While vaccination is considered the most effective means to prevent influenza infection, its seasonal effectiveness varies, depending on the circulating influenza strains. Here, we characterized the circulation of influenza strains in October-2018 and March-2019 around the world. For this, we used nasopharyngeal samples collected from outpatient and hospitalized patients in Israel and data reported in ECDC, CDC, and WHO databases. Influenza A(H3N2) was dominant in Israel, while in Europe, Asia, and USA, A(H1N1)pdm09 virus circulated first, and then the A(H3N2) virus also appeared. Phylogenetic analysis indicated that A(H3N2) viruses circulating in Israel belonged to clade-3C.3a, while in Europe, Asia, and USA, A(H3N2) viruses belonged to subclade-3C.2a1, but were later replaced by clade-3C.3a viruses in USA. The vaccine A(H3N2) components of that year, A/Singapore/INFIMH-16-0019/2016-(H3N2)-like-viruses, belonged to clade-3C.2a1. The circulation of different influenza subtypes and clades of A(H3N2) viruses in a single season highlights the need for universal influenza vaccines. Influenza viruses cause contagious respiratory disease  and are Vaccination is considered the most effective way of preventing influenza infection ,13,14. TSince 2009, A(H3N2) viruses have undergone significant genetic drift (new clades and subclades), with some associated antigenic drift, that has led to multiple changes in the recommended A(H3N2) vaccine virus over the past 12 years . In receThe aim of this study was to review what influenza strains circulated around the world in 2018–19 and to investigate whether they resembled those used in the vaccine. We review influenza circulation in Israel, Europe, Asia, and USA over the 2018–2019 winter, with focus on the unusual pattern observed in Israel. A(H3N2) viruses of clade-3C.3a predominated in Israel from the start of the season, while the same season in Europe, Asia, and USA was biphasic, with A(H1N1) pdm09 viruses dominating for most of the season, with co-circulation of subclade-3C.2a1 A(H3N2) viruses, followed by a second phase, which was dominated by clade-3C.3a A(H3N2) viruses. Consequently, most of the A(H3N2) viruses in circulation differed from the subclade-3C.2a1, A/Singapore/INFIMH-16-0019/2016-(H3N2)-like, vaccine viruses.Respiratory samples from hospitalized and non-hospitalized patients with clinical symptoms of influenza-like-illness (ILI) were analyzed for the presence of respiratory viruses as part of routine testing performed in the Sheba Medical Centre (SMC), Israel. The SMC institutional review board (IRB) approved this research (Helsinki Number 1967-15-SMC). Informed consent was not required for this study.n = 1487) were collected between October 2018 and March 2019 in outpatient clinics located in different geographic parts of Israel as part of annul surveillance conducted by the Israel Influenza Surveillance Network (IISN). In addition, samples were collected from all hospitalized patients at the SMC (n = 7310) that suffered from ILI in 2018–19.Nasopharyngeal samples of patients presenting with ILI were collected into ∑-Virocult  virus transport medium. Samples  was extracted from all samples using MagNA PURE 96 , following the manufacturer’s instructions. All samples were then tested for the presence of influenza viruses (types A and B) and respiratory syncytial virus (RSV), in a single real-time reverse transcriptase polymerase chain reaction (RT-PCR) performed using Ambion Ag-Path master mix  and TaqMan Chemistry (qRT-PCR), on an ABI 7500 platform, as described previously .® 5.0 program  was used to prepare FASTA files for further analysis.Influenza A(H3N2) HA gene-specific RT-PCR primers were used to amplify 1701 base-pair fragments, as described in WHO protocols . The amphttp://tree.bio.ed.ac.uk/software/figtree/ (accessed on 30 March 2020)).The evolutionary relationships for the influenza A(H3N2) HA sequences were inferred using the general time-reversible model, with proportion of invariable sites and gamma plus invariant sites-distributed rate heterogeneity (GTR+G+I), chosen using JModelTest . A BayesTM database of the Global Initiative on Sharing All Influenza Data (GISAID) (www.gisaid.org (accessed on 30 March 2020)). GISAID sequences used in this manuscript are listed in A random selection of 259 influenza A(H3N2) HA gene sequences from Israeli, European, Asian, and USA samples for the years 2009–2019 used in this analysis were downloaded from the EpiFluhttps://www.who.int/influenza/gisrs_laboratory/flunet/en/ (accessed on 30 March 2020)). The data were deposited by National Influenza Centers (NICs) of the GISRS and other national influenza reference laboratories collaborating actively with GISRS.Data for 2018–19 weekly surveillance in the WHO European Region were obtained from The European Surveillance System (TESSy) database which is operaed by the European Center for Disease Control (ECDC): ).Data from seasonal surveillance of influenza in Europe, Asia, and USA were collected from open-access databases. Data for 2018–19 weekly surveillance results from Asia were downloaded from WHO FluNet ), which collects data from both the U.S. WHO Collaborating Laboratories and the National Respiratory and Enteric Virus Surveillance System (NREVSS).For USA data were downloaded for2018–19 weekly surveillance from CDC-FluView  HA gene sequences that encoded full-length HA glycoprotein for viruses collected between October 2018 and March 2019, deposited in the EpiFlumeetings .p < 0.05 was considered statistically statistically significant.The Pearson chi-squared test was applied to evaluate the differences in positive percent between the compared groups—influenza infections in hospitalized and non-hospitalized patients, as described in p < 0.0001). Influenza type B infection was infrequent (<1%)  and hospitalized patients in SMC was mainly caused by A(H3N2) viruses . Influenza A(H1N1) pdm09 viruses were also detected in both patient populations, but at a significantly higher level in hospitalized patients  (nt (<1%) .To examine the compatibility of circulating strains and vaccine strains, a BEAST phylogenic analysis of representative Israeli, European, Asian, and USA samples from the 2018–19 winter, together with samples from the preceding 10 years, and the corresponding vaccine strains was performed . The anan = 597) revealed co-circulation of A(H1N1) pdm09 and A(H3N2) virus subtypes each week, with a clear A(H3N2) dominance in almost every week  and Asia  found A(H1N1)pdm09 dominance for most of the season. However, in some weeks, there was no clear dominance, and towards the end of the season, A(H3N2) dominated  in the USA differed significantly from those seen in both Israel and Europe, with a clear A(H1N1)pdm09 dominance early in the season and a gradual increase in A(H3N2) virus circulation from week 2/2019  and A(H1N1) pdm09, with no clear dominance in many countries  . In addiIn recent years, differences in influenza virus circulation patterns have been reported, including summer outbreaks ,26 and cIn Israel, a comparison of outpatient  versus hospitalized patient influenza infections revealed that while subtype A(H3N2) was dominant in both patient populations, a higher percentage of hospitalized patient samples carried subtype A(H1N1) pdm09. Previous reports showed that influenza A(H1N1pdm09) infection is associated with more severe clinical features and higher intensive care unit admission rate, as compared to subtype A(H3N2) infection ,29, whicAs it is located in the northern hemisphere, Israeli influenza detections during winter seasons usually align with those seen in Europe, Asia, and USA  However,BEAST phylogenetic analysis of Israeli samples from 2009-2019 showed that the majority of 2018–19 A(H3N2) detections belonged to clade-3C.3a, while the 2018–19 A/Singapore/INFIMH-16-0019/2016-like vaccine virus belonged to subclade-3C.2a1; few subclade-3C.2a1 viruses were detected among Israeli patients with ILI symptoms in 2018–19. Such disparity between circulating viruses and the vaccine strain could have resulted in low immunity of populations against clade-3C.3a viruses, allowing significant spread of clade-3C.3a viruses in Israel, Asia, USA, and some countries in Europe, leading to reduced vaccine effectiveness (VE). Indeed, in Israel, we found a very low VE against A(H3N2) influenza virus in the winter of 2018–19 among infants, children, and young adults though, among adults aged ≥45 years, we found higher VE which could be due to previous exposure to clade-3C.3a viruses a few years earlier . SimilarTM data for northern hemisphere influenza circulation during this period revealed that about 8% of the positive influenza A(H3N2) viruses belonged to clade-3C.3a. The long “post-military service” journeys, mainly to South America, Australia, and New Zealand, taken by many Israeli veterans, can reasonably explain a bulk importation of clade-3C.3a viruses into Israel during the summer season of 2018.The supposed clade-3C.3a summer circulation in Israel is supported by its observed circulation in the northern hemisphere during March–September 2018. Analysis of GISAID EpifluRegarding genetic drift, from 2014 until the winter of 2018–19, a single clade-3C.3a A/Switzerland/9715293/2013-like vaccine virus had been used in 2015–16 . Over suIn the 2018–2019 season in Europe, Asia, and USA, A(H1N1) pdm09 viruses dominated early in the season but were displaced by A(H3N2) viruses later. Around week 8/2019 there was a peak in A(H3N2) clade-3C.3a infections in Europe, Asia, and USA, suggesting that A(H3N2) clade-3C.3a viruses effectively out-competed A(H1N1) pdm09 viruses. Specific clades/subclades of the four seasonal influenza viruses, i.e., two type A subtypes and two type B lineages, commonly co-circulate in winter outbreaks/epidemics but with one (or more) subtype/lineage predominating in different hemispheres or sometimes in both hemispheres . The relIt is well known that vaccination against influenza is currently the most effective way to prevent influenza infection and spread. Despite differences in influenza virus circulation patterns between Israel, Europe, Asia, and USA, there remain common global public health challenges crossing boundaries and affecting each continent individually and together. The unusual 2018–19 winter season with co-circulation of viruses in different A(H3N2) genetic clades/subclades, one of which (clade-3C.3a) contained viruses that were antigenically different from the vaccine virus , reinforIn conclusion we show in this manuscript that in Israel during the 2018–19 influenza season the circulating influenza A(H3N2) viruses belonged to clade-3C.3a, while in Europe, Asia and USA they belonged to subclade-3C.2a1, but were later replaced by clade-3C.3a viruses in USA. In contrast, the vaccine A(H3N2) components components for that season, A/Singapore/INFIMH-16-0019/2016-(H3N2)-like-viruses, belonged to subclade-3C.2a1. The circulation of different influenza subtypes and clades of A(H3N2) viruses in a single season highlights the need for universal influenza vaccines.


