBE3 and SBE4 that drive Shh expression in the diencephalon (SBE2) and in the telencephalon (SBE4) [ domains –12 (figuca (zli) ,6. An enn (SBE4) . Most ren (SBE4) .cis-regulation leads to severe neural defects in mammals. Translocations separating SBE2, 3 and/or 4 from Shh, disrupt the function of these enhancers and lead to Shh haploinsufficiency, causing diverse holoprosecencephaly (HPE) phenotypes [cis-regulatory elements that control Shh expression in the CNS.Perturbation of Shh enotypes ,15, poinenotypes . Togethein vitro differentiation of mouse embryonic stem cells (mESCs) to neural progenitor cells (NPCs), we report a new Shh brain enhancer (SBE6) that we show is necessary for proper Shh expression in NPCs and that is active in vertebrate brain and neural tube development in transgenic assays.Using chromatin profiling during the 2.2.1.Sox1 locus [+ cells after transfection or differentiation, cells were trypsinized and resuspended in PBS + 10% medium. Flow cytometric analysis was performed, using the 488 nm laser of a BD FACSAriaII SORP (Becton Dickinson) with 525/50 nm bandpass filters. BD FACSDiva software  was used for instrument control and data analysis.46c mouse embryonic stem cells (mESCs), derived from E14tg2A, contain a GFP insertion into the x1 locus . mESCs wx1 locus . To sort2.2.6 46c mESCs or NPCs, using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol, including a DNaseI (Qiagen) treatment for 15 min at room temperature. cDNA was synthesized from 2 µg purified RNA with superscript II reverse transcriptase (Invitrogen) primed with random hexamers (Promega). Real-time (q)PCR was carried out on a Roche LightCycler 480 real-time PCR system, using a LightCycler 480 Sybr Green detection kit (Roche) as described previously [RNA was prepared from approximately 1 × 10eviously . Primer ightCycler software and normalized to the mean for Gapdh from at least two biological replicates and technical triplicates.The real-time thermal cycler was programmed as follows: 15 min Hotstart; 44 PCR cycles . The relative mRNA expression for each primer set in each sample was measured by the L2.3.6 mESCs or sorted Sox1+ NPCs were prepared and resuspended in NB-R  as previously described [Nuclei from 3 × 10escribed . Microcoescribed ,22. Antiata Collection software.Ten nanograms  of input or ChIP DNA were amplified, using the WGA2 whole genome amplification kit (Sigma). Amplified material was labelled with Cy3 or Cy5 by random priming according to the NimbleGen ChIP-chip protocol (Roche). In total, two or three biological replicates with dye swaps were hybridized for 20 h and washed according to the manufacturer's protocol. A custom 3 × 720 K mouse tiling array  containing 179 493 unique probes from different genomic regions was used, with each probe represented by four replicates. Arrays were scanned on a NimbleGen MS 200 Microarray scanner (Roche), using 100% laser power and 2 µm resolution. Raw signal intensities were quantified from TIFF images, using MS 200 Deadarray and Limma according to the Epigenesys NimbleGen ChIP-on-chip protocol 43 (www.epigenesys.eu/en/protocols/bioinformatics). Scale normalization was used within replicates, to control interarray variability. Each condition was represented by two biological replicates hybridized as dye swap experiments and enrichment scores are defined as log2 ChIP/input signal.Microarray data were analysed in R, using the bioconductor packages B2.4.Evolutionary conservation of the SBE6 region was assessed, using the ‘Vertebrate Multiz Alignment & Conservation/Multiz Alignments and Conserved Elements’ tracks in the UCSC genome browser . This deSBE6.1: Chr5: 28 889 688–28 890 461, SBE6.2: Chr5: 28 893 935–28 895 000 (mm9)rVista [rVista server were selected .rVista  was usedrVista  availablrVista alignment.JASPAR  was used2.5.AGGGGAGAACTTTGTATAGAAAAGTTGGCGCGCCCACCTGCTTCTCTGAGGAASbe6.1 Fw B4 : AGGGGACTGCTTTTTTGTACAAACTTGCTTAGGCCATTGTGCCCACSbe6.1 Rv B1R : AGGGGAGAACTTTGTATAGAAAAGTTGGCGCGCTGAAGTCAAGGGCCTGGTACTSbe6.2 Fw B4 : AGGGGACTGCTTTTTTGTACAAACTTGATCAGCCCTCCAGTTTGACTSbe6.2 Rv B1R : Shh, which have no suspected regulatory activity, and which are the same genomic distance from Shh as SBE6.1 and SBE6.2 are upstream (5′) of Shh.Negative controls used were sequences 3′ of Negative controls:AGGGGAGAACTTTGTATAGAAAAGTTGGCGCGCCGAGTGCAGGTGTTTGTGAASbe6.1 Fw B4: AGGGGACTGCTTTTTTGTACAAACTTGCCTCAACACAGCATTGCCAASbe6.1 Rv B1R: AGGGGAGAACTTTGTATAGAAAAGTTGGCGCGCAGAGAGTGAAGATTCCCAGCTSbe6.2 Fw B4: AGGGGACTGCTTTTTTGTACAAACTTGTGAGGCAGTGTCTATCTTTTGACSbe6.2 Rv B1R: attB4 and attB1r sequences (bold) were included in the PCR primers for use with the Gateway recombination cloning system . The amplified fragment was first cloned into the Gateway pP4P1r entry vector and sequenced using M13 forward and reverse primers for verification. The elements in the pP4P1r vector were combined with a pDONR221 construct containing either a Gata2 promoter-eGFP- polyA or a Gata2 promoter mCherry-polyA cassette [The putative SBE6.1 and SBE6.2 enhancers were cloned by PCR amplification of the relevant fragment and flanking sequence from mouse genomic DNA, using Phusion high fidelity polymerase (NEB) and the following primers:cassette , and rec−1 with DNAse/RNAse free water. Tol2 transposase RNA was synthesized from a NotI-linearized pCS2-TP plasmid using the SP6 mMessage mMachine kit (Ambion), and similarly diluted to 50 ng µl−1. Equal volumes of the reporter construct(s) and the transposase RNA were mixed immediately prior to injections. 1–2 nl of the solution was microinjected per zebrafish embryo at the one- to two-cell stage for up to 200 embryos. Embryos were screened for fluorescence at 1–5 days post-fertilization  and raised to adulthood. Germline transmission was identified by mating of sexually mature adults to wild-type fish and examining their progeny for fluorescence. F1 embryos from three to five F0 lines showing the best representative expression pattern for each construct were selected for confocal imaging. A few positive embryos were also raised to adulthood, and F1 lines were maintained by outcrossing. A summary of the independent lines analysed for each construct and their expression sites is included in electronic supplementary material, table S3. Imaging of zebrafish reporter transgenic embryos was carried out as previously described [Reporter plasmids were isolated using Qiagen miniprep columns and were further purified using a Qiagen PCR purification column (Qiagen), and diluted to 50 ng µlescribed .2.6.2; 5 mM EGTA; 0.02% NP-40 in PBS). After fixation the embryos were washed in PBS containing 0.02% NP-40, before being stained overnight at 37°C in the dark in a solution containing 5 mM K3Fe(CN)6; 5 mM K4Fe(CN)6.3H2O; 2 mM MgCl2; 0.01% sodium deoxycholate; 0.02% NP-40 and 0.1% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside . Embryos were then fixed with 4% PFA and photographed on a Leica MZ FLIII Microscope fitted with a Hamamatsu Orca-ER digital camera and a CRI microcolour filter.The same SBE6.1 PCR amplicon, with attB4 and attB1r sequences included as used for reporter assays in zebrafish (above), were used to generate enhancer-reporter constructs for mouse transgene assays. The amplicon was cloned directly into an hsp68-LacZ vector containing a P4-P1r entry cassette . Transge2.7.in situ hybridization on fish embryos was performed as previously described [Shh hybridization probes are the following:RNA escribed . The seqForward primer (5′-SP6 promoter-sequence-3′) AAGCTGACACCTCTCGCCTA and reverse primer (5′-T7 promoter-sequence-3′) GAGCAATGAATGTGGGCTTT.in situ hybridization was performed with DIG-labelled gene-specific antisense probes as previously described [Mouse escribed . The Shhescribed .2.8.