Zfp423 as a critical Polycomb-dependent transcription factor whose silencing negatively impacts survival. The anti-gliomagenic activity of Zfp423 requires interaction with the SMAD proteins within the BMP signalling pathway, pointing to a novel synergic circuit through which Polycomb inhibits BMP signalling.Malignant gliomas constitute one of the most significant areas of unmet medical need, owing to the invariable failure of surgical eradication and their marked molecular heterogeneity. Accumulating evidence has revealed a critical contribution by the Polycomb axis of epigenetic repression. However, a coherent understanding of the regulatory networks affected by Polycomb during gliomagenesis is still lacking. Here we integrate transcriptomic and epigenomic analyses to define Polycomb-dependent networks that promote gliomagenesis, validating them both in two independent mouse models and in a large cohort of human samples. We find that Polycomb dysregulation in gliomagenesis affects transcriptional networks associated with invasiveness and de-differentiation. The dissection of these networks uncovers  Polycomb-mediated gene regulation has been implicated in gliomas. Here the authors integrate transcriptomic and epigenomic analyses to define Polycomb-dependent networks that promote gliomagenesis, and find that the Polycomb-dependent silencing of the transcription factor Zfp423 hinders survival. Besides the ‘classical' oncogenic properties of EZH2 refs , 15, 16,17192Zfp423 as a critical downstream target of PRC2, whose downregulation negatively impacts survival and whose overexpression impairs gliomagenesis in a SMAD -dependent manner.Here we address this challenge focusing on malignant gliomas, which constitute one of the most significant unmet medical needs and present two particularly relevant features: (i) pronounced heterogeneity among and within tumour types and (ii) convergent evidence of context-dependent Polycomb involvement, which is largely limited to its impact on single genes and has not mechanistically uncovered the diversity of its target pathways in relation to specific glioma subtypes and developmental contexts24252627282930Ink4a/Arf combined with the overexpression of the constitutively active mutant form of the human epidermal growth factor receptor (EGFR) known as EGFR variant III (hereafter indicated as EGFR*), the two most common lesions in human high-grade gliomas (HGGs)Ink4a/Arf−/−; EGFR*) and its unique advantage, as depicted in Ink4a/Arf−/− astrocytes, hereafter Astro; tumorigenic Ink4a/Arf−/− astrocytes expressing EGFR*, hereafter AstroEGFR*; glioma propagating cells (GPCs) derived from primary tumours, hereafter PT; and GPCs derived from secondary tumours, hereafter ST), which constitute a privileged entry point to dissect the transcriptional and epigenomic alterations that accrue during gliomagenesis. Importantly, the Ink4a/Arf-null background allows to study the developmental features of PRC2-dependent oncogenesis independent of its control of the INK4A-ARF-INK4B locusInk4a/Arf−/−; EGFR* combination recapitulates a broad range of HGG, ranging from anaplastic astrocytomas, oligodendrogliomas and oligoastrocytomas to GBM and gliosarcoma  motif enrichment analysis , of which 272 are downregulated and 273 upregulated in PT versus AstroEGFR*. A cluster analysis based on DEGs revealed a clear clustering by cell type  that centred on the transcription start site (TSS), with the typical pattern of great enrichment right upstream and downstream of the sharp dip coinciding with the TSSWhen we analysed the same cell populations for H3K27me3 distribution, comparing the number of PRC2 targets in each class of samples revealed a major overall increase of PRC2 marking in PT, with 2,306 gene hits compared with 668 in AstroEGFR*. We then compared these gene sets with two previous data sets that captured distinct aspects of PRC2 developmental control: (i) the H3K27me3 signature that characterizes cortical neural progenitors (NPs)de novo targets of H3K27me3 39Slit2 is epigenetically silenced in gliomas through DNA methylationin vitro and in vivoBmp4 is known to reduce proliferation and promote astrogenesisde novo H3K27me3 in the AstroEGFR* to PT transition showed a highly significant overlap with the subset of Polycomb targets that are significantly downregulated during cell reprogramming , tumorigenic NP overexpressing PDGF-B (hereafter NP PDGFB) and GPCs derived from primary tumours (hereafter PT) that were able to regrow tumours upon orthotopic transplantation. Consistent with our observations from the Ink4a/Arf−/−; EGFR* model, we confirmed a progressive downregulation of Zfp423 along with the increase in tumorigenic potential  in cortical NP see ref. , to deteotential .Zfp423 had not yet been associated to gliomas, a previous studyZfp423, ZNF423, to be downregulated in human neuroblastoma. We thus assessed the expression of ZNF423 in well-defined human GBM samples that had been previously subdivided according to EGFR expressionhttp://cancergenome.nih.gov/), to investigate the correlation between ZNF423 expression and patient survival. To this end, we interrogated separately low-grade gliomas and GBMs, and stratified samples according to the expression of ZNF423. This analysis revealed a significant direct correlation between ZNF423 expression and survival in low-grade gliomas . Comparable results were obtained by performing a second meta-analysis on a further independent cohort of expression profiles, comprising 157 expression data from Gene Expression Omnibus . Finally, to ground the mechanistic dissection of the role of Zfp423 dosage in gliomagenesis, we resorted back to our initial Ink4a/Arf−/−; EGFR* murine model and interrogated three independent batches of tumorigenic astrocytes  with comparable Zfp423 expression. When challenged in in-vivo experiments upon orthotopic transplantation, we found that AstroEGFR*1 and AstroEGFR*3 yielded gliomas with a shorter survival compared with AstroEGFR*2 . Following confirmation of overexpression . On the contrary, mice transplanted with either AstroEGFR*1 or AstroEGFR*3 astrocytes showed different survivals. Specifically, although AstroEGFR*1, which reached overexpression levels comparable to those in AstroEGFR*2, showed a trend of extended survival that did not reach statistical significance, AstroEGFR*3, despite much stronger Zfp423 overexpression, displayed a shorter survival. To gain insight into the molecular underpinnings of the differential response to Zfp423 overexpression, we followed two complementary approaches.As higher levels of pression , cells wpression . As showZfp423 overexpression  and went on to define the Zfp423-centred transcriptional interactions specific to each pair. To this end, we integrated the results of differential expression obtained from each comparison with a co-expression-based analysis extracted from a large data set of 106 gene expression profiles collected in the GeneMania databaseZfp423 and then define which portion of these predicted circuits were empirically validated in Zfp423-responsive versus Zfp423-non-responsive tumorigenic astrocytes. Consistent with the in-vivo outcome, interrogation of these gene networks showed that Zfp423 overexpression had affected different sets of genes in the three batches of tumorigenic astrocytes , each encompassed different partitions of the three signatures, with a prevalence of the mesenchymal one  with a well-characterized mutant isoform of Zfp423 lacking the SMAD-binding domainZfp423ΔSBD) and assessed their tumorigenicity in vivo. Contrary to the major antigliomagenic effect observed with the overexpression of the full-length protein, overexpression of mutant Zfp423 did not affect the tumorigenicity of AstroEGFR*2 astrocytes  the marked heterogeneity of these tumours, which implies the development of several subgroup-targeted approaches, and (ii) the virtual inaccessibility of early-stage lesions, with the attending inability to trace tumour history and define the critical gene circuits that drive initial establishment of the tumour. Notably, although many mouse models have proven invaluable in dissecting central aspects of this human disease, the experimental features of most of them precluded a thorough characterization of disease dynamics. This applies especially to the chromatin level of regulation, despite its emergence as a fundamental domain of perturbation that is raising major therapeutic hopes. Indeed, although cancer-specific chromatin maps, including for PRC2-dependent H3K27 trimethylation, are rapidly accumulating for both human and murine tumours, they most often describe endpoints of disease progression and are seldom related to transcriptional output in terms of mechanistically tested and functionally validated circuits.Zfp423, that was also under direct PRC2 control and accounted for a significant portion of the transcriptional dysregulation that underlies the transition from tumorigenic astrocytes to GPCs. Importantly, we found that Zfp423 downregulation correlated with a worse prognosis, both in two independent mouse models and in two very large cohorts of human patients. The human homologue of Zfp423, ZNF423, had been previously identified as a positive prognostic factor for neuroblastomaHere we advance significantly our understanding of PRC2 contribution to gliomagenesis with an unbiased characterization of the PRC2-dependent circuits. Specifically, we combined the informative power of a mouse model featuring homogeneous cell populations of differential tumorigenic potential with a computational pipeline for the systematic integration of RNAseq and ChIPseq profiles with discovery tools for TF motif enrichment. This yielded two key insights. First, it enabled us to functionally partition the effect of PRC2 silencing, by showing that two critical developmental domains, one centred on the morphogenetic