In contrast, T cells from WT mice exhibited the CD44low/CD62Lhigh phenotype characteristic of naïve T cells. The onset of autoimmunity and autoinflammation in TRAF6[L74H] mice (two weeks) was much faster than in mice with a Treg-specific knockout of TRAF6 or lacking TRAF6 expression in all T cells (2–3 months) and we discuss whether this may be caused by secondary inflammation of other tissues. The distinct phenotypes of mice lacking TRAF6 expression in all cells appears to be explained by their inability to signal via TNF Receptor Superfamily members, which does not seem to be impaired significantly in TRAF6[L74H] mice.The expression of TNF-Receptor Associated Factor 6 (TRAF6) is essential for many physiological processes. Here we studied the phenotype of TRAF6[L74H] knock-in mice which are devoid of TRAF6 E3 ligase activity in every cell of the body, but express normal levels of the TRAF6 protein. Remarkably, TRAF6[L74H] mice have none of the phenotypes seen in TRAF6 KO mice. Instead TRAF6[L74H] mice display an entirely different phenotype, exhibiting autoimmunity, and severe inflammation of the skin and modest inflammation of the liver and lungs. Similar to mice with a Treg-specific knockout of TRAF6, or mice devoid of TRAF6 in all T cells, the CD4 The expression of TNF-Receptor (TNFR) Associated Factor 6 (TRAF6) is essential for many physiological processes. TRAF6 knock-out (KO) mice are born at sub-Mendelian ratios, most TRAF6 KO embryos dying between embryonic day 14 and birth, for reasons that include defects in neural tube closure . They al+CD8a- splenic dendritic cells  mutant) in which the E3 ligase activity of TRAF6 was inactivated, but not its expression, so that all the other functional domains of the protein were intact, including the C-terminal TRAF domain, which interacts with Pro-Xaa-Glu motifs (where Xaa is any amino acid) present in RANK, other TNFR family members, such as CD40, or proteins that interact with the IL-1R and Toll-Like Receptor signaling complexes, such as IRAK1 and IRAK2 . The ligin vivo raised the question of how many of the essential roles of TRAF6 require its catalytic E3 ligase activity. Here, we present a more detailed analysis of the phenotype of the TRAF6[L74H] mice. Remarkably, we found that these mice do not have any of the phenotypes displayed by the TRAF6 KO mice, but instead develop an equally severe but entirely different phenotype.The finding that the E3 ligase activity of TRAF6 was not essential for IL-1R, TLR or RANK signalling in some cells and The generation of TRAF6[L74H] knock-in mice has been described . TRAF6 mice, we observed increased numbers of splenic Tfh cells, defined by the expression of the surface markers PD1 and CXCR5  cell activation requires the activation of CD4nd CXCR5 , S4B FigThe inducible T cell co-stimulator (ICOS) is another defining marker of Tfh cells, which is induced during T cell activation to maintain the Tfh cell phenotype , 24. ICO+ helper T2 (Th2) cells. We therefore measured the serum levels of a number of cytokines in 16-day mice that are produced by Th2 cells, such as IL-4, IL-5 and IL-13 and the anti-inflammatory cytokine IL-10. Consistent with a Th2 response, there were elevated levels of both IL-5 and IL-10 in the serum of TRAF6[L74H] mice  and their developmental precursors . The mTE+ CD4 and CD8 double negative, double positive and single positive (CD4+ or CD8+) thymocytes  to increase or decrease, so that the phenotypic changes observed may be indirect. Moreover, in the complete absence of a protein, its functions may be replaced by another protein(s), which may obscure, exacerbate or suppress the phenotype, and lead to erroneous conclusions being reached about the function of the enzymatic activity in vivo. For these reasons, the generation of knock-in mice in which the catalytic function of an enzyme is ablated by a single amino acid substitution can provide more definitive information about the functions of the catalytic activity in vivo.TRAF6 KO mice lacking TRAF6 in every cell of the body have been produced in several laboratories –6 and stMost, if not all, of the phenotypic differences between TRAF6 KO mice and wild type mice appear to be explained by the absence of TNF receptor superfamily (TNFRSF) signalling in TRAF6 KO mice, which is either not impaired, or only partially impaired, in TRAF6[L74H] mice. For example, the TNFRSF member RANK is known to be required for the development of lymph nodes , in addiAlthough the TRAF6[L74H] mice did not develop the phenotypes displayed by mice with a knock-out of TRAF6 in every cell of the body, they instead developed autoimmunity and multi-organ inflammation within 16 days of birth, phenotypes that have not been reported in TRAF6 KO mice that survive to this age. The reason why mice lacking TRAF6 expression in every cell do not develop autoimmunity and autoinflammation may be explained by their lack of lymph nodes and GCB cells, and the impaired maturation and activation of their dendritic cells, which appear to be consequences of the absence of TNFRSF signalling in these mice.The phenotype of TRAF6[L74H] mice resembles quite closely the phenotype of mice that do not express TRAF6 in T regulatory cells (Tregs). It is also similar to the phenotype of mice with a specific KO of TRAF6 in all T cells and in which the Tregs are non-functional in vivo , 33. Lik+ Tregs in the spleen and lymph nodes of mice with a Treg-specific KO of TRAF6 was increased 2 to 3-fold, even though they are non-functional [+ Tregs in TRAF6[L74H] mice compared to WT mice  Splenocytes isolated from 16-day old WT and TRAF6[L74H] were stained with DAPI, anti-CD19, anti-TCRβ, anti-CD4, anti-CD8, anti-CD44 and anti-CD62L antibodies. Cells were gated based on FSC-A and SSC-A, doublets were excluded and DAPI- live cells were further analyzed. (A) Representative flow cytometry plots showing the expression of CD19 and TCRβ in DAPI- live cells and the proportion of CD8 and CD4 T cells within the TCRβ+ cells. (B) Total number of CD8 T cells and CD4 T cells in the spleens of WT (n = 8) and TRAF6[L74H] (n = 9) mice. Symbols represent individual biological replicates. Significance between the two genotypes was calculated using the student’s t-test; n.s. indicates that differences were not significant. (C) As in A, except representative flow cytometry plots shows the percentage of GR-1hi CD11b+ (neutrophils) and GR-1lo-med CD11b+ (myeloid cells). (D) As in A, except that representative flow cytometry plots show the percentage of Ter119+ cells (erythrocytes). Individual values, descriptive statistics and results from the statistical analysis are provided in (TIF)Click here for additional data file.S2 Fig Representative images of skin sections from the ears stained with an anti-p21 antibody from WT (A) and TRAF6[L74H] (B) mice. (C) Bar graphs showing semi-quantitative scores of the ear epidermis layer. (D) Image of an ear skin section from one WT mouse and one TRAF6[L74H] mouse stained with TUNEL to detect dead cells (green) and with DAPI to detect nuclei (blue). (E) Quantitation of the percentage TUNEL positive cells relative to the total number of DAPI positive cells. Circles represent individual biological replicates. The circles highlighted in red in E correspond to the WT and TRAF6[L74H] mice shown in D. Significance between the two genotypes was calculated using the Mann-Whitney Test. * denotes p<0.05. Individual values, descriptive statistics and results from the statistical analysis are provided in (TIF)Click here for additional data file.S3 Fig Representative immunohistochemistry image of liver sections processed for staining with anti-PAX5 antibody from WT (A) and TRAF6[L74H] (B) mice.  As in A, B except that lungs sections were processed.(TIF)Click here for additional data file.S4 Fig(A) Splenocytes isolated from 16-day old WT and TRAF6[L74H] mice were stained with anti-TCRβ, anti-CD4, anti-CD25 and anti-FoxP3 antibodies. The representative flow cytometry plots show the percentage of CD25+FoxP3+ Treg cells from the TCRβ+CD4+ population.  As in (A), except that splenic cells were stained with anti-TCRβ, anti-CD4, anti-PD-1, anti-CXCR5, anti-ICOS and anti-Bcl6 antibodies. (B) Plots show the % of PD-1+CXCR5+ TFH cells from the TCRβ+CD4+ population. (C) Representative histograms showing the expression of ICOS and Bcl6 in the TCRβ+CD4+ PD-1+CXCR5+ population in WT and TRAF6[L74H] mice.(TIF)Click here for additional data file.S5 FigA) Thymus weight in 16-day old WT and TRAF6[L74H] knock in mice. (B) Total number of cells in the thymus of WT and TRAF6[L74H] knock in mice. (C) Thymic cells were stained with DAPI, anti-TCRβ, anti-CD4 and anti-CD8 antibodies. Cells were gated based on FSC-A and SSC-A, doublets were excluded and DAPI- live cells were further analyzed. The representative flow cytometry plots show the percentage of TCRβlow and TCRβhigh cells from all live thymocytes. (D) As in C, except that total numbers of TCRβlow and TCRβhigh cells are shown. (E) Representative plots showing the expression of CD4 and CD8 in the DAPI-TCRβhigh population. (F) Plots showing the total numbers of double negative (DN) DAPI-TCRβhighCD4-CD8-, double positive (DP) DAPI-TCRβhighCD4+CD8+, CD8 single positive (SP) DAPI-TCRβhighCD4-CD8+ and CD4 SP DAPI-TCRβhighCD4+CD8- T cells in the thymus of WT and TRAF6 [L74H] mice. In A, B, D and F symbols represent individual biological replicates. Significance between the two genotypes was calculated using the Student T-test or Mann-Whitney test; n.s., not significantly different. Individual values, descriptive statistics and results from the statistical analysis are provided in ((TIF)Click here for additional data file.S6 Fig Representative image showing haematoxylin and eosin (H & E)-stained thymic sections from WT (A) and TRAF6[L74H] (B) mice.  As in A, B except that thymic sections were processed for immunohistochemistry staining using anti-CD3 antibody.  As in C, D except that anti-IBA-1 antibody was used.(TIF)Click here for additional data file.S7 FigA) Body weights of 18–21 week old WT (n = 4) and TRAF6[L74H] heterozygous (n = 3) male mice (B) Spleen weight (left panel) and representative images of spleen size (right panel) of 18–21 week old WT (n = 4) and TRAF6[L74H] heterozygous (n = 4) mice, scale bar = 1 cm. (C) Representative images of axillary and brachial lymph nodes (left panel) and inguinal lymph nodes (right panel) of one WT and one TRAF6[L74H] heterozygous mouse. (D) Splenocyte numbers of 18–21 week WT (n = 4) and TRAF6[L74H] heterozygous (n = 4) mice. (E-Q) As in D, except that splenic immune cell populations were analyzed by flow cytometry. Plots show total numbers of B cells (E), T cells (F), non-T/B cells (G), neutrophils (H), myeloid cells (I), Treg cells (J) and Tfh cells (K). Total numbers of CD4 T cells (L), percentage of CD44+ (M) and CD62L+ (N) from all CD4 T cells and total numbers of CD8 T cells (O) and the percentage of CD44+ (P) and CD62L+ (Q) from all CD8 T cells are shown. Symbols represent individual biological replicates. Statistical significance between the two genotypes was calculated using the unpaired t-test with Welch’s correction; n.s. denotes not significant difference. Individual values, descriptive statistics and results from the statistical analysis are provided in ((TIF)Click here for additional data file.S8 FigA), IgG1 (B), IgG2b (C), IgG2a (D), IgG3 (E), IgE (F) and IgM (G) in the serum of 18–21 week WT (n = 4) and TRAF6[L74H] heterozygous (n = 4) mice. Symbols represent individual biological replicates. Statistical significance between the two genotypes was calculated using the unpaired t-test with Welch’s correction; n.s. denotes that the difference is not significant. * denotes p<0.05. Individual values, descriptive statistics and results from the statistical analysis are provided in Concentrations of IgA ((TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.S7 File(XLSX)Click here for additional data file.S8 File(XLSX)Click here for additional data file.S9 File(XLSX)Click here for additional data file.S10 File(XLSX)Click here for additional data file.S11 File(XLSX)Click here for additional data file.S12 File(XLSX)Click here for additional data file.S13 File(XLSX)Click here for additional data file.