−1 ProteinaseK) followed by ethanol precipitation and washes. Genomic DNA was amplified with the following primers:SBE6.1 Fw: TTTTGGAAGCTTAAATGCCCATSBE6.1 Rv: CCACCACAAGCACATTCATSBE6.2 Fw: GCCTCCATGAAGTCCAATGGSBE6.2 Rv: CCACCCTTGCTACTCAGGAAAmplification was done using DreamTaq Green PCR master mix (ThermoFisher K1081) following the manufacturer's protocol and PCR products were assessed by agarose gel electrophoresis. Amplified products were later sequenced to further confirm homozygous deletions.Cell line deletions were produced, using the Crispr/cas9 system. SBE6.1- and SBE6.2-specific gRNA primers  were cloned into the cas9 plasmid pX458 following protocols from the Zhang laboratory –34. 46C 3.3.1.Shh during neural differentiation. These cells contain a knockin of GFP into the Sox1 locus allowing for the monitoring of neural differentiation and the purification, by fluorescence-activated cell sorting (FACS), of Sox1+ neuroepithelial progenitor cells  [Shh and Nestin increase while Oct4 mRNA levels progressively decrease (b). Analysis of these NPC cells for expression of markers from different regions of the developing brain (c) suggests that these cells do not have a distinct regional identity, though there is some evidence for a slight shift towards a more telencephalic fate (increasing Six3 and Emx2 expression) and away from the hindbrain (decreasing En2 and Gbx2 expression) by day 7 (d).We used the differentiation of 46c mESCs as a model system to identify putative regulatory elements that may become activated concomitant with the expression of ls NPCs; a [17,35,NPCs; a) . Sox1+ cdecrease b. Analysng brain c suggestby day 7 d.FigureShh regulatory region, we assessed the sites of enriched H3K4me1 and H3K27ac in mESCs (where Shh is not expressed) and in Sox1+ NPC after 5 days of neural differentiation. Significant gains of H3K4me1 and H3K27ac were not detected at the known SBE2, 3, 4 or 5 brain enhancers (a). However, a prominent change in the ChIP profile was seen at a small region approximately 100 kb upstream of the Shh TSS. This region has no evidence of active enhancer marks in mESCs but gains both H3K4me1 and H3K27ac upon neural differentiation (a).Genome-wide ChIP has allowed the identification of several post-translational histone modification characteristics of active enhancers including H3K4me1 and H3K27ac . The usenhancers a. Howeventiation a.FigureShh TSS) and SBE6.2 . Interestingly, two other sequences beyond SBE3 also show a gain of active enhancer marks (arrowheads in figure 3a), but are not investigated further here.Analysis of sequence conservation across multiple vertebrate species indicated that this region contains two blocks of evolutionary conservation in mammals and birds, and we named these putative NPC enhancers SBE6.1  regions that are highly conserved. Complementary rVista, JASPAR and RSAT scans of those regions revealed the presence of predicted binding sites for neural transcription factors such as ETV1, SP8, VAX1 and DLX6 .Shh expression in mouse embryos called SLGE (chr5: 28 889 230–28 890 979) [The SBE6.1 sequence is entirely included in a recently described 1.7 kb lung and gut epithelium regulatory element for 890 979) , raising3.2.In situ hybridization for Shh mRNA in wild-type zebrafish embryos reveals expression in the forebrain at 48 and 72 hpf [figure 4a,b; electronic supplementary material, table S3). SBE6.2 however failed to consistently drive reporter gene expression in the forebrain, with forebrain-specific activity noted in only one out of the four independent transgenic lines generated . Therefore, SBE6.1 has a consistent enhancer function and is active in zebrafish forebrain development.To test the regulatory potential of SBE6.1 and SBE6.2, we used a zebrafish Tol2 transposon assay in which the test element is juxtaposed to a minimal promoter driving the expression of either GFP or mCherry reporter gene expression. This assay has been shown to recapitulate the correct expression pattern for the SBE2 enhancer and to detect the loss of this enhancer activity associated with mutation of a SIX3 binding site found in the cases of HPE ,27. In sa,b). X-gal staining could be also detected in few superficial diencephalon cells where Shh is not expressed . However, SBE6.1 also showed activity in the developing ventral mesencephalon with some cells expressing SBE6-LacZ near the hindbrain and as well as in the ventral midline of the mouse embryonic neural tube—all sites of endogenous Shh expression .The ability of SLGE to drive expression in the developing mouse brain is unclear, but it is known to be capable of driving expression in the brain of transgenic rabbits . We therShh expression. However, the strong similarities between the two mouse embryos do support our conclusion that the SBE6.1 enhancer is capable of activity in the developing vertebrate brain, from a forebrain pattern in zebrafish transgenics to a floor plate and ventral mesencephalon expression in mouse transgenic embryos.SBE6.1 is only active in a small number of cells in transgenic embryos, and we cannot at this stage confirm how accurately this recapitulates a subset of endogeneous 3.3.−/− and SBE6.2−/−; electronic supplementary material, figure S2). We generated and analysed two SBE6.1−/− and three SBE6.2−/− independent cell lines. Upon NPC differentiation, the proportion of Sox1-GFP+ cells remained the same between NPCs derived from wild-type and SBE6.1−/− or SBE6.2−/− cells, analysis of Oct4 and Nestin mRNA expression confirmed that differentiation of mESCs into NPC was not perturbed by the loss of either SBE6.1 or SBE6.2 (a). However, in NPCs derived from SBE6.1−/− but not SBE6.2−/− cells, levels of Shh expression were significantly reduced compared with wild-type cells . Average Shh mRNA levels in NPCs differentiated from SBE6.2−/− ESCs were not significantly different relative to wild-type (b).To determine the regulatory activity of SBE6.1 and SBE6.2 in their native context, we used CRISPR/Cas9 to delete these elements from the genome in 46c mESCs  Shh regulatory domain [b). Most of these enhancers were identified using transgenic reporter assays [Shh enhancers cause phenotypes that result from aberrant control of specific aspects of Shh expression in development. Most recently, information on transcription factor motifs in known Shh brain enhancers has been used to search for other similar patterns of motifs in the Shh regulatory domain and has identified a new enhancer that drives Shh expression in a discrete region of the brain [The regulation