cues that enable migration and the other centred on dedifferentiation, were under direct PRC2 control. Importantly, both have been linked to core features of HGG, such as the marked propensity for invasiveness and the aberrant re-expression of elements of the pluripotency networkZfp423 and ZNF423, whose Krüppel-like C2H2 zinc finger clusters mediate interaction with distinct transcriptional partnersZfp423, as only the full-length protein but not its SMAD mutant form was able to prolong survival. This finding is particularly interesting in light of our results demonstrating PRC2-silencing of Bmp4 in our primary tumour samples and of previous data on the aberrant recruitment of PRC2 to the BMPR1B receptor geneThe modular architecture of Ink4/Arf−/−; EGFR* gliomagenesis, which we harnessed at the molecular level to define the mechanisms and context specificity of Zfp423 antigliomagenic activity. As to the former, we found that Ink4/Arf−/−; EGFR* tumorigenic astrocytes, despite their overall similarity, display transcriptional specificities that are differentially retained in the gliomas they originate, suggesting that they represent differentially primed entry points of tumorigenesis. Consistently, this heterogeneity is carried through the primary gliomas, which encompass a relatively wide range of high-grade histopathological subtypes and also recapitulate, to different extents and for different parts, the signatures used to classify human HGG.Finally, our findings also uncovered a significant degree of heterogeneity within Zfp423 exerts the strongest antigliomagenic activity only in the context of the least tumour-primed astrocytes, in which it antagonizes, in an exquisitely SMAD signalling-dependent manner, a very significant portion of the gliomagenic programme that includes Sox2, a well-established glioma oncogene, and several other differentiation-promoting factors that are specifically downregulated in gliomas. However, the context specificity displayed in this experimental model did not prevent the definition of a general prognostic impact for ZNF423 downregulation, which was confirmed in two large independent human cohorts. The context effect of Zfp423 is consistent with accumulating evidence on the extreme heterogeneity that characterizes gliomas, with a growing number of TFs being shown to play critical roles but in highly selected cases, as in the instance of the six TFs that underlie transcriptomic changes uniquely in the mesenchymal subtype HGGFGFR-TACC oncogenic fusion that was identified in three patients from a large cohort of HGG patientsThis transcriptomic dissection of glioma heterogeneity was functionally validated with the observation that Human GBM cell lines were obtained from Rossella Galli's lab  and were grown as described in ref. −1 potassium penicillin+100 U ml−1 streptomycin sulfate (Lonza) in basal medium Eagle's (Gibco)). They were mechanically dissociated with scissors and through pipetting; the dissociated tissue was plated into a T-25 flask and incubated in a humidified incubator at 37 °C with 5% CO2. Medium was changed 3 days after plating and every other day thereafter until day 9 of culture. Cells were passaged every 4–5 days starting from day 10 of culture at a 1:2 or 1:3 ratio. The cells were rinsed with Dulbecco's PBS and detached in trypsin–versene 1:10 ; after detachment, the dissociation into single cells was achieved through pipetting. Trypsin was inactivated by adding FBS  and cells were collected by centrifugation at 280 g for 5 min. The cell pellet was resuspended in fresh astrocyte medium and cells were plated in 6-well plates. EGFR*- or ZFP423/ZFP423dSBD-expressing retroviral particles were generated using the retrovirus producer cell line Phoenix-Eco. Established astrocyte cultures (after passage 2) were infected using the viral supernatant filtered through a 0.45-μm filter. Astrocytes were trypsinized as described before; 5 × 105 cells were resuspended in 3 ml of viral supernatant supplemented with 8 μg ml−1 Polybrene (Sigma) and plated in a well of a 6-well plate. The supernatant was removed and replaced with fresh supernatant at each new collection. Astrocytes were kept in culture for less than ten passages before being used for in-vivo experiments and next generation sequencing profiling.Primary astrocytes, GPCs and NPs were derived and cultured as described previously6667et al.g for 5 min), cells were resuspended in glioma-initiating cell medium , 20 ng ml−1 of both EGF and murine fibroblast growth factor-2, 100 U ml−1 potassium penicillin+100 μg ml−1 streptomycin sulfate in neurobasal medium (Gibco)) and plated in one or two wells of a six-well plate, coated with laminin (Roche). Plates were kept in a humidified incubator 37 °C with 5% CO2; medium was changed 2 days after plating and every other day thereafter. Cells were passaged at 80–90% confluency at a ratio ranging from 1:3 to 1:6. They were detached with Accutase and dispersed into single cells by gentle pipetting; after centrifugation at 300 g for 5 min, cells were resuspended in glioma-initiating cell medium and plated as described above. GPCs were kept in culture for less than ten passages before being used for in-vivo experiments and next generation sequencing profiling. Differentiation was performed according to the protocol published in the same paper. In particular, astrocytic differentiation was performed by adding FBS or BMP4 to the culturing medium, whereas neuronal differentiation was achieved through growth factor withdrawal. The same protocol was used for the differentiation of embryonic neural precursors.GPC cultures were obtained and maintained as described in Pollard EGFR*, Pdgf-β, Zfp423 or Zfp423DSBD retroviral particles were generated using the retrovirus producer cell line Phoenix-Eco. Supernatant was collected and used directly or after ultracentrifugation. Differentiation of NP was performed as described in ref. Experiments involving animals were carried out in accordance with the Italian Laws (D.lgs. 26/2014), which enforces Dir. 2010/63/EU . Specifically, the project was notified to the Ministry of Health before the implementation of the current legislation. As the new legislation does not apply to projects approved before its enactment, for such projects only a notification of the experiments to the Ministry of Health was required (in accordance with the D.L.vo 116/92 (and following additions), which enforced EU 86/609 Directive), and this requirement was dutifully fulfilled.5 AstroEGFR* or GPCs into the caudate of Cd1-Nude HO, 5-week-old female mice . Brains were isolated at the onset of disease symptoms and fixed in 4% buffered formalin. Samples were paraffin embedded, sectioned, stained for haematoxylin and eosin, and histopathologically classified by pathologists.Tumour formation was induced by stereotaxical injection of 5 × 10RNA was extracted from all the lines using the RNeasy Mini Plus Kit (Qiagen) according to the manufacturer's specifications. Quality and concentration of RNA was assessed using a NanoDropSpectrophotometer (NanoDrop Technologies).g for 5 min, then washed in PBS and lysed in RIPA buffer plus protease inhibitors cocktail (Sigma). Lysates were sonicated using the Bioruptor Sonication System (UCD200) for three cycles of 30 s with 60 s pause at high power. Lysates were centrifuged at 13,000 g for 15 min and supernatants were transferred to a new tube. Protein quantification was performed using the Bradford protein assay (BioRad) following the manufacturer's instructions.Proteins were extracted as follows: cells were harvested from the plate and centrifuged at 300 Retrotranscribed cDNAs have been obtained from 1 μg of total DNA-depleted RNA using the superscript VILO retrotranscription kit from Life Technologies according to the manufacturer's instructions.For quantitative reverse transcriptase–PCR (qRT–PCR) analysis, a total amount of cDNA corresponding to 5 ng of starting RNA has been used for each reaction. FAST SYBR green master mix from Life Technologies and 10 μM primer pairs have been used. The qPCR reactions have been performed on an Applied Biosystems 7500 Real-Time PCR machine following the standard amplification protocol (primer list and Taqman Assays ID provided in Library preparation for RNA sequencing was performed using RiboZero and Single Stranded kits (Illumina) according to the manufacturer's instructions. Validation of RNAseq results has been performed through qRT–PCR analysis.For ChIPseq, cells were harvested and resuspended in PBS containing 1% formaldehyde for fixation and quenched with 125 mM glycine. Cells were lysed and sonicated using the Covaris Sonicator, to generate 250 bp DNA fragments. Chromatin was incubated overnight at 4 °C with the antibodies  and H3 total (Abcam)) and recovered the day after using Dynabeads Protein G (Life Technologies). Immunocomplexes were eluted, de-cross-linked and purified using Qiaquick PCR Columns (Qiagen) according to the manufacturer's instructions. DNA libraries were prepared according to ChIPseq Sample Preparation Guide (Illumina) and sequenced on an Illumina GA-II or HiSeq 2000 platform. Validation of ChIPseq results has been performed through qPCR analysis: 5 ng DNA have been used for each reaction. FAST SYBR green master mix from Life Technologies and 10 μM primer pairs have been used. The qPCR reactions have been performed on an Applied Biosystems 7500 Real-Time PCR machine following the standard amplification protocol. Primer lists for qRT–PCR and ChIP–qPCR are provided in How to cite this article: Signaroldi, E. et al. Polycomb dysregulation in gliomagenesis targets a Zfp423-dependent differentiation network. Nat. Commun. 7:10753 doi: 10.1038/ncomms10753 (2016).Supplementary Figures 1-6, Supplementary Tables 1-3, Supplementary Methods and Supplementary ReferencesGene lists from ChIPseq and RNAseq analysisGene lists from IPA analysis.