MAC5A function resulted in stamen malformation and a reduced seed set. The exogenous application of GA considerably exacerbated the defects in mac5a null mutants, including fewer stamens and male sterility. MAC5A was predominantly expressed in pollen grains and stamens, and overexpression of MAC5A affected the expression of homeotic genes such as APETALA1 (AP1), AP2, and AGAMOUS (AG). MAC5A may interact with RABBIT EARS (RBE), a repressor of AG expression in Arabidopsis flowers. The petal defect in rbe null mutants was at least partly rescued in mac5a rbe double mutants. These findings suggest that MAC5A is a novel factor that is required for the normal development of stamens and depends on the GA signaling pathway.The development of floral organs is coordinated by an elaborate network of homeotic genes, and gibberellin (GA) signaling is involved in floral organ development; however, the underlying molecular mechanisms remain elusive. In the present study, we found that MOS4-ASSOCIATED COMPLEX 5A (MAC5A), which is a protein containing an RNA-binding motif, was involved in the development of sepals, petals, and stamens; either the loss or gain of  APETALA1 (AP1) and AP2, while that of petals in the second whorl is determined by the combined activity of class A and B genes AP3 and PISTILLATA (PI). Stamens in the third whorl are determined by the combined effects of class B genes and the class C gene AGAMOUS (AG), which is also solely responsible for the determination of carpels in the innermost whorl. The anthers, which develop in the third whorl, are suggested to be a major site of bioactive GA synthesis during flower development in several plant species ,30AG exp mutants C, suggesed shape D, similae mutant F.Arabidopsis. Both the loss and gain of the MAC5A function frequently resulted in the malformation of floral organs, especially the stamens  and negatively regulates its expression [miRNA164 expression is decreased in the mac5a mutants [rbe was at least partly rescued in the mac5a rbe double mutants  and CUC2, which encode critical transcriptional regulators involved in organ boundary specification [miRNA164 genes to impact the expression of CUC1 and CUC2, which in turn regulate the events required for sepal and petal development [miRNA172 is also reduced in mac5a mutants [miRNA172 mediates the repression of AP2, which is critical for stamen and carpel development [MAC5A and RBE work together to regulate the class A and C genes through an miRNA-dependent pathway.nd whorl ,30. RBE pression . MAC5A ipression . Indeed, mutants . We foun mutants , suggestfication ,35. RBE elopment . On the  mutants . miRNA17elopment ,37. We pArabidopsis by blocking the functions of DELLA proteins [mac5a mutants and MAC5A-overexpressing plants, the upregulated expression of GA biosynthesis and signaling genes  are present in the Arabidopsis genome. The loss of function of RGA and RGL2 is almost sufficient to compensate for floral organ deficiency in ga1-3 mutants, which indicates that both RGA and RGL2 are major repressors of GA response in this process [RGA and RGL2 were significantly increased in mac5a and ox2 plants  and rbe  with a Col-0 genetic background were obtained from the Arabidopsis Information Source . Seeds were surface-sterilized and plated on a 0.5 × Murashige und Skoog basal salt  medium containing 0.8% agar, 1% w/v sucrose, and 0.5 g/L MES (pH 5.8). After sowing, the plates were chilled at 4 °C for 72 h in dark and subsequently incubated at 22 °C under long-day photoperiods . Light intensity was 120 μmol m−2 s−1. After ten days of growth, the plantlets were planted on rock wool cubes and grown with regularly supplied 0.2 × MS solution. To determine the effect of exogenous GA and PAC treatments on floral organs, GA3 and PAC were weekly applied to 20-day-old plants by spraying them with 50 μM or 100 μM GA3  or 20 μM PAC (Sigma-Aldrich) in 0.001% Triton X-100 or Triton X-100 alone.Col-0 plants were used as the wild type. Pollen viability was assessed using Alexander cytoplasmic staining  and obseMAC5A:GUSP construct, an approximately 2-kb long promoter sequence upstream of MAC5A was cloned from Arabidopsis genomic DNA using primer pairs with HindIII and SmaI sites. The CaMV 35S promoter of the pBI121 vector  [MAC5A native promoter that was double-digested with HindIII/SmaI. MAC5A cDNA was obtained from ABRC and sequenced. Full-length MAC5A cDNA without a stop codon was cloned into a KpnI/SpeI site of the vector pBS:35S:GFP [35S:MAC5A-GFP. The MAC5A-GFP fusion gene was cloned from pBS:35S:MAC5A-GFP using primer pairs with BamHI and SacI sites and cloned into the pBI121 vector to replace the GUS gene, generating pBI121:35S:MAC5A:GFP. Thus, GFP was fused to the C-terminus of MAC5A. pBS:35S:GFP was digested by XbaI and SacI to obtain the open reading frame (ORF) of the GFP gene. EGFP fragments were inserted into the XbaI/SacI site of pBI121 to generate pBI121:35S:GFP. The primers are listed in To create the pBI121:CA, USA)  was repl:35S:GFP , generatArabidopsis Col-0 plants were transformed using Agrobacterium tumefaciens via the floral dip method [Agrobacterium strain that harbored pBI121:35S:GFP, pBI121:35S:MAC5A-GFP, or pBI121:MAC5A:GUSP.p method . We usedMAC5A:GUSP vector was cleared with chilled 90% acetone. GUS activity was assessed by incubating plant tissues in 100 mM NaPO4 (pH 7.2), 5 mM 5-bromo-4-chloro-3-indolyl-D-glucuronide, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, and 0.25 Triton X-100 at 37 °C. Samples were cleared using 70% ethanol after staining.The GUS expression analysis was conducted as previously reported . The chlTotal RNA was isolated from inflorescences using an RNeasy Plant Mini Kit , and cDNA was prepared using PrimeScript Reverse Transcriptase . Quantitative reverse-transcription PCR (qRT-PCR) was performed using a StepOne Real-Time PCR System  and SYBR Premix Ex Taq (TAKARA Bio). Actin (NM_112764) levels were used to normalize the expression patterns between samples. qRT-PCR primers used in the study are shown in MAC5A and RBE were cloned into the EcoRI/BamHI I sites of pGADT7 and pGBKT7 vectors , respectively. Bait and prey plasmids were cotransformed into yeast stain AH109 [The ORFs of in AH109 . Empty vMAC5A and RBE without the stop codon were cloned into the KpnI/SpeI site of the vector pBluescript II SK:35S:GFP (pBS:35S:GFP) [MAC5A-GFP and pBS:35S:RBE-GFP. Vectors for the BiFC assay were constructed by replacing GFP in the vector pBS:35S:GFP with the N-terminus (154 amino acids) or C-terminus (80 amino acids) of YFP, generating pBS:35S:nYFP and pBS:35S:cYFP, respectively [MAC5A and RBE without stop codons were amplified using a primer pair with the KpnI and SpeI sites and cloned into pBS:35S:cYFP and pBS:35S:nYFP, respectively. The primers are shown in Full-length cDNAs of 35S:GFP) , generatectively . The ORF


Influenza viruses continue to be a major public health threat due to the possible emergence of more virulent influenza virus strains resulting from dynamic changes in virus adaptability, consequent of functional mutations and antigenic drift in surface proteins, especially hemagglutinin (HA) and neuraminidase (NA). In this study, we describe the genetic and evolutionary characteristics of H1N1, H3N2, and influenza B strains detected in severe cases of seasonal influenza in Thailand from 2018 to 2019. We genetically characterized seven A/H1N1 isolates, seven A/H3N2 isolates, and six influenza B isolates. Five of the seven A/H1N1 viruses were found to belong to clade 6B.1 and were antigenically similar to A/Switzerland/3330/2017 (H1N1), whereas two isolates belonged to clade 6B.1A1 and clustered with A/Brisbane/02/2018 (H1N1). Interestingly, we observed additional mutations at antigenic sites  as well as a unique mutation at a receptor binding site (S200P). Three-dimensional (3D) protein structure analysis of hemagglutinin protein reveals that this unique mutation may lead to the altered binding of the HA protein to a sialic acid receptor. A/H3N2 isolates were found to belong to clade 3C.2a2 and 3C.2a1b, clustering with A/Switzerland/8060/2017 (H3N2) and A/South Australia/34/2019 (H3N2), respectively. Amino acid sequence analysis revealed 10 mutations at antigenic sites including T144A/I, T151K, Q213R, S214P, T176K, D69N, Q277R, N137K, N187K, and E78K/G. All influenza B isolates in this study belong to the Victoria lineage. Five out of six isolates belong to clade 1A3-DEL, which relate closely to B/Washington/02/2009, with one isolate lacking the three amino acid deletion on the HA segment at position K162, N163, and D164. In comparison to the B/Colorado/06/2017, which is the representative of influenza B Victoria lineage vaccine strain, these substitutions include G129D, G133R, K136E, and V180R for HA protein. Importantly, the susceptibility to oseltamivir of influenza B isolates, but not A/H1N1 and A/H3N2 isolates, were reduced as assessed by the phenotypic assay. This study demonstrates the importance of monitoring genetic variation in influenza viruses regarding how acquired mutations could be associated with an improved adaptability for efficient transmission. Seasonal influenza virus epidemics are a source of respiratory disease burden which results in substantial numbers of deaths each year . Four coThis study was approved by the Mahidol University Central Institutional Review Broad (MU-CIRB) Thailand on 10 January 2018 .Sputum samples were collected from hospitalized patients (between 18 and 65 years old) in the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, who presented signs and symptoms of a severe respiratory infection and tested positive for influenza via the QuickNaviTM Flu + RSV rapid test kit . The sputum samples were kept in universal transport medium (UTM)  per the manufacturer’s manual for further laboratory tests. Viruses from each specimen were isolated in Madin-Darby Canine Kidney (MDCK) cells . In brief, sputum samples in UTM were vigorously vortex and centrifuged at 3000 rpm for 10 min