of y domain  , typically associated with active enhancers, in an Shh expression in different developmental contexts. Given the large size of the gene desert upstream of Shh, where many of these enhancers are located, there is the potential for this region to harbour many more cis-regulatory elements and, given the complexity of brain development, many of these may be enhancers active in the brain. Indeed, regulatory segmentation built from ChromHMM or Segway using ENCODE data from various mouse primary tissues indicates the presence of several regions with chromatin signatures indicative of enhancer activity in mouse brain at E14.5—a period of mouse development when neurogenesis is ongoing (a). This includes the genomic regions containing the known neural enhancers SBE2–4, but also a region that corresponds to SBE6. The many other regions called as likely active enhancers using the analysis from just four tissues  at one embryonic stage (a) suggests that the Shh regulatory region may harbour many tens of as yet unannotated enhancers. We note that the new sites detected by these high-throughput methods (marked with arrowheads as #1 and #2 in figure 6a) correspond to peaks of H3K4me1/H3K27ac that are induced during the differentiation of 46c mESCs to NPCs . A similar analysis of chromatin profiling data from the Roadmap project also indicates the signature of an active neural enhancer at the position of SBE6 in material from different regions of the human brain and particularly in ganglionic eminence derived neurospheres (figure 6b). This analysis also indicates many other potential regulatory elements active in different brain regions.Our analyses presented here add to the growing number of functionally validated enhancers directing  ongoing a. This iic stage a suggestShh lung and gut epithelium regulatory element SLGE [figure 6a; electronic supplementary material, figure S3) and gastric tissue (figure 6b). Important sequences required for enhancer function work as assemblies of transcription factor motifs [SOX9 and PAX6, there are cis-regulatory elements driving expression in multiple developmental sites, and in which disease-associated variants have been identified that ablate enhancer function in one tissue but leave the other sites of expression unaltered [It is interesting that the genome coordinates of SBE6.1 are completely contained within those reported for the ent SLGE . Transgeent SLGE . Here, wr motifs . SBE6.1 r motifs . Moreovenaltered ,42. Furt


It has affected multiple countries over time. The best strategy to reduce the burden of influenza is through vaccination whose efficacy varies with respect to the circulating strains. This study was performed to better understand the molecular evolution of influenza A(H3N2) and assess vaccine efficacy in Cameroon. Complete sequences of three gene segments were obtained from 2014 to 2016 influenza seasons in Cameroon. Hemagglutinin (HA), Neuraminidase (NA) and matrix (M) genes of 35 A(H3N2) virus strains were amplified and sequenced. Predicted vaccine efficacy was measured using the Pepitope model. Phylogenetic analysis of the HA gene showed that all Cameroonian strains had evolved away from the 3C.1-A/Texas/50/2012-like clade. Globally, 2014 virus strains clustered with the 2015–2016 vaccine strain, 3C.3a-A/Switzerland/9715293/2013, whereas 2015 and 2016 virus strains clustered with the 2016–2017 vaccine strain, 3C.2a-A/HongKong/4801/2014. In order to determine the genotypic drug susceptibility to neuraminidase inhibitors and amantadine, the NA and M2 protein coding sequences were analyzed. There was no strain with characteristic mutation for resistance to neuraminidase inhibitors, per contra; all strains possessed the substitution S31N, peculiar of resistance to adamantanes. There was drift in influenza A(H3N2) dominant epitopes B (2014 and 2015) to epitopes A (2016) with a theoretical efficiency in vaccine ranging from low to moderate. The presence of several antigenic site mutations among H3N2 virus strains between 2014–2016 influenza seasons in Cameroon confirms the progressing evolution of circulating H3N2 strains.The first outbreak of influenza A(H3N2) occurred in 1968 and caused the third flu pandemic of the 20 Influenza A virus is a major cause of acute respiratory disease in humans and is responsible for approximately 250,000–500,000 deaths annually worldwide . PandemiInfluenza A nomenclature is based on the genotypic properties of two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Currently, 18 HA and 11 NA subtypes have been recognized . There aThe best strategy to reduce the burden of influenza is through vaccination whose efficacy varies with respect to the circulating strains. AlthougRespiratory samples were collected between January 2014 and June 2016 from outpatients with clinical evidence of influenza-like illness (ILI), defined as person with sudden onset of fever >38°C and cough or sore throat, at 12 sentinel sites involved in influenza surveillance in Cameroon. Naso-pharyngeal and/or oropharyngeal swabs were collected and put in 2 ml cryovials containing virus transport medium and stored at 4°C before transportation to the Pasteur Centre of Cameroon (PCC) for analysis. All samples were stored anonymously. This study was specifically approved by the Cameroon National Ethics committee (N° 2016/08/798/CE/CNERSH/SP).Viral RNA was extracted then from 140 μL of clinical samples with Qiagen RNA kit according to the manufacturer’s instructions into a final volume of 60 μL elution buffer. Extracts were analyzed for the presence of influenza by a real-time reverse transcription-polymerase chain reaction (RT-PCR) in an ABI Prism 7300 or 7500 thermocycler . Positive samples for influenza H3N2 with Ct below 30 after sub-typing were eligible for sequencing. A convenient sample size of 35 was randomly selected from the flu database based on their geographic origin and distribution over time. Samples co-infected with other influenza subtypes were not included.2, 1μL of 10μM forward and reverse primer, 0.25 μL of Taq DNA polymerase 5U/μL and 2μL of first PCR product. This mixture was run using the following program for all gene segments: 94°C for 5min, 30 cycles at  and 72°C for 5min. PCR products were run in a gel and bands corresponding to the appropriate size were sent for sequencing at GENEWIZ UK, LTD  with the corresponding primer sets.The first PCR was performed using Superscript III One Step RT-PCR System  and a set of primers . The folSequences were edited and assembled with CLC Main Workbench version 5.5. The sequences of reference strains of known clades and northern hemisphere vaccine strains recommended by WHO  includedInfluenza A(H3N2) virus sequences included in the analysis were submitted to Genbank under accession numbers KY653817 to KY653919. The prediction of potential N-liked glycosylation sites was performed with an online server: NetNGlyc 1.0. . This seepitope method [epitope measures the antigenic distance between the vaccine strain and the dominant circulating strains, and it is calculated as follows: (number of mutations in the dominant epitope)/(number of amino