Peste des petits ruminants virus (PPRV). Differences in susceptibility to goat plague among different breeds and water buffalo exist. The host innate immune system discriminates between pathogen associated molecular patterns and self antigens through surveillance receptors known as Toll like receptors (TLR). We investigated the role of TLR and cytokines in differential susceptibility of goat breeds and water buffalo to PPRV. We examined the replication of PPRV in peripheral blood mononuclear cells (PBMC) of Indian domestic goats and water buffalo and demonstrated that the levels of TLR3 and TLR7 and downstream signalling molecules correlation with susceptibility vs resistance. Naturally susceptible goat breeds, Barbari and Tellichery, had dampened innate immune responses to PPRV and increased viral loads with lower basal expression levels of TLR 3/7. Upon stimulation of PBMC with synthetic TLR3 and TLR7 agonists or PPRV, the levels of proinflammatory cytokines were found to be significantly higher while immunosuppressive interleukin (IL) 10 levels were lower in PPRV resistant Kanni and Salem Black breeds and water buffalo at transcriptional level, correlating with reduced viralloads in infected PBMC. Water buffalo produced higher levels of interferon (IFN) α in comparison with goats at transcriptional and translational levels. Pre-treatment of Vero cells with human IFNα resulted in reduction of PPRV replication, confirming the role of IFNα in limiting PPRV replication. Treatment with IRS66, a TLR7 antagonist, resulted in the reduction of IFNα levels, with increased PPRV replication confirming the role of TLR7. Single nucleotide polymorphism analysis of TLR7 of these goat breeds did not show any marked nucleotide differences that might account for susceptibility vs resistance to PPRV. Analyzing other host genetic factors might provide further insights on susceptibility to PPRV and genetic polymorphisms in the host.Ovine rinderpest or goat plague is an economically important and contagious viral disease of sheep and goats, caused by the  Peste des petits ruminants (PPR), also known as ovine rinderpest or goat plague, is an acute, highly contagious viral disease of goats and sheep, caused by the Peste des petits ruminants virus (PPRV), a morbillivirus in the family Paramyxoviridae. The disease is characterized by high fever, nasal and ocular discharges, pneumonia, necrotic and ulcerative lesions of the mucus membranes and inflammation of the gastro-intestinal tract PPR is generally considered a more serious disease in goats than sheep, however, increased susceptibility of sheep, goat and outbreaks involving both sheep and goats have been equally reported The natural susceptibility to PPRV in goats could be attributed to several host-derived or virus-derived factors. One such host-derived factor could be the differential presence or distribution of specific viral receptors in these species, such as the signalling lymphocyte activation molecule (SLAM) that has previously been observed to be associated with PPRV and other morbilli viruses such as measles virus and canine distemper virus Toll like receptors (TLR) are type 1 transmembrane proteins expressed in almost all cell types and activate the innate immune system upon sensing pathogen associated molecular patterns (PAMPs). Intracellular TLR that sense viral nucleic acids include TLR3 (double stranded RNA), TLR7 and TLR8 (single stranded RNA) and TLR9 (CpG motifs in DNA) E. coli.In addition to the differential expression profiles of TLR, ligand induced downstream cytokine profiles and/or levels could also play a role in the innate disease resistance of a species or breed. For example, mastitis is an economically important inflammatory disease of the udder that has been shown to be more prevalent in the Holstein breed of cows than in Jersey cows http://www.icar.org.in/en/node/4688). Barbari is a common breed of goat reared for meat and milk production in northern India. Tellicherry, Kanni and Salem black are indigenous breeds of goats prevalent in southern India India has 23 genetically well characterized indigenous breeds of goats  from four breeds of goats and water buffalo resulted in differential viral replication kinetics and inflammatory cytokine profile including IFNα, IFNγ and TNFα with differential activation of TLR3 and TLR7. Analysis of single nucleotide polymorphisms (SNPs) in the complete gene sequences of TLR7 between goat breeds did not show any differences that could account for this.3.0 mean tissue culture infective dose (TCID50) of PPRV and the virus load analyzed at 24 h post infection (PI) by qRT-PCR, using primers specific to the PPRV-H gene and TCID50. PBMC from Barbari and Tellicherry goats supported significantly (p<0.01) higher PPRV replication than those from Kanni and Salem Black  compared to Barbari/Tellicherry goats  in nine individual animals per breed (n = 9). The Kanni and Salem Black breeds revealed significantly higher basal TLR3 (p<0.001) and TLR7 (p<0.001) transcripts than Barbari goats while there were no significant difference (p>0.05) between Tellicherry and Barbari breeds . To undeem Black  with thery goats .50 was not determined for poly I:C treated and PPRV infected PBMC since the 40-Ct values of PPRV H mRNA expression levels across these breeds were not significantly different  replication of PPRV than goat PBMC (p<0.001). Approximately 2 log10 difference in the virus yield was evident between buffalo and goat PBMC upon PPRV infection  could reduce the replication by 451 fold .To confirm the role of TLR7 signalling in IFNα induction by PPRV unequivocally, goat and buffalo PBMCs were treated with a predetermined optimum concentration (10 µg/ml) of a TLR7 antagonist IRS 661  24 h prior to imiquimod treatment or PPRV infection. IFNα mRNA levels were significantly reduced by IRS 661 treatment, when PBMC were stimulated either with Imiquimod or PPRV . To furtTo determine whether the differential IFN and pro-inflammatory cytokine production between Kanni/Salem vs Barbari/Tellicherry breeds of goats are dependent on single nucleotide polymorphisms (SNPs) in TLR genes, we examined the complete gene sequence of TLR7. The TLR7 gene was amplified and seven overlapping PCR amplicons were sequenced to obtain the full length TLR7 gene using the goat TLR7 sequence from GenBank as a template (Accession # GU289401). The sequences obtained for Barbari, Tellicherry and Kanni goat breeds were submitted to GenBank . Nucleotide variations were detected across goat breeds. Sequence analysis revealed five nucleotide changes in the TLR7 coding region and two nucleotide changes in the 3′ untranslated region (3′UTR). Changes were observed at nucleotide positions 777 , 2082 , 2730 (C/T in Barbari while C/C in other breeds), 3006 (A/A in Tellicherry while A/G in other breeds) and 3069 (G/A in Barbari while G/G in other breeds) that corresponded to amino acid positions 259, 694, 910, 1002 and 1023. However, all of these changes were synonymous and no amino acid changes were observed between different goat breeds. In addition, two changes in the 3′UTR, at nucleotide positions 151  and 161 (G/A in Barbari while G/G in other breeds) were also observed.Following eradication of rinderpest, focus has shifted to PPRV as a potential virus for eradication efforts. The single serotype of PPRV, availability of an efficacious live attenuated vaccine, sensitive diagnostic tests and the economic impact of this disease, coupled with its ability to spread into new geographical regions, render it an attractive target Goats have been reported to be more susceptible to PPRV than sheep, while cattle and buffalo do not contract clinical disease Single and double stranded RNA are recognized by TLR7/8 and TLR3, respectively. TLR3 is a key sensor of viral infection, as most viruses will