and the supernatant was collected and kept on ice. Approximately 100 µL of supernatant was added into 80 to 90% confluent monolayer of MDCK cells in 400 µL Minimum Essential Medium Eagle (MEM)  media supplemented with L-glutamine, antibiotic-antimycotic , and 1 µg/mL trypsin-tosyl phenylalanyl chloromethyl ketone (Trypsin-TCPK) . The infected MDCK monolayer was observed daily for the appearance of cytopathic effect (CPE). Cell culture containing viruses were harvested, centrifuged and hemagglutination assay was performed to determine the hemagglutinin titer of the cultured supernatants. The supernatants containing the virus was aliquoted and stored at −80 °C for further experiments. The positive cultured viruses were passaged up to three times to obtain sufficient virus titers to perform virus identification, NAI assay, and whole genome sequencing. To assess the titer of isolated viruses, hemagglutination assay was performed as previously described . The tes® Viral RNA Extraction Kit  according to the manufacturer’s instructions. One-step RT-PCR was performed to differentiate A/H1N1, A/H3N2, and influenza B based on WHO recommended protocol [https://bioedit.software.informer.com, accessed on 10 January 2020). The nucleotide sequences described in this study have been deposited into the GenBank database  in the MEGA 6 software .A fluorescence-based NA inhibition (NAI) assay was used to determine the sensitivity of influenza viruses to the viral NA inhibitor as previously described . Brieflyhttps://swissmodel.expasy.org, accessed on 15 January 2020) [https://discover.3ds.com, accessed on 15 January 2020).Three-dimensional (3D) structural model of the H1N1 protein structure was constructed and its putative interactions with sialic acid were predicted. The impact of mutations on sialic acid binding site was illustrated. The structure of influenza hemagglutinin from the A/California/07/2009 (H1N1) (PDB ID: 3UBE), A/Victoria/361/2011 (H3N2) (PDB ID: 4O51), and B/Brisbane/60/2008 (PDB ID:4FQM) were used as a template for protein modeling. The model was constructed using SWISS-MODEL (ry 2020) . Then, try 2020) . The strThe median age of influenza infected patients was 52 years old. Female accounted for 76.7% of influenza infected patients. In all diagnosed severe cases, approximately 80% of these cases had a chronic medical condition. Regarding the age distribution data for all diagnosed cases, 43% of severe cases were associated with those aged 60 or older . In the Of 30 laboratory confirmed cases, the influenza type/subtype identified comprised 9, (30%), 14 (46.7%), and 7 (23.3%) of A/H1N1, A/H3N2, and influenza B, respectively. Out of 30 sputum samples, 20 influenza virus isolates were obtained and subjected to molecular-genetic analysis. We identified 7 A/H1N1, 7 A/H3N2, and 6 B/Victoria viruses. The identification numbers, strain names, collection dates, and GenBank accession numbers of all 20 viruses detected in this study are shown in To better understand the evolutionary trend of A/H1N1 viruses in Bangkok, Thailand, during the period of 2018–2019, all gene sequences of the seven influenza A (H1N1) isolates from our study were analyzed alongside the global strains. The phylogenetic tree concatenates from all eight segments of the influenza A/H1N1 strains , as wellThe homology-based modeled HA mutated structure in comparison with A/California/07/2009 (H1N1) revealed that the observed mutation among H1N1 strains in our study are unlikely to alter the overall tertiary structure of HA A. SubstiThe phylogenetic tree from the concatenated segment from the seven influenza A/H3N2 strains , as wellAll six influenza B isolates clustered in clade 1A3-DEL of the Victoria lineage and are closely related to B/Washington/02/2009 . This clSensitivity to oseltamivir of the current influenza virus isolates were assessed by NAI assay. The results showed that all influenza A/H1N1 and A/H3N2 isolates were sensitive to the anti-neuraminidase drug, oseltamivir. All strains of these viruses showed sensitivity to oseltamivir within the normal IC50 range of 0.1–1.5 nM ,19. HoweTo better understand the evolutionary trend of the current circulating influenza viruses in Thailand, the whole genome sequences of influenza A/H1N1, A/H3N2, and influenza B isolates from our study  were anaWhole genome sequencing revealed that the H3N2 isolates belonging to genetic clades 3C.2a2 and 3C.2a1b, could be represented by the Southern Hemisphere vaccine strains A/Switzerland/8060/2017(H3N2) and the newly announced A/South Australia/34/2019 (H3N2) , respectNone of the A/H1N1 isolated viruses were sensitive to neuraminidase inhibitors. The sequence analysis of the NA gene reveals one potential NA drug-resistance substitution (V116A) but not other amino acid substitutions such as I117V, Q136K, D151A, Y155H, R156K, D198V, I222R, R224K, Q226H, E227D, E227Q, H274Y, R293K, N294S, E425G, and I436N ,29. The A few limitations of the study may have affected the accurate representation of circulating serotypes. Firstly, a small sample size may not be adequate to make definitive conclusions. Secondly, the samples were collected only in Bangkok, which may not represent the epidemiology of influenza viruses for the entire country. Thirdly, most patients in our study had an underlying health condition that increases the risk of severity. Thus, further study with a larger sample size in a healthy population is needed with sputum collected from major regions around Thailand. This would then allow a more accurate representation of the circulating strains. With regards to the severity of the cases, it is not possible to conclude which amino acid changes confer the virus’s ability to infect and replicate. For instance, amino acid changes could confer stronger immunodominance when favorable structural changes in antigenic sites occur due to changes in the molecular bonds; changes could also confer no structural change but contribute to another pathogenesis mechanism. Whole genome sequencing allows a full analysis of the virus’s genome and serves as a basis for further research to be conducted. Ultimately the severity may be a result of host factors such as age, immunocompromised state, vaccine coverage, or genetic susceptibility .Finally, with the current trend of pandemics occurring approximately every 39–40 years , whole g


Scientific Reports 10.1038/s41598-020-66690-7, published online 18 June 2020Correction to: The Acknowledgements section in this Article is incomplete.“This work was supported by National R&D (Grant Nos. NRF-2017K1A3A7A09016309 and NRF-2019R1A2C1007491) and Radiation Technology R&D (Grant No. NRF-2017M2A2A6A01016849) program through the National Research Foundation of Korea funded by the Ministry of Science and ICT. DSH acknowledges support from the U.S. Department of Commerce, the NIST Radiation Physics Division, the Director’s office of NIST, and the NIST Center for Neutron Research.”should read:“This work was supported by National R&D  and Radiation Technology R&D (Grant No. NRF-2017M2A2A6A01016849) program through the National Research Foundation of Korea funded by the Ministry of Science and ICT. DSH acknowledges support from the U.S. Department of Commerce, the NIST Radiation Physics Division, the Director’s office of NIST, and the NIST Center for Neutron Research.”


Rousettus aegyptiacus), raising the question of whether this bat species is a suitable host for IAV. Here, we studied the susceptibility, pathogenesis and transmission of avian and bat-related H9N2 viruses in this new host. In a first experiment, we oronasally inoculated six Egyptian fruit bats with an avian-related H9N2 virus (A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2)). In a second experiment, six Egyptian fruit bats were inoculated with the newly discovered bat-related H9N2 virus (A/bat/Egypt/381OP/2017 (H9N2)). While R. aegyptiacus turned out to be refractory to an infection with H9N2 avian-type, inoculation with the bat H9N2 subtype established a productive infection in all inoculated animals with a detectable seroconversion at day 21 post-infection. In conclusion, Egyptian fruit bats are most likely not susceptible to the avian H9N2 subtype, but can be infected with fruit bat-derived H9N2. H9-specific sero-reactivities in fruit bats in the field are therefore more likely the result of contact with a bat-adapted H9N2 strain.Influenza A viruses (IAV) of subtype H9N2, endemic in world-wide poultry holdings, are reported to cause spill-over infections to pigs and humans and have also contributed substantially to recent reassortment-derived pre-pandemic zoonotic viruses of concern, such as the Asian H7N9 viruses. Recently, a H9N2 bat influenza A virus was found in Egyptian fruit bats ( Low-pathogenic avian influenza viruses (LPAIV) of subtype H9N2 are globally widespread in poultry and endemic in China and Eurasian countries ,2. WhileSturnira lilium) and a South American flat-faced fruit-eating bat (Artibeus planirostris), respectively [Eidolon helvum) in Ghana [Rousettus aegyptiacus) in Egypt. Interestingly, the hemagglutinin (HA) and neuraminidase (NA) encoding segments of this particular virus were closely related to avian H9N2-viruses [The first evidence of influenza A and influenza A-like viruses originating from different bat species was found in 2012 and 2013: two novel influenza A-like viruses, provisionally designated as H17N10 and H18N11, were identified via next-generation sequencing in the feces of a little yellow-shouldered fruit bat  with either a poultry-origin H9N2 strain of clade G1 (A/layer chicken/Bangladesh/VP02-plaque/2016) or the fruit bat-derived strain A/bat/Egypt/381OP/2017 (H9N2). The latter group was cohoused with naïve contact bats (24 h post-infection) to further elucidate H9N2 bat flu virus transmission.In order to specifically investigate susceptibility, pathogenesis and virus shedding, we oronasally infected two groups of Egyptian fruit bats , was obtained from the Friedrich-Loeffler-Institut (FLI) virus repository [Rousettus aegyptiacus) from both experiments were reared at FLI and tested negative for influenza A reactive antibodies via immunofluorescence analysis (IFA) using the inoculation viruses. Six bats in each experiment were anesthetized by inhalation of 5% isoflurane in O2 and oronasally inoculated with 100 µL H9N2 (A/layer chicken/Bangladesh/VP02-plaque/2016) distributed equally into each nostril and mouth at a concentration of 104.7 TCID50 per animal   , respect7 (H9N2) . The six7 (H9N2)  were co-Organ pieces (approximately 2 × 2 × 2 mm) were transferred into 2 mL collection tubes prepared with a stainless-steel bead (diameter 5 mm) and 1 mL of DMEM supplemented with antibiotics . Homogenization was performed using a TissueLyser II instrument  for 2 min at 300 Hz. Supernatants for RNA extractions were acquired following centrifugation at 13,000 rpm for 2 min.RNA extraction of all oral and rectal swabs, as well as organ samples after euthanasia from both experiments was performed via the NucleoMag Vet kit  according to manufacturer’s instructions on a Biosprint 96 platform .RT-qPCRs of organs, oral and rectal swabs in the first experiment (avian origin H9N2) were performed as described before .In order to detect A/bat/Egypt/381OP/2017 (H9N2) viral RNA in the second experiment, a specific primer and probe system was designed . For proBased on RT-qPCR data, selected organs from bats sacrificed at 7 dpi were trimmed for paraffin-embedding: nasal atrium (non-respiratory region), nasal conchae (respiratory and olfactory region), duodenum, jejunum, colon and rectum of inoculated #5 and #6, as well as transmission #11 and #12. Tissue sections were stained with hematoxylin and eosin (HE). Immunohistochemistry for viral antigen detection using a primary antibody against the M protein of IAV (ATCC clone HB-64) was performed on consecutive slides as described earlier . The sevAnimal samples were initially diluted in medium , whereupon 200 µL was transferred into the allantoic cavity of embryonated chicken eggs (three eggs per sample) followed by incubation for five days at 37 °C. Afterwards, the eggs were placed at −80 °C for 30 min to ensure the death of the chicken embryo, whereupon the egg was opened to allow harvesting of the allantoic fluid. The fluid was centrifuged at 3500 rpm for 10 min, followed by a TRIzol LS reagent -based RNA extraction (according to manufacturer’s instructions). Existence of A/bat/Egypt/381OP/2017 (H9N2) viral RNA and thereby evidence for successful propagation of A/bat/Egypt/381OP/2017 (H9N2) was finally verified via RT-qPCR as described before.3.3 TCID50/mL was mixed with the same volume of diluted sera. Each serum was prepared in triplicate in a 96-well plate. After incubation for 2 h at 37 °C, the samples were transferred into a second 96-well plate prepared 24 h earlier containing 100 µL medium on 24 h grown MDCK II cells. Viral replication was assessed after an incubation period of 5 days  via visualization of the cytopathic effect. Validation was achieved by titration of the virus dilutions. In the second experiment, seroconversion was investigated via IFA as well. For that reason, MDCK cells were seeded in a 96-well plate and were grown for 24 h. Afterwards, cells were infected with A/bat/Egypt/381OP/2017 (H9N2) for 24 h followed by cell fixation with 4% paraformaldehyde (PFA), as well as permeabilization with 0.5% Triton-X-100 in PBS. Cells were then stained for 1 h with a dilution series from 1:10–1:256 of the appropriate animal serum, followed by three PBS-washing steps and the incubation with a secondary goat-α-bat IgG (H + L) antibody  for 1 h. Subsequently, cells were washed three times with PBS again, followed by the addition of an Alexa Fluor 488-coupled chicken-α-goat IgG (H + L) third antibody for 1 h to allow visualization under the fluorescence microscope after a final three-times PBS-wash procedure.All serum samples were heat-inactivated at 56 °C for 30 min and animal sera from both experiments were analyzed by a commercial enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against the IAV nucleoprotein (NP) according to manufacturer’s instructions . In addition, a virus neutralization assay was performed in the first experiment. In brief, 50 µL of medium containing H9N2 (A/layer chicken/Bangladesh/VP02-plaque /2016) at a concentration of 10Rousettus aegyptiacus) with 104.7 TCID50 per animal of A/layer chicken/Bangladesh/VP02-plaque /2016 (H9N2), neither clinical signs nor any significant changes in body weight or body temperature could be observed during the experiment. In addition, no viral RNA was detected in organs, oral or rectal swabs. Samples below 10 viral genome copy numbers per mL  were considered negative. At necropsy, no macroscopic lesions were evident. Analysis of sera until 21 dpi by IAV-antibody-ELISA and serum neutralization assay revealed no evidence of seroconversion. Taken together, these observations strongly suggest that this H9N2 subtype of avian origin fails to replicate in Egyptian fruit bats.Following oronasal inoculation of Egyptian fruit bats  per animal of A/bat/Egypt/381OP/2017 (H9N2) established a productive infection in these animals. At 1 dpi, one out of six oral swabs, as well as two rectal swabs from the inoculated animals, were positive for the H9N2 bat flu viral genome using RT-qPCR  viral RNA.Fecal samples of animal cage one were positive for A/bat/Egypt/381OP/2017 (H9N2) viral RNA at 4 and 7 dpi and samples from cage two were positive at 3, 4, 6 and 8 dpi, with maximum viral loads of 4 × 10Histopathology identified an oligo-focal, minimal to mild rhinitis in all animals evaluated at 7 dpi  and 6 days post-contact . Additionally, inoculated animal #6 showed a moderate transmigration of granulocytes and lymphocytes as well as intraluminal cellular debris within the nasolacrimal duct A. ContacSince it has been shown previously that A/bat/Egypt/381OP/2017 (H9N2) efficiently replicates in embryonated chicken eggs , we atteAs shown in As shown by an indirect immunofluorescence assay, all directly inoculated animals sacrificed at day 21 had seroconverted against A/bat/Egypt/381OP/2017 (H9N2), whereas all contact animals remained seronegative . InteresEidolon helvum) [Rousettus aegyptiacus). In addition, the infection dose of 104.7 TCID50 per animal used in this experimental inoculation could be another factor, potentially in combination with the application route. However, oral swabs taken from naturally infected Egyptian fruit bats were more likely to be tested positive for viral RNA than rectal swabs, highlighting that oral epithelial cells of Egyptian fruit bats are able to replicate IAV [Our study shows for the first time that oronasal inoculation using LPAIV of subtype H9N2 (A/layer chicken/Bangladesh/VP02-plaque/2016) does not result in any virus replication or specific seroconversion in an experimental setup with Egyptian fruit bats. This suggests that these fruit bats are not susceptible to an avian-origin H9N2 influenza A virus infection. This was of special interest since there was, contrastingly, a clear indication of H9-specific antibodies in African fruit bats . Therefo helvum) , this obcate IAV .Despite the fact that bat borne H9N2 showed strong genetic similarities to avian H9N2 viruses, the phylogeny revealed that in contrast to the HA and NA, all inner segments were very divergent from the recent AIV ,14. TherOverall, although inoculation of Egyptian fruit bats with A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2) did not result in a productive infection of these animals, inoculation and thereby infection of the same species with A/bat/Egypt/381OP/2017 (H9N2) was successful. First indications for a successful infection were detected by a permanent temperature monitoring of four infected and four contact animals, which only showed subtle differences between both groups, that would have been unnoticed by monitoring of the rectal temperature alone. Therefore, monitoring of the body core temperature independent of any manipulation of the animal is important for challenge experiments where only mild or even no clinical signs are to be expected in the infected animals. Although we were not able to detect viral RNA related to A/bat/Egypt/381OP/2017 (H9N2) in oral/rectal swabs and organ samples from the direct contact animals, we demonstrated mucosal epithelium degeneration via histopathologic analysis in contact animal #6 , indicatIn addition to virus isolation and propagation, we observed that all animals directly inoculated with A/bat/Egypt/381OP/2017 (H9N2) and euthanized at 21 dpi displayed seroconversion. Although we could not demonstrate threshold-exceeding seropositivity of all animals in the ELISA that were seropositive in the IFT, we nevertheless saw obvious differences in the data values of these animals compared to the naïve animals . Taken tRecently published observations concerning the replication of another bat influenza A virus, H18N11, in various animal models proved that H18N11 is being transmitted among and is able to replicate in bats, whereas replication in mice and ferrets was only weakly detectable . Thus, bThis hypothesis could thereby explain why the same species of flying foxes are susceptible to infection with A/bat/Egypt/381OP/2017 (H9N2), but not to A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2). However, due to the rapid adaptation processes of influenza viruses in general, the zoonotic potential of these viruses should not be underestimated and cannot be excluded without further experiments. Therefore, future studies will focus in more detail on the different influenza virus infections of fruit bats and the necessary adaptation processes. Our study provided first insights into the host range of different H9N2 viruses and the adaptation of bat-origin H9N2 to Egyptian fruit bats. Future studies will focus on the minimum infectious dose or the reactivity of the host following bat-origin H9N2 infection.