acids in the dominant epitope). The mathematical formula linking vaccine efficacy for influenza A(H3N2) and Pepitope is given by E = −2.47 × Pepitope + 0.47 for [epitope between the vaccine and circulating strains is null, the influenza A(H3N2) vaccine efficacy is 47% [The predicted vaccine efficacy of the influenza A(H3N2) seasonal influenza virus was estimated using the Pe method –23. Pepiy is 47% . This moy is 47% .A total of 103 gene segments from the 35 virus strains were analyzed . Nucleotide and amino acid sequences for the 3 selected genes were compared within the Cameroon virus strains and with the WHO vaccine strains, WHO reference strains and available sequences of other African virus strains.The HA coding sequences and amino acid sequences of the 35 Cameroonian virus strains were analyzed. Comparison of genes was performed on the strains circulating during the 2014 (N = 6), 2015 (N = 17) and 2016 (N = 12) seasons and sequences from the northern hemisphere vaccine and reference strains. Phylogenetic analysis showed that all Cameroonian virus strains had evolved away from the 3C.1-A/Texas/50/2012-like clade by acquiring several amino acid substitutions , Table 1The 2014 virus strains differed from the 2015 and 2016 virus strains at three antigenic sites: antigenic site A, which contained 2 amino acid substitutions , antigenic site B showed 3 substitutions  and antigenic site C showed a Q311H substitution when compared to the 3C.1-A/Texas/50/2012-like clade . IsolateThe NA sequence analysis showed that all Cameroonian strains evolved away from the 3C.1-A/Texas/50/2012-like clade . StrainsThe NA genes of the 2014 virus strains differed from those of the 2015 and 2016 virus strains by substitutions of several amino acids: E221D and T267K were found in all 2015 and 2016 virus strains. Whereas, I67V, A110D, V313A and V412I substitutions were found only in 2014 virus strains.Analysis of the M gene showed that 2014 virus strains clustered with the 2016–2017 vaccine strain, 3C.2a-A/HongKong/4801/2014, while 2015 and 2016 virus strains did not cluster with any reference strain . GloballIn order to determine the genotypic antiviral drug susceptibility of the A(H3N2) virus strains included in this study we analyzed the NA and M2 protein coding sequences for the reported genetic markers of resistance against neuraminidase inhibitors  and adamantanes (amantadine and rimantadine). Mutations E41G, R118K, E119V/I/Q, Q136K (but 1 Q136H), D151A/D/E/V, R152K, I222T, R224K, Q226H, E227D, H274Y, E276D, R292K, N294S, S331R, R371K and E119V+I222L/V in the NA protein have been reported to cause reduced susceptibility to NAIs –32. NoneAmino acids at positions 26, 27, 30 and 31 of the transmembrane region of the M2 protein are the residues frequently associated with amantadine- and rimantadine-resistant strains . Amino aThere were 9 predicted glycosylation sites in the HA1 of 2014 virus strains as well as one 2015 isolate (A/Cameroon/15V-8790/2015) at amino acid positions 8(NST), 22(NGT), 38(NAT), 45(NSS), 63(NCT), 133(NGT), 165(NVT), 246(NST) and 285(NGS), whereas 2015 and 2016 virus strains had 2 additional glycosylation sites at position 126(NWT) and 158(NYT) respectively due to A128T and K160T substitutions.epitope method was used to evaluate how closely the vaccine strain resemble the circulating strain . For the 2015 and 2016 seasons, HA1 sequences showed antigenic drift mainly in epitopes A. The Pepitope of 0.1579 for 2015 strains  predicted a vaccine efficacy against these strains of 17.02%  of that of a perfect match with the A/Switzerland/9715293/2013 vaccine strain. For 2016, Pepitope of 0.0526 from 9 strains  predicted a minimum vaccine efficacy against these strains of 72.36%  of that of a perfect match with the A/HongKong/4801/2014 vaccine strain. Influenza A(H3N2) demonstrated antigenic drift from epitopes B to epitopes A.To assess the effect of the accumulated mutations in the HA1 domain on predicted vaccine efficacy in a given year, the Pg strain . Theoretnegative , 22. Forepitope value of 0 gave an estimated worst-case vaccine efficacy against these strains of 100%. Moreover, clade 3C.2 strains of A(H3N2) in 2015 season and the 2016–2017 vaccine strain, A/HongKong/4801/2014, showed a value of 0.0526, indicating a worst-case vaccine efficacy against these strains of 72.36%.The 2015–2016 vaccine strain A/Switzerland/9715293/2013, which belongs to clade 3C.3, was also examined for its ability to match the A(H3N2) strains of the same clade circulating in 2014. The resulting PThis is the first report of genome characterization of influenza H3N2 in Cameroon. We analyzed the coding sequences of 3 gene segments of influenza A/H3N2 virus strains from 2014 to 2016 influenza seasons in Cameroon and compared them with those of the WHO reference strains, WHO recommended vaccine strains and with The HA gene sequences indicated that all the Cameroon strains had evolved away from the 3C.1-A/Texas/50/2012-like clade by acquiring the genetic markers N145S, Y186G, P198S and F219S which has been identified as being characteristic of clades 3C.2 and 3C.3 , 34; theet al. in the Southern hemisphere [epitope > 0.19 observed, and eventually resulting in a negative vaccine efficacy. The vaccine change to the A/Switzerland/9715293/2013-like strain for the following season resulted in more than 80% of the circulating H3N2 strains not covered by the selected vaccine. Notwithstanding the poor results with the previous influenza seasons, the 2016–2017 selected vaccine strain showed moderate efficacy when compared to the circulating virus strains. It is noteworthy that vaccination against influenza virus, is not a common phenomenon in the Cameroon population, however a better selection of annual vaccine with the use of Pepitope as suggested by other authors, [Theoretically, a clear H3N2 vaccine mismatch was observed during the 2014–2015 influenza seasons as reported by Tewawong misphere . Severalauthors,  will impauthors, .The acquisition of a mutation in influenza A viruses conferring resistance to an antiviral agent may occur as a result of drug selection, spontaneous mutation or through genetic reassortment with another drug resistant influenza A virus . NeuramiAmantadine resistance reported in most H3N2 viruses worldwide was confirmed in the Cameroonian virus strains by an S31N substitution in the M2 protein , 27. Higet al., glycosylation and deglycosylation are important viral mechanisms to (i) mask antigenic epitopes and thus inhibit binding to neutralizing antibodies, (ii) adjust receptor-binding affinity, or (iii) regulate virulence of influenza [As reported by Mostafa nfluenza . The CamThe presence of several antigenic site mutations with high frequency among H3N2 virus strains between 2014–2016 influenza seasons confirms the progressing evolution of circulating H3N2 strains. These results lay more emphasis on the relevance of routine influenza surveillance as well as the genetic and antigenic characterization in facilitating the prompt and efficient selection of the influenza strains which will be included in the flu vaccine. This data will be very valuable in tracking the phylogenetic evolution of influenza viruses within sub-Saharan Africa.