produce dsRNA at some stage of its life cycle. TLR7 is highly expressed in immune cells like plasmacytoid dendritic cells (pDC), which produce substantial amounts of type I IFNs in response to viral RNA. In our study, the basal levels of TLR3 and TLR7 were significantly higher in the PBMCs of PPRV resistant goat breeds, Kanni and Salem Black. Engagement of both TLR3 and TLR7 with the synthetic ligands poly I:C and imiquimod respectively, led to the suppression of PPRV RNA and infectious virus yield in PBMC of goats. This indicates that TLR3 and 7 play a role in the recognition of PPRV RNA by goat PBMC, though the role of cytosolic RNA sensors like Retinoic acid-inducible gene 1 (RIGI) and Melanoma differentiation-associated protein 5 (MDA5) have not been analyzed in this study and cannot be ruled out. Almost complete abrogation of viral gene expression was observed after stimulation by poly I:C. This may be because poly I:C can also be recognized by other sensors, including RIGI, MDA5 and Protein kinase R (PKR) If factors other than the receptor expression for PPRV determine clinical disease, this should be at the level of virus replication and clearance by innate and adaptive responses. The cell surface receptor for PPRV, the SLAM is expressed at lower levels in buffalo than goats Cytokines play a pivotal role in the induction and modulation of immunological responses. TLR signaling events lead to the activation of nuclear factor kapp-light-chain-enhancer of activated B cells (NFκB) and interferon regulatory factor (IRF), which switch on expression of a specific panel of pro-inflammatory cytokines and chemokines such as TNFα, IL6, IL8 and Regulated on activation, Normal T cells (RANTES) Consistent with the inflammatory cytokine environment induced by PPRV infection, expression of the immunomodulatory cytokine IL10 was also observed, but its levels were high in the PPRV susceptible goat breeds, Barbari and Tellicherry. IL10 is a key regulatory cytokine with immunosuppressive properties that helps to regulate an uncontrolled inflammatory response The goat TLR7 gene is 3.4 Kb long, with a 3141 nucleotide open reading frame (ORF), coding for 1046 amino acids. Nucleotide sequence homology studies have shown a close relationship with other ruminant species, particularly sheep TLR7 We also found a role for basal and induced TLR3 expression levels in PPRV infection in our study. In addition to TLR, variations in downstream intracellular signaling molecules such as MyD88 and MD2 may also play a role. In conclusion, our studies suggest that higher basal levels of TLR3/7 and augmented innate antiviral immune responses upon infection may afford resistance to PPRV infection in Kanni and Salem breeds of goats compared to Barbari and Tellicherry breeds. Compared to goats, elevated type I IFN levels after PPRV infection in water buffaloes may afford reduced virus replication and possibly early virus clearance. LRR prediction revealed goat TLR7 has 21 LRRs and buffalo TLR7 has 15 LRRs. The difference in LRR numbers could be a critical factor in determining the signaling responses of goat and buffalo TLR7. Future studies may provide insights into understanding the immunogenetic mechanisms underlying variations in the immune response to PPRV.Limulusam ebocyte lysate assay, Sigma Aldrich, St. Louis, MO) and found to be free of endotoxins.All animal studies have been conducted as approved by the Ethics Committee of the Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 051, India. Apparently healthy, 12–18 month old, Barbari, Tellicherry, Kanni and Salem Black breeds of goats of either sex were maintained under similar conditions at the Livestock Research Station, Tamil Nadu Veterinary and Animal Sciences University, India. These animals were not vaccinated against PPRV and had no record of any other disease during the course of the study. PBMC from nine animals of each breed were used for TLR mRNA expression analysis, while PPRV infection and TLR stimulationstudies were carried out on PBMC from five animals of each breed. Live attenuated PPRV (strain AR-87) was obtained from the department of veterinary microbiology, Madras Veterinary College, India and has been described elsewhere Blood was collected aseptically from the jugular vein into sterile ethylene diamine tetra acetic acid (EDTA) coated vacutainer tubes  and processed for PBMC isolation. Briefly, 5 ml of anti-coagulated blood was diluted in equal volumes of RPMI-1640  medium containing antibiotic and antimycotic solution, overlaid on 2.5 ml of Histopaque, specific gravity 1.077  and centrifuged at 1500×g for 25 min. Mononuclear cells were collected from the interface and washed three times in RPMI-1640 by centrifugation at 200×g for 10 min. Viability was determined by trypan blue dye exclusion method. PBMC were stimulated with predetermined doses of TLR3 and TLR7 agonists, poly I:C and imiquimod (R837), respectively. Cells were harvested at 24 h for cytokine transcript analysis.3.0 TCID50 of PPRV. Virus yields from TLR 3/7 stimulated and un-stimulated PBMC were assessed at 24 h PI by SyBr Green quantitative real time reverse transcription polymerase chain reaction (qRT-PCR), using primers specific for the PPRV-H gene 50/mL In a separate experiment, TLR ligand stimulated PBMC were infected with 1018 primers using the High Capacity cDNA Archive kit . Basal expression levels of TLR3 and TLR7 mRNA in PBMC were determined using gene-specific primers and TaqMan probes (FAM-NFQ)   as permanufacturer’s instructions. RNA concentration and purity was determined using the BioPhotometer plus . Two µg of total RNA was reverse transcribed with Oligo (dT)bad, CA) . qRT-PCRCytokine gene expression levels were compared by SyBr Green qRT-PCR using gene specific primers  (primer sequences available upon request). Un-stimulated PBMC were used as control. Corrected Ct was calculated as:−1/slope) was determined. Results were expressed as 40-Ct values −ΔΔCt) over the respective basal levels of mock-induced PBMCs after normalizing for the endogenous control gene and using the corrected Ct.Ct + (Nt – Ct’)×S/S’, where Ct is the mean sample Ct, Nt is the mean of the house keeping genes GAPDH/actin from the control group, Ct′ is the mean of the GAPDH/actin from treatment, S is the target gene slope, and S’ is the GAPDH/actin slope. The slope values were calculated using serial dilutions of cDNA and the respective Ct values for each dilution and PCR efficiency , IFNα  and IFNγ  were used to determine cytokine concentrations in the culture supernatants of TLR ligand stimulated and/or PPRV infected PBMC. ELISA was performed according to the manufacturer’s instructions and values were obtained spectrophotometrically on an ELISA reader  at 492 nm. Mock infected cell culture supernatant served as a control. TNFα and IFNγ levels were expressed as the corrected optical density [OD] of TLR-ligand stimulated or PPRV infected culture supernatants from which the OD of mock infected supernatants is subtracted. IFNα concentrations in the experimental samples were extrapolated from the values generated from standards.Blood samples were collected from Barbari, Tellicherry, Kanni and Salem black breeds of goat and genomic DNA was isolated using the Blood DNA isolation kit . The concentration of extracted DNA was determined using biophotometer plus . Seven pairs of overlapping primers were designed to amplify the full-length TLR7 gene (primers available upon request) and the PCR fragments were directly sequenced in both directions using the Big Dye Terminator v3.1 cycle sequencing kit . Sequences were assembled into a 3.4 kb contig, which contained a 3141 bp open reading frame. Sequence contigs of TLR7 from each animal were further subjected to multiple alignments to identify nucleotide variations, using the Lasergene software . Heterozygous nucleotides were scored manually across samples from different breeds by visualizing the individual chromatogram in Chromas Lite 2.01 . Each polymorphic nucleotide was further analyzedfor its amino acid position and change.50 determination was compared by two-way ANOVA with Bonferroni test for multiple comparisons. ELISA values for each cytokine and PPRV-H gene levels upon IFNα treatment were compared by one-way ANOVA with Bonferroni test for multiple comparisons. Means were considered significantly different when the p value was <0.05.Statistical analysis was performed with the GraphPad Prism software. The 40-corrected Ct values of TLR3 and TLR7 mRNA, fold changes in the ligand induced cytokine mRNA expression, PPRV H gene levels and virus yield estimated by TCID