This study aimed to determine the prevalence and localization of complement factor C4d in renal biopsies from patients with lupus nephritis (LN), as well as its associations with the disease’s clinico-pathological features. The correlation between arteriolar C4d deposition and renal microvascular lesions (RVLs) was further analyzed.A total of 325 biopsy-proven LN patients were enrolled, and their clinico-pathological data were collected. C4d staining of renal biopsies was performed by immunohistochemistry. The associations between C4d deposition and the clinico-pathological features were further analyzed.P < 0.01). Patients with arteriolar C4d deposition were more likely to develop RVLs (91.2%) compared to those with no arteriolar C4d deposition , especially with two or more types of RVLs (P < 0.001). During the mean follow-up of 55.8 months, arteriolar C4d was related to worse renal outcomes . Multivariate Cox hazard analysis showed that co-deposition of arteriolar C4d and C3c was an independent risk factor  for predicting renal outcomes.C4d deposition was present in most (98.8%) renal specimens in our cohort. These deposits were localized in the glomeruli (98.2%), tubular basement membrane (TBM) (43.7%), arterioles (31.4%), and peritubular capillary (33.8%). Patients with TBM C4d staining had higher disease activity (measured with the Systemic Lupus Erythematous Disease Activity Index) and higher National Institutes of Health pathological activity and chronicity indices (all C4d deposition was common in renal tissues from LN patients. TBM C4d deposition was related to the disease activity, and arteriolar C4d deposition was associated with RVLs and worse renal outcomes. Systemic lupus erythematous (SLE) is an autoimmune disease with a wide variety of clinical manifestations and serological abnormalities . Lupus nComplement activation plays a key role in LN pathogenesis . The claC4d is a fragment of C4 generated during activation of the complement pathway and is regarded as a hallmark of classical or lectin complement pathway activation . Its depHerein, we assessed the prevalence and anatomic localization of C4d deposition in renal biopsy specimens from a well-defined LN cohort. The associations between C4d deposition in different kidney compartments and clinico-histopathological features and outcomes of LN were further analyzed.Clinical and renal histopathological data of 325 patients with renal biopsy proven LN diagnosed between January 2000 and June 2008 from Peking University First Hospital were collected. All the patients included fulfilled the 1997 American College of Rheumatology revised criteria for SLE . PatientDisease activity was assessed with the Systemic Lupus Erythematous Disease Activity Index (SLEDAI) , 18. AllSerum antinuclear antibodies were detected using the indirect immunofluorescence assay . Anti-double-stranded DNA (anti-dsDNA) antibodies were detected using the Crithidia luciliae indirect immunofluorescence test (EUROIMMUN). Serum C3 and C4 were determined using the rate nephelometry assay . Anti-cardiolipin antibodies (aCL) were detected using enzyme-linked immunosorbent assay (EUROIM MUN).The renal biopsy specimens were routinely examined by light microscopy, direct immunofluorescence, and electron microscopy techniques. Renal histopathology data were reviewed and reclassified according to the International Society of Nephrology and Renal Pathology Society 2003 LN classification system  by two eC4d was immunohistochemically labeled in formalin-fixed paraffin-embedded tissue (4-μm thick). Rabbit anti-human C4d polyclonal antibodies  were used as primary antibodies. The intensity of Glomerular C4d (G-C4d) was semi-quantitatively graded (0–3) , 25. TubThe fresh frozen tissue sections were stained immediately after the renal biopsy with fluorescein isothiocyanate-labeled rabbit anti-human IgG, IgA, IgM, C3c, and C1q antibodies . The results were graded from 0 to 4 according to the intensity of fluorescence.P value threshold of 0.05. Results are expressed as hazard ratios (HR) with 95% confidence intervals (CI). Statistical significance was considered at P < 0.05.Statistical software SPSS 18.0  and GraphPad-Prism 5.0  were used for statistical analyses. Quantitative data are expressed as mean ± s.d. or median with range (minimum and maximum) or interquartile range. Spearman’s rank-order correlation tests were performed to compare various lesions. Differences in semiquantitative data were tested with the Kruskal–Wallis test or Mann–Whitney U-tests. Bonferroni’s correction was used for multiple comparisons. Kaplan–Meier curves were generated to analyze patients’ prognosis. Univariate survival analysis was carried out using the log-rank tests. The Cox regression model was applied to identify prognostic factors associated with renal outcomes. The proportional hazards assumption was tested by using Schoenfeld residuals. No violations were found for any of the covariates based on a A total of 325 LN patients were enrolled. Their clinico-pathological data are presented in All 325 patients received oral prednisone therapy. Among them, 28 patients received prednisone alone, 254 patients received cyclophosphamide , 27 patients received mycophenolate mofetil, 12 patients received leflunomid, 3 patients received azathioprine, and 1 patient received cyclosporine.The average follow-up time was 55.8 months (range from 4 to 360 months). During this period, three patients died of heart failure and one patient died of severe infection. A total of 37 patients reached the end point, including 35 with ESRD and 2 with Scr doubling.C4d staining was present in 321/325 (98.8%) LN samples Table 2.P < 0.001); it was more often present in patients with classes III, IV, and V disease than class II. The rates of glomerular, arteriolar, and PTC C4d deposition were not significantly different among the subgroups.The intensity or distribution of C4d staining among different sub-classes of LN are shown in P = 0.035), leukocyturia (P = 0.002), and acute kidney injury (P = 0.001) were more frequent in TBM C4d-positive patients, who also had higher levels of urinary protein, Scr, and SLEDAI value, and a lower serum C3 value.The detailed analyses between C4d deposition in different renal areas and clinical and laboratory features are shown in Renal histopathological findings showed that C4d deposition in TBM, arterioles, and PTCs area had higher NIH activity indices (AI) and chronic indices (CI) than those without C4d deposition in the corresponding areas. Detailed data were presented in P = 0.004). Similarly, those with A-C4d deposition had a higher rate of RVLs (91.2%) compared to those negative for A-C4d , especially multiple RVLs (P < 0.001), which is most obvious in type IV LN (P = 0.001)  deposition and RVLs was explored. In our study, 267 (82.2%) patients presented with RVLs. Among them, 34.8% of patients had A-C4d deposition, which was a much higher rate than in those without RVL  Figure 3P = 0.003) or ICD lesions . No significant differences were found for AS, NNV, and TRV lesions. Furthermore, among 58 patients with TMA lesions, 25 had acute TMA, 14 had chronic TMA, and 19 had both acute and chronic (A+C) TMA lesions. Arteriolar C4d deposition was not found significantly different amongst the groups: 44.0% (11/25) in acute TMA subgroup, 42.1% (8/19) in A+C TMA, and 64.3% (9/14) in chronic TMA cases (P = 0.385). Neither C4d deposition nor the presence of renal TMA lesions correlated with aCL status: aCL was positive in 2.8% (1/36) in TMA group and 11.1% of (18/162) patients without TMA (P = 0.208). ACL was positive in 3.4% (2/59) of arteriolar C4d positive patients and in 12.2% (17/139) of arteriolar C4d negative patients (P = 0.065).With regard to RVL subtypes, patients with TMA lesions had the highest rate of A-C4d deposition (48.3%), followed by patients with ICD (38.3%), NNV (38.5%), and AS (34.5%). Significantly more C4d deposition was found in patients with TMA lesions  or ICD lesions  compared to those without TMA lesions , C3c deposition , IgG deposition , IgM deposition , and IgA deposition  compared to those without A-C4d deposits. Patients with both C4d and C3c staining had significantly higher scores of CI (P = 0.024), tubular atrophy (P = 0.049), and interstitial fibrosis (P = 0.027) than those who were only C4d positive. Similar results were also found in the C4d+C1q, C4d+IgG, C4d+IgM, and C4d+IgA double positive groups  presented with worse renal survival rates than those who were negative (We found that patients with arteriolar C4d deposition (P = 0.034) deposition and co-deposition of C4d and C3c  were risk factors for poor renal outcomes  in LN patients, which was mildly higher than the previous reported values ranging from 86.8 to 92% , 15, 33.in situ classical complement pathway activation induced by immune complexes . The diverse conclusions between different papers from various cohorts might be attributed to the different sample size and the method of C4d evaluation  deposition in glomeruli did not differ between active and chronic disease in LN . In contIn summary, complement factor C4d deposition was common, but its prevalence varied among different renal compartments in patients with LN. TBM-C4d deposition was associated with more severe clinico-pathological features, and arteriolar C4d deposition was closely related to RVLs renal outcomes. Further studies are needed to clarify its actual role in the pathomechanism of LN.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Research and Ethics Board of the Peking University First Hospital [Approval reference: No. 2017 (1333)]. The patients/participants provided their written informed consent to participate in this study.YD, LW, and YT planned the project, selected patients, interpreted results, and wrote the paper. XY performed analysis of the renal pathology. ZQ and FY planned the project and revised the paper. All authors contributed to the article and approved the submitted version.This work was supported by Grants of National Natural Science Foundation of China (No. 81870479) and Pekin University International Hospital Research Grant .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Homozygous eed-deficient mutants present a normal body plan development but display strong defects at the level of the digestive organs, such as reduced size of the pancreas, hepatic steatosis, and a loss of the intestinal structures, to die finally at around 10–12 days post fertilization. In addition, we found that PRC2 loss of function impairs neuronal differentiation in very specific and discrete areas of the brain and increases larval activity in locomotor assays. Our work highlights that zebrafish is a suited model to study human pathologies associated with PRC2 loss of function and H3K27me3 decrease.Polycomb repressive complex 2 (PRC2) mediates histone H3K27me3 methylation and the stable transcriptional repression of a number of gene expression programs involved in the control of cellular identity during development and differentiation. Here, we report on the generation and on the characterization of a zebrafish line harboring a null allele of  Polycomb group (PcG) proteins are epigenetic repressors involved in a variety of biological processes, including stem cell renewal and control of cell identity during differentiation and development ,2,3. PcGDanio rerio) is a widely used vertebrate model for studying gene functions in development, morphogenesis as well as in physiopathological conditions. Furthermore, this organism is particularly suited to study the role of the PRC2 complex and H3K27me3 methylation. Indeed, it has been previously shown that in contrast to mice, loss of function of several core subunits of the PRC2 complex, including Ezh1 [ezh2 and suz12a; suz12b homozygous mutants die at around 12 days post fertilization (dpf), after body plan formation and tissue specification [ezh1 homozygous mutants are viable and fertile [eed to investigate the role of PRC2 function and H3K27me2/3 