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms. Seasonal influenza A viruses (IAV) cause tens of thousands of deaths and tens of billions of dollars in economic costs every year in the U.S. alone, despite widespread pre-exposure and vaccination. IAV persists within the human population by evading herd immunity through the continual accumulation of immune escape substitutions in a process known as antigenic drift. Unfortunately, the specific mechanisms that facilitate IAV antigenic drift remain unclear, making it difficult to design more efficacious, escape-proof vaccines. Here, we used a high-definition population sequencing approach to understand how IAV tolerates the accumulation of immune escape substitutions during antigenic drift without losing fitness. We found that the virus rapidly acquired N-linked glycan structures on the hemagglutinin (HA) protein following escape from a panel of neutralizing antibodies. Glycosylation of HA alleviated the deleterious effects of immune escape substitutions on interactions with host cell receptors. These results expand our understanding of the mechanisms that facilitate IAV immune evasion, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms. Influenza A virus (IAV) persists in human populations by continuously evolving to escape herd immunity. Protective immunity is predominantly mediated by neutralizing antibodies (Abs) specific for the viral surface glycoprotein hemagglutinin (HA). HA mediates both target cell attachment, by binding terminal sialic acids (SA) on cellular membrane components, and fusion between viral and cellular membranes following virion internalization. Neutralizing Abs mainly target the five highly variable immunodominant antigenic sites on the globular head domain of HA, blocking either HA-mediated attachment or fusion –4. Virusversus antibody [Influenza virus escapes from neutralizing antibody via a number of defined mechanisms involving HA mutations. The simplest is an amino acid substitution in a cognate epitope that diminishes antibody affinity . Less coantibody –13. The antibody ,14–19.The ability of viruses to exploit these escape routes is limited by their costs to viral fitness. The sialic acid receptor binding site is nestled among the immunodominant antigenic sites, and escape substitutions often alter viral fitness by changing HA receptor binding avidity. Amino acid substitutions that change the net charge of the globular domain typically alter cell binding avidity ,20. We pHere, we use the primerID method for error-correcting virus population sequencing to reveal a surprising new role for N-linked glycosylation in facilitating IAV immune escape. Beyond its canonical role in blocking Ab binding, we show that glycosylation can also compensate for fitness costs imposed by escape substitutions elsewhere on HA, thus increasing the viability and subsequent emergence potential of Ab-escape variants.We previously modeled IAV antigenic evolution by sequentially selecting for virus escape from different mouse anti-HA monoclonal Abs (mAbs) under over-neutralizing conditions using the allantois-on-shell (AOS) system . Twelve th sequential selection in the AOS system [An alternative possibility that we did not examine in detail is that the accumulation of antigenic escape substitutions imposed significant costs, but strong selection for compensatory mutations or reversions in SV12 rapidly restored fitness during the initial rounds of SV12 expansion in eggs; the original SV12 isolate had been expanded 2–3 times in the absence of antibody in embryonated chicken eggs after the 12S system . To testCompensatory mutations that increased the fitness of SV12 may not have been identified with conventional Sanger sequencing if multiple variants were selected that collectively were a significant proportion of the population, but that individually remained below the wild-type SV12 consensus frequency at each codon. To identify compensatory mutations or reversions in HA that potentially emerged following sequential antigenic escape, we used the primerID method for error-correcting virus population sequencing to identify minor sequence variants that emerged during the limited expansion passaging of SV12 ,23.−3–10−4 non-consensus events/nt), by correcting the high-frequency PCR and base-calling errors that occur during standard next-generation sequencing. PrimerID can also correct for variant frequency skewing due to PCR resampling imbalance [PrimerID sequencing is based on tagging individual viral cDNAs with random unique sequence barcodes during reverse transcription ,23. Thesmbalance ,23.−3) show consistent results whether an amplicon/primerID or a shotgun sequencing approach were used  that encompassed the receptor binding pocket and most of the SV12 substitutions in the original expanded SV12 virus stock, we performed paired-end sequencing of primerID libraries on the Illumina MiSeq platform and analyzed data using a custom software pipeline. Sequencing revealed amino acid substitutions at 7 sites that emerged at frequencies of >0.1% during 2–3 passages of SV12. These substitutions were (including their frequencies) K123N (1.7%), K123E (1.1%), N133T (25%), N133S (8%), K144N (9%), K144E (0.5%), N145D (0.3%), K174E (0.3%), K222T (0.17%), and G225D (3.7%)  . LinkageEach substitution was observed at sites with potential functional roles in SV12 . SubstitThree of the amino acid substitutions that emerged in the original expanded SV12 virus stock  created new potential N-linked glycan sites (PNGS) within the HA sequence . To determine whether these substitutions indeed affected the glycosylation status of virion-associated HA, we intrWe next tested whether compensatory glycosylation site additions increased the replicative capacity of rSV12-HA in multi-step growth assays in eggs . The impKon) and dissociation (Koff) rates of purified WT PR8, rSV12-HA, rSV12(K123N)-HA, rSV12(N133T)-HA, and rSV12(K144N)-HA virion preps binding to the model sialic acid receptor 3-SLN using bio-layer interferometry (BLI) (A clue to how these glycan additions restore SV12 fitness came from their close proximity to the HA receptor binding pocket . We hypory (BLI) , Table 1ry (BLI) .4 EID50  to initiate each passage. We harvested each passage at 16 hours post-infection (h.p.i.) to minimize defective interfering particle formation. After the third passage, we sequenced the entire HA coding region of all six passaged populations, along with the parental seed stocks, using an optimized primerID protocol,  of the compensatory mechanisms that we observed, we repeated the passage experiment using recombinant, reverse genetics-derived versions of either WT PR8 or SV12-HA (rSV12-HA) virus as the parental virus. We generated each virus in 293T cells from transfected plasmids and then passaged each virus in triplicate in chicken eggs, using a dose of 10col, see . To overBoth SV12 and PR8 (WT) parental populations harbored numerous missense mutations at frequencies above background in HA . Five suBy contrast, the parental rSV12-HA population