Newborn hearing screening is a type of screening test for the early detection of hearing loss. It can recognize with good accuracy newborns affected by hearing impairment allowing an early diagnosis and intervention and avoiding cognitive and linguistic deficits -6.The incidence of bilateral sensorineural hearing loss (SNHL) in Sicily is 2.35 cases per 1000 newborns; this value increases to 2.95 if we consider also unilateral SNHL -9.A correct newborn hearing screening programme is based on different protocols depending on the presence/absence of audiologic risk factors:• Newborns without risk factors: -3Initial Hearing Screening (Step I): The initial screening should be performed using Transient-Evoked Otoacoustic Emissions (TEOAEs) in the birth centers as close to discharge as possible, preferably 12 hours or more after birth. It is recommended that an infant be referred for a re-screening (step 2) if s/he does not pass the initial screening or results cannot be obtained in one or both ears.Re-screening (Step II): The re-screening should be performed in a second level center using TEOAEs and Automated Auditory Brainstem Response (AABR). If an infant does not pass the re-screening or if results cannot be obtained in one or both ears, s/he shall be referred to the regional third level center for diagnostic audiological evaluation.• Newborns with risk factors JCIH 2007) [ 7-10]10]Initial Hearing Screening (Step I): The Initial Hearing Screening should be performed in a second level center using TEOAEs and AABR. If an infant does not pass the initial screening or if results cannot be obtained in one or both ears, s/he shall be referred to the regional third level center for diagnostic audiological evaluation.Actually TEOAEs have a sensitivity of 100% and a specificity of about 70-95%. A higher TEOAEs specificity value depends on ,11:• Timing of TEOAEs recording• Trained and qualified personnel• PASS/REFER criteriaAudiologic screening does not identify:• Post-natal SNHL  , mainly related to genetic causes ,13• ANSD (Auditory Neuropathy Spectrum Disorder): the main risk factors associated to ANSD are severe jaundice, prematurity, respiratory distress, ototoxic drugs , genetic mutations (e.g. OTOF gene). The diagnosis of ANSD is usually based on the combination of absent or abnormal ABR with normal TEOAESs and/or cochlear microphonics (CM) .


To test blood interferon signature as a screening tool for type I interferonopathies in children with early-onset SLE and vasculopathy.IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) was quantified by standard RTPCR techniques. An IFN score was calculated for each patient using the median fold change of gene expression related to a healthy control. Patients were selected based on the presence of the following features: i) atypical or incomplete SLE-like symptoms occurring in infancy or in preprepubertal age; ii) vasculopathy  iii) panniculitis with or without lypodystrophy iv) interstitial lung disease in the context of systemic inflammation.We collected blood samples from a cohort of pediatric rheumatologic patients and scored them according to a qPCR based IFN gene signature assay. Expression of 6 type I IFN-related genes . 14 out of 24 patients had a positive signature with an IFN score ranging from 3.5 to 23,7. Based on the clinical presentation and the result of the IFN score we further analyzed two patients for mutations affecting TMEM173 gain of function dominant mutations. With the present ongoing study we anticipate that testing blood for an interferon signature is a valuable screening tool for the identification of type I interferonopathies in selected children with rheumatic diseases.Several studies have shown how mutation in genes involved in type I interferon (IFN) pathway can be responsible of severe inflammatory syndromes such as Aicardi-Goutières syndrome (AGS) and spondyloenchondrodysplasia with immune disregulation (SPENCD). STING associated vasculopathy with onset in infancy (SAVI) and a familial inflammatory syndrome with systemic lupus erythematosus (SLE)-like manifestations are among the last clinical phenotypes that have been linked to an increase of type I IFN - in both cases as a result of


Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus. SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to control SRLV spread in sheep and goats. In the last century we have witnessed the emergence of acquired immunodeficiency syndrome, multidrug-resistant tuberculosis and tick-borne related diseases as a result of the interactions between humans and zoonotic pathogens in a pathway including wildlife and domestic animals .Small ruminant lentiviruses (SRLV) infection is present in sheep and goats from Europe , AmericaEnv and LTR genomic regions have been related to cell tropism by modifying the receptor usage  and “empty” p-GL4.10 [luc2] were used as positive and negative controls, respectively. At the same time, 20 ng of pGL4.73 [hRluc/SV40] Vector (Promega) were cotransfected, so that the firefly activity was standardized according to the Renilla luciferase activity. After 24 h, cells were harvested with Passive Lysis 5X Buffer (Promega), and firefly and renilla luciferase activity were measured following manufacturer’s instructions. Results were expressed as: Relative Luminiscence Units (RLU) firefly luminescence/RLU Renilla luminescence.LTR basal activity from strains A4 , KV1772 CAEV   virions Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using Ev1 and 496 infected DSF, FSF and OSF extracts. For Western blot analysis, monoclonal antibodies against p25  was used undiluted. Anti-mouse IgG peroxidase-conjugated (Pierce) was used as secondary antibody. In addition, sera from two experimentally infected sheep with SRLV strains from genotype A and B respectively were used to reveal protein production. Reactions were developed by chemiluminescence .n = 6) were serially diluted in DMEM 2% FBS in a 96-well plate and incubated 1:1 (vol/vol) with 100 TCID50 of strain Ev1. Six wells were used per serum dilution. After mixing and incubating overnight at 4 °C, the mixture was added to sheep fibroblasts. After 2 h at 37 °C, 5% CO2, the supernatant containing the virus and serum mixture was removed and after washing DMEM 2% FBS was added. Following a 7 day incubation at 37 °C and 5% CO2 cytopathic effect  was screened by microscopy. The NtAb titer was defined as the reciprocal of the serum dilution in which 50% of the culture wells showed no signs of infection.Heat inactivated sera from ELISA positive red deer  red deer and one mouflon out of 10 analyzed. Using a secondary antibody able to react against wild ruminant species , increased the number of seropositive red deer to 20 out of 141 (14%) and, moreover, allowed the detection of SRLV antibodies in 3 out of 36 roe deer (8%) and one mouflon, but not in fallow deer. Combining results using protein G and EG5 conjugates SRLV antibodies were detected in 26 out of 259 (10%) red deer, 3 out of 36 roe deer and one out of 16 analyzed mouflon.Single