methylation in zebrafish development and organogenesis. Homozygous −/−eed mutants die at around 10–12 dpf with strong defects at the level of the digestive organs, including reduced size of the pancreas, hepatic steatosis, and loss of the intestinal structures. However, this relatively late lethality allowed us to focus on the expression of markers of the developmental brain. We found that PRC2 loss of function alters neuronal differentiation in very specific and discrete areas of the brain. Furthermore, locomotor activity assays highlighted the role of PRC2 in the zebrafish larval behavior. Altogether, our observations showed that this eed-deficient zebrafish line could constitute a valuable model to study the impact of H3K27me3 decrease and PRC2 loss of function in development as well as in several human pathologies.Here, we describe the use of the zebrafish model to investigate the function of Eed. Zebrafish nsgenics  are mainnsgenics . The choZebrafish were maintained in compliance with the French and European Union guidelines for the handling of laboratory animals . The experimental procedures carried out on zebrafish were reviewed and approved by the local Ethics Committee, CEEA 75 Nord Pas-de-Calais, and the French Ministry of Higher Education and Research .eed were designed and assembled as previously described [eed TALEN target site was selected using the online TAL Effector-Nucleotide Targeter tool  in exonEed-specific TALEN constructs were engineered using the TALEN Golden Gate assembly system described by Cermak et al.  using thCapped mRNAs were synthetized using SP6 mMESSAGE mMACHINE kit (Ambion) from linearized plasmid templates. mRNAs (50–100 pg) were injected into 1-cell zebrafish embryos using a FemtoJet microinjector .For genotyping, embryos, larvae or caudal biopsies of larvae were incubated in 10 μL PCR extraction buffer  and placed at 50 °C for 4 h prior to proteinase K inactivation at 95 °C for 5 min. Genotype analysis was performed by PCR on 2.5 μL of samples using the primer set TAL_eed_5′ (CAGCCTAAAGGTGAGGCCGC) and TAL_eed_3′a (CGATCTCGACCCGGAACAC) followed by PCR product digestion with the BseNI restriction enzyme. An alternate genotyping strategy consists of the PCR amplification of genomic DNAs with the primer set TAL_eed5_new GTGTGCAGGAGGATCATGGTCAG) and TAL_eed3_new (GTCCGTCACTCACTCTGTTGCTG) followed by a 2% agarose gel electrophoresis without enzymatic digestion.To genotype paraformaldehyde-fixed embryos and larvae, DNA was extracted using sodium hydroxide and Tris . Single Sequence determination  was performed after cloning of the PCR products into pCR-XL-TOPO  according to the manufacturer’s instructions.+/−eed incross was transected within the pigment gap distal to the circulating blood for genotyping purposes [For the establishment of survival curves, the tip of the caudal fin of embryos from an purposes . The emb2O2. Larvae were stained with oil red-O  for 15 min. Stained larvae were washed with PBS, stored in 70% glycerol, and imaged on a bright-field Leica MZ125 stereomicroscope equipped with a Leica DFC295 digital camera. After imaging, DNA was extracted using sodium hydroxide and Tris and genotyped.Zebrafish larvae were fixed overnight in 4% paraformaldehyde and pigments were bleached 2 h in 1% KOH, 3% HSmurf assays were conducted according to Dambroise et al.  by placiFor histological analyses, tail biopsies were used for genotyping before paraformaldehyde fixation of the larvae. Larvae were embedded in paraffin and cut into 5 μm-thick sections. These were mounted on sylanated glass slides, deparaffinated, rehydrated, and stained with hematoxylin and eosin for histological analysis as previously described .Standard length and eye diameter were measured according to Parichy et al.  on anestprss1 probe were performed by equal intensity pixel counting using ImageJ.Measurements of the surface of the pancreas labeled by in situ hybridization with a −/−eed mutant larvae using ImageJ. Each image was anonymized and transformed into an 8-bit grayscale picture before counting the hepatic nuclei and the number of vesicles.Quantification of macrovesicular in the liver was performed on at least 3 consecutive hematoxylin and eosin-stained slides from 4 wild-type and 4 For all biometric measurements, the software GraphPad Prism was used to build the graphs and to perform the statistical analyses.For confocal imaging, tail biopsies were transected for genotyping before paraformaldehyde fixation of the larvae. After fixation in 4% paraformaldehyde, zebrafish larvae were maintained on glass-bottom dishes in 1.5% agarose. Fluorescent images were captured with an LSM 880 microscope and an AiryScan detector . A Z-stack slice was made every 5 µm.Antisense-RNA probes were generated by RT-PCR from total mRNA extracted from zebrafish larvae at 5 dpf using the RNeasy Mini Kit , following the manufacturer’s protocol. After reverse transcription , cDNAs were amplified by PCR using the probe-specific primers, coupled to the T7 sequence for forward primers and the SP6 sequence for reverse primers. DIG-labeled antisense-RNA probes were synthetized using the DIG RNA Labeling Kit (SP6) , following the manufacturer’s instructions.The primers used for probe generation were:ISHa_eed_F: TAATACGACTCACTATAGGG GCTGTGGCGGCTGGACTCTGISHa_eed_R: GATTTAGGTGACACTATAG CGCCAGATGGAGGCGTCGTISH_fabp2_F: TAATACGACTCACTATAGGG ATGACCTTCAACGGGACCTGGISH_fabp2_R: GATTTAGGTGACACTATAG AGCCCTCTTGAAAATCCTCTTGGCISH_ace2_F: TAATACGACTCACTATAGGGCTGTTGGAGAGATCATGTCGCTTTCTISH_ace2_R: GATTTAGGTGACACTATAGTGTCTTCCTCAAAGGCTTTGTTCACTISHa_fabp10a_F: TAATACGACTCACTATAGGG AAAGCATGGCCTTCAGCGGGAISHa_fabp10a_R: GATTTAGGTGACACTATAG GTCAGCGTCTCCACCATTTCTCCAISH_prss1_F: TAATACGACTCACTATAGGG TGCTCACTGCTACAAGTCCCGTISH_prss1_R: GATTTAGGTGACACTATAG CCCGAGCTTAGTTGGAGTTCATGGTISH_phox2bb_F: TAATACGACTCACTATAGGG GGGCCTAACCCGAACCCTACCTCISH_ phox2bb _R: GATTTAGGTGACACTATAG GAGCGCACATCGCAGTCTATCGGISH_pcna_F: TAATACGACTCACTATAGGG GGCAACATCAAGCTCTCACAISH_pcna_R: GATTTAGGTGACACTATAG AAATCCCACAGATGACAGGCISH_ccna2_F: TAATACGACTCACTATAGGGGGAAGGATGTCAACACAAGGAAGISH_ccna2_R: GATTTAGGTGACACTATAGGAGAGAACTGTCAGCACCAGATGISH_ccnd1_F: TAATACGACTCACTATAGGGTTTGCTGCGAAGTGGATACCAISH_ccnd1_R: GATTTAGGTGACACTATAGAACAGTTTGGGCGTGCTGAGTISH_sox2_F: TAATACGACTCACTATAGGG GTCCGAGAGCGAGAAGCGACCISH_sox2_R: GATTTAGGTGACACTATAGGCTGTAGGTGGGCGAGCCGTISH_mycn_F: TAATACGACTCACTATAGGGTGCGACTCCAAACGGGAGGCAISH_mycn_R: GATTTAGGTGACACTATAGGCTCACTTCGGGCGCTTTGACTISH_neurod1_F: TAATACGACTCACTATAGGGTCGAGACGCTCCGACTAGCCAAISH_neurod1_R: GATTTAGGTGACACTATAGGCGTCGAGCCCGCGTAAAGAISH_mag_F: TAATACGACTCACTATAGGGCCGTGAGGGTGTTCAGTGTGTGTISH_mag_R: GATTTAGGTGACACTATAGCGTCTCCCGTGCCTTCCTCTbmi1a were synthesized with the DIG RNA Labeling Kit (SP6/T7) (Roche) on 1 μg of linearized cDNA clone MGC:56403 IMAGE:5605189 .RNA probes for +/−eed incrosses at 5 dpf, were hybridized with digoxigenin-labeled antisense-RNA probes at 70 °C. After extensive washing, the probes were detected with anti-digoxigenin-AP Fab fragment , followed by staining with BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) alkaline phosphate substrate. The embryos were imaged using a Leica MZ10F stereomicroscope equipped with a Leica DFC295 digital camera.Whole-mount in situ hybridizations were performed according to Thisse and Thisse . BrieflyHistone extraction and Western blot analyses were performed from 10 larvae as described previously .Primary antibodies used were mouse anti-H3K27me3 , rabbit anti-H3K27me2 , rabbit anti-H3K27me1 , rabbit anti-H2AK119ub , rabbit anti-H3K9me3 , rabbit anti-H4K20me3 , rabbit anti-H3K27ac  and rabbit anti-H3 . The secondary antibodies were peroxidase conjugated anti-mouse antibody  and peroxidase conjugated anti-rabbit antibody .RNA extraction and RT-PCR experiments were conducted as previously described . cDNA waDr_eed_cDNA_5′: GGAAACGAGATGCCGAACAADr-eed_cDNA_3′a: CAGCCGGATCTCTCCCTGAGDr_cDNA_ube2a_F: TGACTGTTGACCCACCTTACAGDr_cDNA_ube2a_R: CAAATAAAAGCAAGTAACCCCGDr_beta-actin_cDNA_5′: CGTGACATCAAGGAGAAGCTDr_beta-actin_cDNA_3′: ATCCACATCTGCTGGAAGGTqPCR_fasn_5′: CCAGCCATAAGAACGTCAGCCGAGAqPCR_fasn_3′: CACCTTCCCGTCACACACCTCGTqPCR_srebf1_5′: ACAGCGCGGCTAATGGCAGGqPCR_srebf1_3′: TGCCCAGGAGCCGACAGGAAqPCR_pparg_5′: GCACAGGCGCTTCAGTGTTCAGqPCR_pparg_3′: CCAGCGAGTGCGTGTCGTCCqPCR_nr1h3_5′: ACCCAGCGATTGACAGCATCACCTqPCR_nr1h3_3′: CCTGCACGTTTGGTCGGTCTGCTPCR reactions were performed as follow: 95 °C 4 min,  35 cycles, 72 °C 10 min.ube2a levels.The quantitative qPCR reaction was performed in triplicate using a QuantStudio 3 Real-Time System  using SYBR Green Supermix . Relative mRNA expression of each gene was normalized to +/−eed incrosses in plates that were handled minimally before placement in a Zebrabox chamber  equipped with an infrared light-emitting floor and a top-mounted infrared camera allowing video recording of the whole plate under both light and dark conditions. Larval behavior measurements were achieved using the ZebraLab software with a detection threshold set at 35 and an xmin set at 3 . After recording, the larvae were euthanized, and their genotype was determined.Behavioral studies were performed on 5 dpf larvae from The locomotor activity assays were conducted in 48-well plates. The protocol consists of a 10 min initial acclimating period in the dark, followed by six alternating 10 min light and dark phases.Thigmotaxis assays were conducted in 24-well plates. The timeline of the thigmotaxis assay protocol consists of a 6 min acclimatization step in the light followed by a 4 min dark challenge period. The arena was divided into two areas, inner and outer, of the equivalent surface. The diameter of the inner area corresponds to the total diameter of the well divided by HIKESHI (heat shock protein nuclear import factor hikeshi) gene. The protein is composed of 441 amino acids and contains six WD40 domains adopting a β-propeller architecture forming multiple van der Waals interactions and hydrogen bonds with a domain located at the N-terminus of EZH2 [eed gene is located on chromosome 1. It is organized in 12 exons positioned at the 5′ of the hikeshi gene, thus defining an evolutionarily conserved synteny block. The gene also codes for a 441-amino-acids-long protein sharing 91.4% identity with its human ortholog and containing six WD40 domains as well . Before this mid-blastula transition (MBT) stage, all developmental events depend on the maternally provided gene products ,53. Usiny RT-PCR c and appy RT-PCR b. Finalle tested d.eed mutations in zebrafish, we designed TALENs targeting the exon 3 within the sequence coding for the first WD40 repeat  and Glp (EHMT1) histone lysine methyltransferases have been shown to target H3K27 in vivo [−/−eed homozygous larvae display a slight increase in global H3K27ac levels at 9 dpf , the liver , and the exocrine pancreas  in 5-dpf old larvae by in situ hybridization  [−/−eed transgenics at 9 dpf. Indeed, the number of cells forming the thickness of the intestinal wall and visible through GFP expression is decreased in eed-deficient zebrafish as of 9 dpf in (GFP) ,64,65. Fof 9 dpf a. Then, −/−eed mutants is strongly reduced and lacks folds at the level of the intestinal bulb , srebf1 (sterol regulatory element-binding transcription factor 1), pparg (peroxisome proliferator-activated receptor gamma), and nr1h3  at 9 dpf. Then, in spite of showing an increase in lipid accumulation, RNA levels for lipogenic factors remain either unchanged (srebf1 and nr1h3) or reduced (fasn and pparg) in −/−eed larvae  of the mutant transcript (phox2bb (paired like homeobox 2Bb), a transcription factor expressed by a subset of cells in the posterior regions of the brain. In −/−eed mutants, phox2bb appears additionally expressed ectopically in the retina  and sox2 (SRY-box transcription factor 2); the proliferation marker pcna (proliferating cell nuclear antigen); mycn , which is involved in several signaling pathways promoting cell growth, proliferation and metabolism of progenitor cells in different developing organs and