carried nearly three times as many missense mutations (14 vs. 5) at frequencies >0.1% , primarivs. hitchhiking in amino acid variant emergence.Emergence of an amino acid variant within a population may result from positive selection driven by specific beneficial effects of the variant on fitness. Alternatively, the variant may be neutral or deleterious but hitchhike with another variant under positive selection to attain higher frequency. The potential for intragenic hitchhiking is high with IAV due to extreme rarity of intragenic recombination during infection . PrimerIWe assessed the extent of genetic linkage between the five high frequency variants that emerged in all three rSV12-HA passage populations  . ExaminaThese findings suggest that each emergent, mutually-exclusive substitution was selected individually for its ability to compensate for the fitness defects of SV12, and that their combination in the same variant may substantially reduce fitness. The data, however, are also consistent with the predicted infrequent recombination within IAV genome segments and a strong role for clonal interference in governing the emergence of beneficial alleles during adaptation ,30.To test whether combining compensatory HA glycan additions decreases viral fitness, we generated a N133T, K144N double mutant ) using reverse genetics. After confirming the double mutant did incorporate an additional glycan compared to the single mutants by SDS-PAGE , we examIAV has persisted in humans despite widespread vaccination and infection, while vaccination has driven other RNA viruses with similarly high mutation rates, such as measles virus and poliovirus, to near-extinction. Understanding the specific features of IAV that enable effective immune escape is critical for designing more effective vaccines and therapeutics.Our findings reveal a novel role for glycosylation in viral immune evasion, independent of its well-known steric effects on epitope shielding –19. PrevImportantly, the fact that we selected for glycan addition in the absence of Ab selection does not diminish the profound effects that these glycans have on the antigenicity of HA. In particular, the predominant variant selected, N133S/T, also emerged during the circulation of seasonal H1N1 in humans, and has been demonstrated to have enormous effects on antigenicity and sensitivity to neutralization . These rin vitro found that the HA globular head was highly tolerant of both random substitutions and 5-amino acid block insertions [Defining the specific properties of the HA molecule that facilitate mutational tolerance is an area of intense study. Studies sertions ,27,31,32sertions . Alternasertions ,35. Our sertions ,13,36,37in vitro mutation rates and rapid Ab-based selection of influenza viruses in vivo and the relatively slow rates of population-level antigenic evolution seen with influenza viruses[If rapid compensation of antigenic escape mutations is important for maintaining fitness during antigenic drift, then defining compensatory mechanisms is essential. Many antigenicity-determining residues scattered in the HA globular domain also strongly influence avidity and specificity for sialic acid receptors, and thus Ab escape substitutions at these residues can negatively affect receptor interactions. As a rea viruses,38. Our The availability of multiple mutational pathways to restore fitness increases the odds that a fit variant will emerge under selection . Our disOur unexpected discovery of a new role for glycosylation in compensating for IAV immune evasion highlights the power of virus population sequencing for uncovering novel mechanisms of viral adaptation through high-definition forward genetics screening. The ability to accurately measure changes in the viral population through methods such as primerID can provide a comprehensive, unbiased profile of the genetic pathways to increased fitness under defined selection conditions.First, the ability to generate a multi-read consensus of the original cDNA sequence reduces the background nucleotide error rate from ~1% to ~0.01%, greatly increasing the sensitivity of minor variant detection. In theory, this background represents the bona fide mutation frequency within the population, combined with the mutation rate of the reverse transcriptase used to generate the cDNA pool (~0.01%)[Second, assembly of amplicon reads into ancestral cDNA sequences eliminates the distortion of measured variant frequencies by the stochastic PCR re-sampling that occurs during conventional amplicon sequencing. Third, the number of unique primerID barcodes used to generate consensus sequences represents the actual depth of the viral population sequenced. Fourth, the ability to phase substitutions into authentic haplotypes enables quantitation of linkage between substitutions within a given genome segment. Finally, primerID requires far less virus input than other methods of highly accurate population sequencing such as CirSeq, facilitating a wider range of experimental applications, including the analysis of samples collected from infected animals or human subjects [The primerID approach offers several advantages over conventional deep sequencing protocols.  (~0.01%). Second,subjects ,42.Altogether, we reveal a surprising new role for glycosylation in facilitating IAV immune evasion by compensating for the fitness costs of antigenic escape mutations. These results broaden our understanding of the critical function of glycosylation during antigenic drift and shed light on the unique mutational plasticity of influenza HA. Further, these studies highlight the remarkable potential of highly accurate virus population sequencing and high-definition forward genetics for exploring viral evolution.We obtained chicken eggs from a commercial vendor.50 titers were determined using the Reed–Muench method.The original SV12 stock was generated as previously described , and pas50 of each virus were inoculated into 10 day old embryonated eggs in triplicate. Allantoic fluid was collected 48 hours post infection, and titers determined based on end-point dilution using MDCK cells as described above.To compare peak titers of molecular clone-derived mutants, 500 TCID50/cell. After 1 hour, virus supernatant was removed and replaced with Gibco MEM supplemented with 7.5% fetal bovine serum, after washing cells with PBS containing CaCl2 and MgCl2. Six hours post infection, cells were washed twice with PBS, and then exposed to lysis buffer containing 1% sodium dodecyl sulfate, 50mM Tris-HCl pH 7.5, 10mM dithiothreitol (DTT), 15U/mL DNase1, and mini-complete protease inhibitor. Lysate was then digested either with EndoH or PNGase F or left untreated. Control or digested lysates were then immunoblotted using the mouse HA2 chain specific mAb RA5-22[MCDK cells were infected with rSV12-HA and each molecular clone-derived mutant at an MOI of 5 TCIDAb RA5-22.4 AOS ID/egg at 35°C. At 16 hours post infection, infected allantoic fluid was harvested, clarified and titrated using the AOS method for the subsequent passage [Recombinant PR8 and SEQ12HA viruses were serially passaged three times in 10-day old