peptide ELISA analyses indicated that peptides 126M1 and 126M2 were responsible for the seropositivity in most of the animals . None of the animals that were seropositive to the peptide ELISA were seropositive to the Elitest with protein G or commercial secondary antibody conjugates. In contrast, 2 red deer that were seronegative to the peptide ELISA were seropositive with the modified Chekit test using EG5 conjugate.Seropositive deer were mainly found (15 out of 20 seropositive deer) in commercial farms which represents the largest sample source, followed by hunting campaigns with 4 seropositive animals. Seropositive roe deer were from hunts and the seropositive mouflon was from a zoo.Seropositive animals were tested for the presence of neutralizing antibodies obtaining titres ranging from 3 to 40.Some bands of the expected size were obtained following PCR amplification using different sets of primers Table . HoweverAfter being unable to demonstrate virus presence in wild ruminants by PCR in vivo, we decided to shift to in vitro experimental infection and transduction experiments. For this, as indicated in the particular experiments, DSF, FSF and BDM cultures were obtained and infected with SRLV strains belonging to genotype A (Ev1 and Swiss A4) or genotype B (CAEV-Co and Ov496). After 16 h, CPE was evident in cells from both species  only in the case of Ev1 virus. Since this effect may correspond to a virus over load, viral stocks were titrated again and infection was repeated with similar results.To clarify if these results correspond to a differential ability of SRLV in entering the cell, entry assays using CAEV-AP virions pseudotyped with envelopes from different SRLV strains were carried out. Although all the tested strains were able to enter into red deer and fallow deer cells, there were differences among them. Red deer cells were more susceptible to Seui, Ev1 and CAEV-Co strains in that order, while FSF mainly allowed the entrance of Ev1 strain from genotype A. Strains Roccaverano, and to a lesser extend 697, showed the lowest values in both cell types Figure .Figure 3Once demonstrated the SRLV ability to enter into red deer and fallow deer cells, we checked further steps of the viral cycle such as integration and RNA production. PCR at 16 h post-inoculation showed positive results in all cases confirming, the permissive entry and the presence of viral DNA within red deer and fallow deer cells. Regardless of the high transcriptional activity of the LTR in fibroblastic cells from deer and fallow deer Figure , RNA proIn spite of these positive correlates of lentiviral productive infection, RT activity in the supernatants was negative in all cases at 7 and 10 days, suggesting low or no production of viral particles to the supernatants Figures A and B. In the case of strains A4 and CAEV-Co, viral RNA was detectable in the supernatants 7 days post-inoculation only in CAEV-Co infected cells. As above, supernatants from these infected-cells were used to infect GSM cells. After 7 days, viral RNA increased in the supernatant for CAEV-Co strain but remained undetectable for A4 strain Figure E.Furthermore, infected DSF and FSF were passed weekly by trypsinization and after the first passage strains Ev1, 496 and CAEV-Co showed increased production only in DSF, being the strain A4 not able to replicate. At the second passage, Ev1 was able to destroy the cellular monolayer whereas strain 496 was adapted to DSF causing persistent infection similar to ovine fibroblasts. Virus production after passages in FSF remained always negative .The emergence of the devastating HIV epidemics in the human population following cross species transmission from the natural non-human primate host, in which these lentiviruses cause non-pathogenic persistent infections , highligTransgression of the species-specific barrier by SRLV has been explored in cattle through experimental infection of cows with CAEV-Co strain . SRLV inHere, we show positive serological reaction against different SRLV antigen preparations in red deer, roe deer and mouflon, being fallow deer negative. However, this serological reaction was not accompanied by the detection of SRLV sequences by PCR in vivo. In vitro, skin cell cultures from red deer and fallow deer did not produce detectable levels of SRLV Gag or RT proteins in spite of the presence of syncytia and viral DNA and RNA. Infection was rescued by transferring supernatants from red deer skin fibroblasts infected in vitro, to fully permissive ovine fibroblasts suggesting the production of low levels of virus. Although highly dependent on the infecting strain, virus adaptation could be induced in DSF through periodical cell passages in vitro. In contrast, fallow deer cells did not support SRLV infection despite supernatant transfer or cell passages in agreement with serological findings. These results may suggest that SRLV are at different steps in the viral adaptation to red deer and fallow deer. In the last case, host may have evolved to mount an effective long-lasting restriction mechanism of lentiviral replication in vivo. Neutralizing antibodies were detected in ELISA positive sera from red deer however, they unlikely play an important role in the viral infection restrictive pathway as described in caprine and ovine species , 50. SerIntra-individual SRLV viral reservoir is represented by proviral integration into monocytes that after maturation to tissue macrophages become permissive to viral replication. Wild ruminants could act as inter-individual reservoir potentially responsible for the re-emergence of infection in domestic small ruminant flocks . The higOdocoileus hemionus) genome [gag region were compared with VMV-like isolates and preliminary analyses indicate some degree of structural similarity between both sets of sequences that might explain the presence of serological reaction in the absence of an exogenous lentivirus in vivo. Unfortunately, it is unknown whether red deer develop a detectable antibody production against CERV.Endogenous retroviruses from cervids (CERVs) have been described in Mule deer  genome , confirm) genome , 58. How) genome . InteresAntigenic cross-reactivity with CAEV in humans has been reported likely due to the consumption of contaminated caprine dairy products  potentiaLack of viral proteins in cultured cells from wild ruminants could be a consequence of the abnormal processing of the Env protein in these cell types. This has been previously described in sheep choroid plexus cells infected with CAEV-like virus . HoweverThe host innate immunity may play an important role in counteracting infection independent of the presence of antibodies which are generated following antigenic presentation in the presence or absence of viral replication. Remarkably, cell factors involved in innate and adaptive immunity may evolve much quicker than those required for cell survival likely contributing to lentiviral resistance. Intracellular restriction mechanisms described in small ruminants include tripartite motif containing (TRIM5α) and APOBEC3, both leading to proteasomal degradation of viral proteins and therefore sharing the MHC-I antigenic presentation pathway that could result in antibody production in the absence of viral replication. On the other hand, tetherin, which impedes normal virus burden at final steps of the infection cycle, may interfere virus release without affecting proviral load or RNA transcripts. Therefore a future study on SRLV restriction due to intrinsic factors from wild ruminants is warranted.In summary, our results suggest that cells from the Cervidae family are susceptible to SRLV infection in vitro but factors involved at post-entry steps are controlling infection spread in vivo. These results may indicate that cervids have a bystander role in SRLV epidemiology.