tissues; two genes coding for cyclins, ccna2 (cyclin A2) and ccnd1 (cyclin D1); neurod1 , a marker of neuronal precursors; and mag (myelin-associated glycoprotein), a marker of glial precursors  and glial precursors (mag), whole-mount RNA in situ hybridizations showed a reduction in the labeled areas indicating that the numbers of these precursors are diminished in the hindbrain or midbrain of −/−eed larvae, respectively. Altogether, our results suggest that PRC2 loss of function disturbs the differentiation and the development of a subset of neurogenic cells in the midbrain-hindbrain region and in the retina without dramatically affecting cell proliferation, as judged by pcna expression. Thus, remarkably, while eed loss of function drastically affects bmi1a expression, the effect of the mutation on the expression of other markers such as ccna2, mycn, neurod1, or mag remains relatively thin and affects discrete areas of the brain.Analysis of expression profiles of the stemness markers eed function alters the differentiation of neurogenic cells at 5 dpf. Then, to investigate whether eed deficiency could also affect larval behavior, we performed locomotor and thigmotaxis assays. Zebrafish larvae from heterozygous +/−eed incrosses were individually disposed of into dish wells. The plates were handled minimally before placement in a Zebrabox chamber  equipped with an infrared light-emitting floor and a top-mounted infrared camera allowing video recording of the whole plate under both light and dark conditions. Larval behavior measurements were achieved using the ZebraLab software . After recording, the larvae were euthanized and their genotype determined by RFLP.Loss of −/−eed larvae are hyperactive compared with wild-type larvae at 5 dpf. This hyperactive phenotype of −/−eed mutant is particularly prominent and significant during the dark cycles  is a common behavioral endpoint used in preclinical studies employing rodents but also applied to the zebrafish model ,75. Animat 5 dpf e,f.−/−eed mutants do not present an increase in their exploration behavior when compared to wild-type siblings.Thus, in spite of having a hyperactive phenotype, the suz12a and suz12b. Then, eed is the sole zebrafish gene coding for a PRC2 subunit present in all alternate complexes. Consequently, since EED is required for EZH1/2 methyltransferase activity, it is expected that inactivation of the zebrafish eed gene will result in the loss of activity of all PRC2 complexes.PRC2 is a histone-modifying protein complex conserved from fruit flies to humans and able to deposit H3K27me1/2/3 epigenetic marks responsible in turn to local chromatin compaction and gene repression. Genome-wide studies using human or mouse embryonic stem cells showed that PRC2 and H3K27me3 marks are enriched at promoters of numerous developmental genes, leading to the proposal that PRC2 could be involved in the maintenance of the pluripotency of stem cells by keeping developmental genes repressed ,17,18,19eed allele harboring a 14 bp deletion within the third exon. This deletion conducts to a frameshift within the coding sequence leading to the production of a putative protein lacking all the WD40 domains, predicted to be non-functional, and therefore likely producing an eed null allele. Heterozygous +/−eed fish are viable, fertile, and do not present visible gross phenotypes. By opposition, −eed homozygosity leads to larval death at around 12 dpf. This time point is similar to what was observed for zebrafish lacking the function of other PRC2 components, such as for ezh2 mutants [suz12a; suz12b double-mutants [Eed knockout mouse phenotype [Eed mutation fail to gastrulate normally, eed embryonic expression is not required for the establishment of the zebrafish body plan. This relatively late lethality makes the zebrafish a compelling model to study PRC2 function during the development of various tissues without the requirement of conditional knockout strategies. It also reinforces the idea that PRC2 function plays little or no role in the implementation of developmental decisions but is mainly required to maintain these developmental instructions.Using the TALEN technology, we generated an  mutants ,38 or su-mutants , but conhenotype . While m−/−eed mutants display various alterations of the digestive organs. These defects include a smaller pancreas at 5 dpf, hepatic steatosis characterized by an increase in lipids and macrovesicles in the liver, and the loss of maintenance of the intestine structures particularly marked at the level of the intestinal bulb. Studies in mice also pointed out the role of Eed in intestinal development [Eed in postnatal intestinal crypts decreases the stem cell pools, reduces the proliferation potential, increases the secretory cell differentiation, and results in smaller mouse intestines. Zebrafish larvae hatch with a yolk sac, which is depleted at 6–7 dpf. Then, after that time, their growth relies on external food uptake. If left unfed, the larvae will usually die of starvation at around 12 dpf [−/−eed homozygous mutants account for their larval death at 12 dpf. Furthermore, the marked −/−eed phenotype at the level of the digestive organs closely resembles the phenotype of the −/−ezh2 homozygous mutants we have previously described [Zebrafish elopment . Indeed,d 12 dpf . Thus, iescribed . These ieed-deficient larvae at 9 dpf revealed a dramatic decrease in H3K27me2/3 marks, as expected. However, −/−eed larvae retained some H3K27me2 and H3K27me3 residual methylation marks. We assume that the remaining H3K27me2/3 signals were deposited by the maternal PRC2 activity and retained in slowly dividing or quiescent cells. Maternal PRC2 contribution was previously demonstrated by showing that maternal-zygotic −/−MZezh2 mutants display a much stronger phenotype than zygotic −/−ezh2 mutants, with small eyes, accumulation of blood near the yolk extension, stringy heart, heart edema, and absence of pectoral fins at 2 dpf [Eed and Suz12 knockout mouse ESC lines, as well as knockdown experiments in mouse ESCs, showed that the inhibition of PRC2 activity is responsible for a global loss of H3K27me2 and H3K27me3 levels, but at best for a partial reduction in H3K27me1 marks [−/−eed larvae indicating that in the absence of PRC2 function, H3K27 monomethylation is achieved by other histone methylation routes. It has been shown that the histone lysine methyltransferases G9a (EHMT2) and Glp (EHMT1), in addition to being potent H3K9 methyltransferases, are able to monomethylate H3K27 both in vitro and in mouse ESCs [Investigations on bulk histone modifications in at 2 dpf . EZH1 anat 2 dpf ,58,78,79e1 marks ,80,81. Ouse ESCs ,57,82. T−/−eed larvae indicating H2K119 monoubiquitylation is maintained in the absence of PRC2 function, presumably thanks to ncPRC1 action. This situation is similar to what was observed in ESCs, where Ezh1/Ezh2 double knockout was reported to do not affect H2K119ub levels [−/−eed mutants also explains the milder phenotype observed in eed-deficient fish when compared to −/−rnf2 mutants. In zebrafish, Rnf2 is the only catalytic subunit common to all PRC1 subcomplexes [rnf2 gene function lack H2K119ub marks, display several defects including jaw malformation, pericardial edema, diminished blood circulation, absence of pectoral fins, severe craniofacial phenotype, and die at around 4–5 dpf [MZezh2 zebrafish mutants [−/−rnf2 and −/−MZezh2 mutants display more severe phenotypes than ezh2 and eed homozygous zygotic mutants do. Furthermore, it has been demonstrated that ncPRC1 complexes are able to propagate H2AK119ub during cell division via their recruitment to the chromatin through the binding of the RYBP/YAF2 subunits to H2AK119ub marks [Polycomb repression is achieved by the combined action of both PRC2 and PRC1. While PRC2 methylates H3K27, PRC1 monoubiquitylates H2AK119. Multiple PRC1 subcomplexes have been shown to coexist in cells. These complexes are commonly referred to as canonical (cPRC1) or non-canonical (ncPRC1), based on their dependence on H3K27me3 recruitment to target loci ,83. We hb levels . H2K119uomplexes . Zebrafi 4–5 dpf ,86. Howe mutants . In thisub marks .K27M mutation primarily occurs in H3F3A (>70%), one of the two genes coding for the histone variant H3.3, with a lower frequency in HIST1H3B and HIST1H3C encoding H3.1, and more rarely in HIST2H3C coding for H3.2 [K27M mutated histone binds to EZH2 and inhibits the methyltransferase activity of the PRC2, leading to a dramatic decrease in global H3K27me3 marks and alterations in gene expression programs [K27MH3F3A mutations, DIPGs also contain genetic alterations targeting canonical signaling pathways, most frequently TP53 loss of function (about 60%) and gain-of-function mutations or amplifications in platelet-derived growth factor receptor alpha  [K27MH3F3A, in glioma genesis remains poorly understood. In this context, we took advantage of our eed-deficient zebrafish line to investigate the effects of PRC2 loss of activity on neuronal differentiation and proliferation. We found that in −/−eed larvae, expression of some stemness (bmi1a), as well as neuronal (neurod1) or glial (mag) precursor markers, is altered, indicating that neural differentiation is impaired in the absence of functional PRC2 activity. Interestingly, a number of human DIPGs have been shown positive for BMI1 expression, which then appears as a potential therapeutic target [pcna expression profile remained unchanged in the brain of −/−eed larvae suggesting that PRC2 loss of function and reduction in H3K27me3 marks do not affect the proliferation potential of neural cells. Thus, we propose that in DIPGs, the K27MH3F3A mutation is responsible for the maintenance of an undifferentiated state, whereas other mutations might contribute to proliferation.Sequencing studies on pediatric high-grade gliomas, including glioblastoma multiformes (GBMs) and diffuse intrinsic pontine gliomas (DIPGs), identify recurrent somatic lysine to methionine substitutions at position 27 (K27M) in histone H3. The H3for H3.2 ,90,91,92programs ,95,96,97programs . In addiout 40%) ,98, as wout 40%) . Howeverc target ,101,102.eed loss of function results in gene expression alterations in relatively thin and discrete areas of the brain. For instance, ectopic expression of phox2bb is specifically found in the retina, whereas neurod1 expression is lost in a subset part of the hindbrain. Then, the effects of PRC2 loss of function appear strictly cell-specific and context-dependent, differentially affecting cell fates. These subtle changes in gene expression would indubitably escape to global transcriptomic analyses. Thus, our observations also outline the importance of performing whole-mount in situ hybridizations, but not only global transcriptomic analyses, for the characterization of zebrafish lines harboring mutations in polycomb group genes. In this context, it is worth emphasizing that the loss of eed function gives different phenotypes depending on the considered organ. It affects neuronal differentiation in specific brain regions, the development of the pancreas, the maintenance of the intestine wall structures, or the metabolism of lipids in the liver. This variation in effects and phenotypes might reflect the fact that Eed controls different gene expression programs in the different organs. It also reinforces the idea that a better deciphering of PRC2 function would rely on transcriptomic analyses at the single-cell level.Surprisingly, we also showed that while expressed ubiquitously in the brain of wild-type larvae, eed−/− larvae also present a hyperactive phenotype, raising the possibility that the eed-deficient zebrafish line could constitute a pertinent model to study the neurological defects leading to behavioral disabilities in COGIS.Cohen-Gibson syndrome  is an overgrowth disorder characterized by dysmorphic facial features, advanced bone age, skeletal abnormalities, associated with variable intellectual disability, and caused by de novo mutations in EED ,103. To