embryonated chicken eggs (Charles River) at a titer of 10 passage .5 copies of cDNA templates, a universal forward primer (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACAC-3’) and amplicon-specific reverse primers: PR8_SV12 Amp 1R 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNATGGGAGCATGCTGCCGTTA-3’, PR8_SV12 Amp 2R 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNGGGAGACTGCTGTTTATAGC-3’, PR8_SV12 Amp 3R 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNCAGAGTCCTTTCATTTTCCA-3’, PR8_SV12 Amp 4R 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNATATCTCTGAAATTCTAATC-3’.In experiments using recombinant viruses, hemagglutinin was sequenced using four overlapping amplicons spanning the entire coding sequence. Viral RNA was extracted from allantoic fluid using the QiaAmp Viral RNA Mini Kit. Viral RNA was reverse transcribed with Accuscript Hi-Fi Reverse Transcriptase (Agilent) with the following primers bearing random 12-mer barcodes (All Ns hand-mixed to achieve ~25:25:25:25 ratio): PR8_SV12 Amp 1F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNCAGGAAAATAAAAACAACCAAA ATG-3’, PR8_SV12 Amp 2F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNCACCAAAGAAAGCTCATGGCCC-3’, PR8_SV12 Amp 3F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNCAACACGAAGTGTCAAACACCC -3’, PR8_SV12 Amp 4F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNCAGGATTTCTGGACATTTGGAC -3’. The reverse transcription reactions were purified using the PureLink PCR Purification kit (Invitrogen) using the high-molecular-weight cutoff buffer. First-round PCR was performed using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with an estimated 10Sample indices were finally added using KAPA HiFi HotStart PCR Kit, 2.5μL of the first round PCR products and indexed primers (5’- CAAGCAGAAGACGGCATACGAGAT(8-mer sample ID)GTGACTGGAGTTCAGACGTGTG-3’). Amplicons were gel purified using the Qiagen Gel Extraction Kit and quantified on a Nanodrop 1000. Amplicons were pooled into a single library, quantified with the KAPA Library Quantification Kit, and sequenced using the MiSeq Reagent Kit V3 with 325x325 paired-end cycles. The raw sequencing data is available at the NCBI Short Read Archive under accession numbers SRR5428773-SRR5428801.The primerID library preparation methods used for the original SV12 stock  differedhttp://hannonlab.cshl.edu/fastx_toolkit/). Overlapping paired-end reads were merged into a single read using PANDAseq (v. 2.9)[http://broadinstitute.github.io/picard/), and retains only the reads that have the most common mapped start and stop coordinates using BEDTools (v. 2.19.1)[For the sequence analysis of passaging experiments using the recombinant PR8 and SV12 viruses, an optimized analysis pipeline was employed. In this pipeline, the first 4 bases of R2 reads were trimmed using fastx_trimmer from the FASTX Toolkit . Merged  (v. 2.9) with opt (v. 2.9) with opt. 2.19.1). Reads w. 2.19.1) in orderSequence analysis of the original SV12 stock library , generatTo determine linkage between high frequency variants (MAF > 0.5%), we performed a Fisher’s exact test for every pair of variants within each replicate and we derived a combined p-value from these individual replicate p-values using Fisher’s method.http://github.com/niaid/primer-id-progs.All software used for the optimized primerID analysis is available on Github: Libraries for the parental and passaged viruses were constructed as previously described. Briefly, full-length genomes were amplified from purified viral RNA using a multisegment RT-PCR protocol. IlluminWe used Streptavidin Dip and Read Biosensors  to measure binding of viruses to sialic acid. Graded amounts of biotinylated 3’-Sialyl lactosamine PAA-biotin aka 3’SLN (GlykoTech) were captured on an avidin coated biosensor. Virus binding experiments were performed by an Octet QKe biolayer interferometer (ForteBio). All of the viruses were diluted in PBS, pH = 7.4, 0.01% BSA, 0.002% Tween-20, (kinetic buffer) for binding to 6’SLN to 1pM in presence of 10μM Oseltamivir  and Zanamivir  to block viral NA activity. Binding of virus was measured at 30°C for 10 min followed by a 10 min dissociation measurement.S1 Fig(A and B) and all common variants in three replicates for passage 3 wild-type and sequential 12 viruses (C and D). Red line indicates the expected 1:1 ratio.HA libraries were prepared for both Nextera and amplicon sequencing, and analyzed with ViVan and PrimerID pipelines, respectively. Variant frequencies were compared between the final outputs of both methods for parental wild-type and sequential 12 viruses (TIFF)Click here for additional data file.S2 FigA, B) Combined minor amino acid variant frequency is shown for each position of HA amplicon 2 region in SV12 parental sample for unmerged reads (before applying PrimerID), PrimerID consensus reads, and Nextera shotgun sequencing, evaluated by ViVan. (B) is a zoomed-in view of (A).  Fold enrichment of variant frequency over median variant frequency for the amplicon region, shown for the same three methods in (A) and (B). (D) is a zoomed-in view of (C).((TIF)Click here for additional data file.S3 FigImmunoblot of purified virions as indicated electrophoresed under non-reducing conditions using the HA2 specific mAb RA5-22.(TIFF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIFF)Click here for additional data file.S3 TableVariant amino acids were evaluated pairwise to determine the frequency at which any two substitutions were observed in the same primerID consensus read. The combined counts across the two biological replicates that were sequenced are listed for each pairwise amino acid variant combination.(TIFF)Click here for additional data file.S4 TableEach primerID consensus read was assessed for its inferred amino acid identity at positions 133, 136, 144, 145, and 225 . The number of primerID consesus reads that contained the indicated amino acid identities at these positions is listed. Variant amino acids are highlighted in red, and combinations that contain two variant amino acids are in bold face. This table only displays amino acid variant combinations that were observed in at least two primerID concensus reads across all populations examined.(TIFF)Click here for additional data file.S5 TableCombined p-values from three replicate populations as determined by Fisher’s method.(TIFF)Click here for additional data file.


Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Chronic hepatitis B virus (HBV) infection is a major cause for HCC. Hepatitis B virus X (HBx), one of four proteins encoded by HBV genome, plays a vital role in the pathogenesis of HBV-induced HCC. However, the molecular mechanisms of HBx-triggered HCC remain largely undetermined. Here we revealed that the expression of Brain-expressed X-linked 2 (BEX2) and Osteopontin (OPN) were elevated in liver tissues of HBV transgenic mice and human HCC specimens. Moreover, a positive correlation between BEX2 and OPN was exhibited in samples from HCC patients with HBV infection. The protein levels of BEX2 and OPN were both higher in HBV-positive HCC specimens compared to that of HBV-negative HCC specimens. HBx potentiated OPN expression through up-regulation of BEX2. Importantly, the depletion of BEX2 suppressed tumorigenic potential of HCC cells with highly expressed HBx. We demonstrated the important role of BEX2 in HCC pathogenesis, and BEX2 may be a novel therapeutic target for HCC patients with HBV infection. The newly identified HBx/BEX2/OPN signaling cassette is implicated in the pathogenesis of HBV-induced HCC. Hepatocellular carcinoma (HCC) is one of the major malignancies and is the third leading cause of cancer-related death in the modern world , 2. ChroHBV genome encodes four proteins, including the polymerase (P), core protein (C), envelope protein (S), and X protein (HBx) , 11. So Brain-expressed X-linked 2 (BEX2) is one of BEX family members and plays an important function in the development of nervous system . Recentlin vitro and in vivo. Therefore, this newly discovered mechanism contributed to unveil the molecular genesis of HCC. Targeting BEX2 may be a promising strategy for treatment of HBV-induced HCC.In this study, we found the elevated expression of BEX2 and OPN in liver tissues of HBV transgenic mice and human HCC specimens, and there was a potential positive correlation between BEX2 and OPN in HCC. Moreover, HBx up-regulation of OPN was mediated by BEX2. Importantly, BEX2 was critical for the growth of HCC cells with highly expressed HBx HBV transgenic mouse model was used to explore the potential molecular events in the development of HBV-induced HCC. In line with the previous study, liver tumors were observed in HBV transgenic mice at the age of 14 months Figure . α-fetopTo extend our findings to clinical relevance, the expression of BEX2 and OPN was checked in 30 pairs of specimens from HCC patients. We found that BEX2 and OPN were elevated in 70% (21/30) and 73% (22/30) HCC specimens in comparison with their adjacent normal tissues, respectively Figure . FurtherTo investigate whether there is a putative regulatory relationship among HBx, BEX2, and OPN, firstly HBx was overexpressed in HepG2 cells. Ectopic expression of HBx dramatically augmented the protein levels of BEX2 and OPN Figure . qRT-PCRTo investigate the role of BEX2 in HBV-induced HCC, we silenced BEX2 in HCC cells and examined their colony formation. Knockdown of BEX2 significantly inhibited the colony formation of MHCC97H cells Figure . Likewisin vivo role of BEX2 in the oncogenesis of HCC cells, the tumorigenic capacity of MHCC97H cells harboring shRNA against negative control or BEX2 was evaluated in a nude mice model. Depletion of BEX2 significantly retarded the tumorigenicity of MHCC97H cells, as indicated by tumor initiation, survival, and tumor volume . Lipofectamine 2000 and 4-12% Bis-Tris NuPAGE gels were from Invitrogen . Antibodies against OPN, BEX2, HBx, Ki-67, and CD34 were from Abcam . Antibody against cleaved-caspase 3 was from Cell Signaling Technology . Antibody against β-actin and all the secondary antibodies were from Santa Cruz Technology . Antibodies against cyclin D1 and GAPDH were from Abclonal .2 at 37°C.HepG2 cells were from American Type Culture Collection . MHCC97H cells were from Liver Cancer Institute, Zhongshan Hospital, Fudan University, China. HepG2.2.15cells were kindly provided by Wei Wang . Cells were cultured in Dulbecco modified Eagle’s medium (DMEM) with 10% FBS and 1% penicillin/streptomycin in 5% COA total of 6,000 cells were seeded in 100 mm dishes and cultured for 10 or 14 days, and then fixed with methanol for 15 min and stained with 0.1% crystal violet. Cell colonies stained were photographed and counted using photoshop CS6 software.The shRNA sequence targeting human BEX2 was cloned into pLL3.7 vector through Hpa I and Xho I sites. The shRNA sequences were as follows:BEX2 forward: 5’-TGGACATAATGCATAGGCTTTTCAAGAGAAAGCCTATGCATTATGTCCTTTTTTC-3’, BEX2 reverse: 5’-TCGAGAAAAAAGGACATAATGCATAGGCTTTCTCTTGAAAAGCCTATGCATTATGTCCA-3’. 293T cells were cotransfected with pLL3.7-shRNA and the packaging vectors . Forty-eight hours later, supernatants were collected and filtered with a 0.45 μm filter for infection of MHCC97H cells. The infection rate was assessed according to the expression of green fluorescent protein after incubation with virus for 48 h.All siRNA oligonucleotides were synthesized from GenePharma . Cells were seeded in 6-well plates and transfected with indicated siRNAs according to the manufacturer’s protocol. The siRNA sequences were as follows: Negative Control (NC): 5’-UUCUCCGAACGUGUCACGUdTdT-3’; HBx: 5’-CCGACCUUGAGGCAUACUUdTdT-3’.Cells were washed with PBS and harvested with lysis buffer , and 10% glycerol), then boiled for 10 min and centrifuged at 13,000 rpm for 5 min. The levels of the indicated proteins were detected by western blotting described previously . The relTotal RNA was extracted from cells with TRIzol reagent (Invitrogen). One microgramRNA was subjected to reverse transcription reaction using ReverTra Ace® qPCR RT Master Mix . Amplification was performed for 40 cycles using cDNA as the template with TransStart Green qPCR SuperMix  in a quantitative real time PCR reaction. TATA-binding protein (TBP) serves as an internal control. The primer sequences were as follows: OPN, forward: 5’-GAAGTTTCGCAGACCTGACAT-3’, and reverse: 5’-GTATGCACCATTCAACTCCTCG-3’; BEX2, forward: 5’-AAAGAGGAACGAGCGTTAAACA-3’, and reverse: 5’-TCACTAACATTCAAAGGTAGGGC-3’; TBP, forward: 5’-GAGCCAAGAGTGAAGAACAGTC-3’, and reverse: 5’-GCTCCCCACCATATTCTGAATCT-3’.HBV transgenic mice (C57BL/6J-Tg (Alb1HBV) 44Bri/J) contain HBV genome S, pre-S, and X domains under the mouse albumin promoter. The mice spontaneously developed HCC at the age of 14 months . The livSerum collected from HBV transgenic and wide-type mice were subjected to biochemical analysis. The level of AFP was measured with Fetoprotein assay kit . The levels of ALT and AST were examined in the department of laboratory medicine of Peking Union Medical College Hospital. The level of OPN was examined using Quantikine Mouse/Rat OPN ELISA kits  according to the manufacturer’s protocol .Human HCC specimens were obtained from patients who underwent surgery at Peking Union Medical College Hospital. Tumor tissues were extracted with lysis buffer and then subjected to immunoblotting. All the patients provided written informed consent. All the procedures were performed under the permission of the Peking Union Medical College Hospital Ethics Board.5 MHCC97H cells expressing shBEX2 or scramble shRNA in 100 μl of DMEM were subcutaneously inoculated into the right posterior back region of nude mice. The subcutaneous tumor model was established as described previously [Immunodeficient nude mice  were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. Eight male mice were in each cohort. A total of 4×10eviously .Immunohistochemical staining was performed as described previously . In brieP<0.05.The Kaplan-Meier log-rank test was used for the analysis of tumor development and mice survival. The two-tailed Student’s t-test was used for the analysis of differences between two groups in GraphPad Prism software. Data represents the mean ± SEM of triplicate samples. Statistical significance was defined as