Distance decay, the general reduction in similarity of community composition with increasing geographical distance, is known as predictor of spatial variation and distribution patterns of organisms. However, changes in fungal communities along environmental gradients are little known. Here we show that distance decays of soil-inhabiting and root-associated fungal assemblages differ, and identify explanatory environmental variables. High-throughput sequencing analysis of fungal communities of beech-dominated forests at three study sites across Germany shows that root-associated fungi are recruited from the soil fungal community. However, distance decay is substantially weaker in the root-associated than in the soil community. Variance partitioning of factors contributing to the observed distance decay patterns support the hypothesis that host trees stabilize the composition of root-associated fungi communities, relative to soil communities. Thus, they not only have selective impacts on associated communities, but also buffer effects of changes in microclimatic and environmental variables that directly influence fungal community composition. A major goal of ecological research is to characterize processes responsible for spatial variation in organism communities134678910The similarity of communities declines with increasing distance1212Numerous environmental factors shape soil161719209242526273132Fagus sylvatica L.) across a north-south transect in Germany. We tested two hypotheses: that due to the strong plant control in its recruitment, RAFCs will be less affected by distance decay than SFCs, and hence environmental parameters  will affect SFCs more than RAFCs.Despite evidence that the two fungal communities differ in habitat and diversity, it is unclear whether their spatial patterns are driven by similar or different processes, which limits our understanding of community shifts and their functional ecosystem-level effects. We have addressed this uncertainty by analyzing root-associated and soil fungal communities in 57 plots in three temperate forest areas dominated by European beech  were obtained from 114 root and soil samples from 57 beech-dominated plots spread across three areas along a north-south transect across Germany. Subsequent quality filtering resulted in 454,641 sequences . After removing plant sequences in the root dataset and chimeric sequences in both datasets, 249,746 sequences  remained. The sequence read normalization procedure resulted in 95,760 sequences representing 1,415 reads per soil sample and 265 reads per root sample. Merger and further clustering of the two datasets resulted in 5,090 fungal operational taxonomic units (OTUs) including 3,528 rare OTUs (with ≤3 reads). Removal of the rare taxa had no significant effect on the composition of the fungal community . Consequently, only the 1,562 abundant fungal OTUs were used to test our hypotheses.Overall 88% of the 1,562 fungal OTUs obtained could be assigned to a fungal phylum. Members of the phylum Basidiomycota represented the majority of the fungal communities . NMDS orIn the soil fungal community OTU richness and Shannon diversity were higher than in the root community . Most of the root-associated fungal OTU were also found in the SFC . In total, 1,037 OTUs (around 66%) were unique to soil and only 99 (6%) were unique to roots .Lactarius, Russula and Inocybe and 60 additional fungal genera belonged to the shared fungal communities  on the fungal communities  explained 10.1% and 7.5% of the SFC and RAFC variance, respectively, and the RAFC responded to changes in precipitation. These findings are consistent with expectations as precipitation and temperature are linked to soil moisture, which correlates with soil fungal community composition1144In conclusion, the significant difference in distance decay between the soil-inhabiting and root-associated fungal communities is consistent with dispersal limitation theory, and partly attributable to effects of spatial changes in soil properties. Consequently, no cosmopolitan distribution patterns were detected in either root-associated or soil fungal communities. Since our study was limited to three biogeographic areas, further investigations are required to address the assembly rules of fungal communities across larger scales including temporal patterns. Moreover, our results also indicate the importance of the host tree species and future studies need to address the interactive effects of soil physico-chemical properties, host plant root traits and exudation patterns, to improve mechanistic and functional understanding of the rhizosphere microbial communication and shifts in community composition. In general our results provide a stimulating insight for new ideas of theoretical models related to fungal meta-communities, biogeography and landscape ecology.The study was performed in the Biodiversity Exploratories: three study areas along a 600 km geographical transect, located in the south-west (Swabian Alb), centre (Hainich-Dün) and north-east (Schorfheide-Chorin) of GermanyTable S34917In early May 2011 soil was sampled from each of the 57 experimental plots at all study areas, by collecting 14 soil cores of 5 cm diameter and 10 cm depth (after removing organic litter) at points 1, 7, 13, 19, 31 and 37 m from starting points of two 40 m transects (north-south and east-west). The cores obtained from each plot were sieved , mixed into a composite sample, and 50 g of each composite sample was stored at −80 °C for molecular analysis. After sieving, the root samples were collected and pooled separately. About 2 g samples of roots were washed in water that had been deionised and sterilised using a USF Seralpur System  with a DCF CHS92DE Delta Supor Filter  at 4 °C, then frozen in liquid nitrogen. The root samples were stored temporarily at −80 °C then freeze-dried using a P4K-S System  and PK4D vacuum pump  starting at −60 °C rising to −20 °C for four days and finally stored at room temperature.Microbial genomic DNA was extracted from two independent 0.5 g frozen subsamples of each composite soil sample using MoBio Power Soil DNA isolation kit  following the manufacturer’s recommendations. The two soil DNA extracts per sample were pooled and their DNA concentrations were quantified using a NanoDrop UV-Vis spectrophotometer . Although the ITS primers are known for their taxonomic bias towards Ascomycota and Basidiomycota by favoring certain sequence lengths during PCR, they are the established barcodes for identifying fungal communities from environmental samples4, 1 μl dNTP Mix (10 mM) 0.5 μl Pfu DNA polymerase , 2 μl of each primer (10 μl) and 4 μl of DNA template (diluted 1:10). We performed touchdown PCR following the same procedures as for soil DNA. All samples were amplified in triplicate, and products were purified using the Qiagen gel extraction, as above, following the manufacturer’s instructions. After the amplification DNA concentrations were measured using a NanoDrop UV-Vis spectrophotometer and the PCR products of each triplicate were pooled equimolarly. A unidirectional pyrosequencing from the ITS4 end of the amplicons was performed using a 454 titanium amplicon sequencing kit and the Roche GS-FLX 454 pyrosequencer  following the instructions of the manufacturer at the Göttingen Genomics Laboratory (Germany).The freeze-dried root samples were ground in a MM2 ball mill , and DNA was extracted from them following the same procedure as for soil samples using the MoBio Power Soil DNA isolation kit. The fungal ITS rDNA fragment was amplified using the ITS3 primer pairThe soil- and root-based 454 ITS sequences were processed and quality-filtered on multiple levels using MOTHUR software51http://www.jennajacobs.org/R/rarefaction.html) we obtained a table with values for each curve, so we could retrieve associated sequence values (1415 sequences per sample SEW47_soil) for our calculated numbers of OTU (169.63 SEW47_soil). The following formulas summarize our procedure:A crucial requirement in any comparison of microbial community datasets is sampling to species saturation or, if this is not possible, using the same or normalized sample numbers . We decided to normalize the datasets based on rarefaction curves rather than sample numbers because we expected the RAFC to be a subset of the SFCIn our specific case:Finally, the normalization process was repeated 10 times for both soil (1415 sequences per sample) and roots (265 sequences per sample) by random removal of sequence reads using the subsample command as implemented in MOTHURIn order to select the most appropriate dataset for our analysis, we performed Procrustes-based tests of the ten datasets, by applying the protest function2 (1:2.5) was determined using a glass electrode. Ground mineral soil samples (<100 μm) were analyzed for total carbon (C) and nitrogen (N) by dry combustion with a Vario Max CN analyzer . After removing organic C by combusting samples for 16 h at 450 °C, inorganic C was determined using the same method. Organic C concentrations were then calculated from the differences between total and inorganic C concentrationsAll soil analyses were performed with air-dried samples sieved at <2 mm. The pH of supernatants of duplicate suspensions of the soil samples in 0.01 M CaClSoil texture was determined using the pipette methodAir temperature and precipitation data were extracted, for grid cells for each of our study plots, from the Bioclim subset of the Wordclim datasetForest plant community inventory data  was extracted for each of our study plotsp < 0.001, suggesting nearly identical ordination). We also tested the need for re-normalization of the abundant fungal OTU data matrix. We compared the congruence of the NMDS plots based on the dominant fungal OTU data matrix and on a re-normalized abundant OTU data matrix using Procrustes correlation analysis. We found that this normalization step did not affect the fungal community composition . Hence, all subsequent analyses were performed using the fungal community matrix excluding singletons, doubletons and tripletons.Statistical analyses were performed using R software (version 3.1.1)The proportions of unique OTUs and those shared by the soil and root compartments were visualized as Venn diagrams using the R package VennDiagramTo examine spatial variation of the fungal community and relationships of its composition to environmental factors, we computed Mantel statisticsIn order to understand the relative contribution of geographical location, vegetation, climatic and soil conditions in shaping the fungal communities in soil and roots we performed variance partitioning. For this purpose we used the capscale command as implemented in veganAccession code: The raw ITS rDNA sequences have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under study accession number SRP070568 (PRJNA312066).How to cite this article: Goldmann, K. et al. Divergent habitat filtering of root and soil fungal communities in temperate beech forests. Sci. Rep.6, 31439; doi: 10.1038/srep31439 (2016).


Traditional modes of investigating influenza nosocomial transmission have entailed a combination of confirmatory molecular diagnostic testing and epidemiological investigation. Common hospital-acquired infections like influenza require a discerning ability to distinguish between viral isolates to accurately identify patient transmission chains. We assessed whether influenza hemagglutinin sequence phylogenies can be used to enrich epidemiological data when investigating the extent of nosocomial transmission over a four-month period within a paediatric Hospital in Cape Town South Africa. Possible transmission chains/channels were initially determined through basic patient admission data combined with Maximum likelihood and time-scaled Bayesian phylogenetic analyses. These analyses suggested that most instances of potential hospital-acquired infections resulted from multiple introductions of Influenza A into the hospital, which included instances where virus hemagglutinin sequences were identical between different patients. Furthermore, a general inability to establish epidemiological transmission linkage of patients/viral isolates implied that identified isolates could have originated from asymptomatic hospital patients, visitors or hospital staff. In contrast, a traditional epidemiological investigation that used no viral phylogenetic analyses, based on patient co-admission into specific wards during a particular time-frame, suggested that multiple hospital acquired infection instances may have stemmed from a limited number of identifiable index viral isolates/patients. This traditional epidemiological analysis by itself could incorrectly suggest linkage between unrelated cases, underestimate the number of unique infections and may overlook the possible diffuse nature of hospital transmission, which was suggested by sequencing data to be caused by multiple unique introductions of influenza A isolates into individual hospital wards. We have demonstrated a functional role for viral sequence data in nosocomial transmission investigation through its ability to enrich traditional, non-molecular observational epidemiological investigation by teasing out possible transmission pathways and working toward more accurately enumerating the number of possible transmission events. Despite the existence of infection control policies and protocols, nosocomial transmission of respiratory viruses is a common problem that can occur in virtually any health-care setting –7. The cTraditionally, investigation of influenza nosocomial transmission has required a combination of confirmatory influenza diagnostic testing  and epidemiological investigation. PCR-based molecular diagnostic assays are generally limited in their capacity to classify etiologic agents beyond the type/subtype level. While this is acceptable for the diagnosis of uncommon hospital acquired infections (HAIs), for more common causes of HAIs such as influenza it is desirable to apply assays that yield enough data to more accurately distinguish unique patient transmission chains. In conjunction with epidemiological data such assays could be effectively used to test whether patient infections have a nosocomial origin , 14–17.Continued innovation in pathogen sequencing techniques have enabled the rapid and cost-effective generation of greater volumes of molecular genetic data from routine diagnostic assays . For rapRecently, influenza nosocomial transmission investigations have employed molecular techniques to corroborate comprehensive epidemiological data sets –7, 34. T0) [The potential analytical power afforded by being able to use sequence data to accurately infer close epidemiological linkages has been highlighted in large-scale Influenza sequencing studies that identified sequence variations that arose within single individuals during infection and were then transmitted to multiple individuals residing in the same household/hospital ward –38. The 0) , 42–47.Among the most commonly applied of these new tools are those implemented in the computer program Bayesian Evolutionary Analysis Sampling Trees (BEAST) . The BayDespite these notable analytical advances in recent years only a few studies have investigated influenza nosocomial transmission at the nucleotide level. Most nosocomial infection studies have simply focused on observing patient Influenza like illness (ILI) symptoms and/or conducted confirmatory influenza molecular tests. In South Africa there is no reliable information available on the frequency of nosocomial respiratory virus transmissions. There is similarly sparse information on the proportions of HCWs who are vaccinated against influenza and other respiratory viruses. It is, however, assumed that vaccine compliance is low amongst South African HCWs, as is the worldwide trend, which is concerning given that increased influenza vaccine coverage amongst HCW correlates with decreased incidence of ILI in hospitalised patients –57.In order to implement a cost effective vaccination strategy and evaluate the effectiveness of infection control procedures in an environment where influenza nosocomial transmission is assumed to be common, the burden of nosocomial disease needs to be evaluated. Here we assess whether an influenza sequence phylogenetic analysis could, by enriching basic epidemiological data, assist with investigating transmission chains and the frequency of nosocomial transmission within Red Cross War Memorial Childrens Hospital (RXH) in Cape Town South Africa. Over a 4-month period, influenza A positive patient samples from various wards in the hospital were obtained and partial influenza hemagglutinin sequences were generated for each patient. Possible transmission chain/channels were determined through a combination of basic patient admission data and the analysis of Maximum likelihood and time-scaled Bayesian phylogenetic trees, to identify potentially epidemiologically linked transmission chains. The inclusion of a time-scaled phylogenetic analysis allowed us to estimate times to most recent common ancestors of epidemiologically and phylogenetically linked samples with statistical support to evaluate the likelihood of phylogenetically related infections sharing a common transmission source.Archived influenza positive patient RNA samples were obtained from the National Health Laboratory Service (NHLS) within Groote Schuur Hospital in Cape Town, South Africa, which routinely conducts respiratory virus diagnostic PCR testing for surrounding public hospitals including RXH using the Seeplex ® RV7 respiratory virus detection system (Seegene). RXH is the largest tertiary paediatric hospital in South Africa and manages approximately 260 000 patient visits per year. Most of its patients are from poor communities, a third of which are less than one year old. Although > 98% of patients are referred to the hospital from the Western Cape region of South Africa, referrals from the rest of South Africa are also common as a result of the unique and high level of paediatric care available at the hospital. For the period spanning 1 April to 31 July 2011, a total of eighteen samples were obtained. Fourteen were confirmed Influenza A, pandemic H1N1 (H1N1pdm) positive RNA samples from pediatric patients admitted to RXH, which formed the focus of our study. A further four samples, served as geographic controls and were obtained from three other hospitals within the same region during the same time period; 1 from Groote Schuur Hospital (GRS), 1 from 2-Military Hospital (2MIL) and 2 from Somerset Hospital (SOM). Clinical history and hospital admission dates were abstracted from hospital records of the RXH patients from whom virus samples were obtained. This study was approved by the University of Cape Town Human Research Ethics Committee (UCT research ethics number: 305/2012). To ensure patient anonymity, study samples were unlinked by being issued with a study identification number. For the purpose of this investigation the transmission case definitions were as follows: (1) a suspected hospital-acquired influenza infection (HAI) case was defined as an influenza PCR positive individual who did not present to the hospital with respiratory symptoms or was confirmed to be influenza negative by laboratory testing within three days of admission ; and (2)cDNA was synthesized using a random hexamer primer set with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to manufacturer’s instructions. All influenza A genomic segments were amplified using a multisegment PCR with the universal primer set, MBTuni-12 (5’-ACGCGTGATCAGCAAAAGCAGG-3’) and MBTuni-13 (5’-ACGCGTGA TCAGTAGAAACAAGG-3’) and PCR conditions adapted from the RT-PCR method previously described . InfluenPotential nosocomial transmission clusters were identified by examining the phylogenetic relationships among the sequences in our dataset. Fourteen HA sequences from RXH and four geographic controls from surrounding hospitals were analysed together with homologous H1N1pdm HA sequences sampled from elsewhere in South Africa (n = 53) (GenBank accession numbers JF745847 to KT036481) during the same flu season and a representative set of sequences from other continents (n = 34), all available in GenBank -like virus, accession number: KF527477) were determined using BioEdit Version 7.2.5.http://beast.bio.ed.ac.uk/) [http://beast.bio.ed.ac.uk/Tracer). The resultant trees were summarised using Tree annotator (http://beast.bio.ed.ac.uk/TreeAnnotator) after discarding the first 10% of trees in the Marcov chain as burn-in. This summarisation process generated a maximum clade credibility (MCC) tree with branches supported by an associated posterior probability of > 0.95 considered as having significant statistical support. The ML and MCC trees were visualized using FigTree .Time-scaled Bayesian MCMC trees were constructed using BEAST available at (.ac.uk/)  and were.ac.uk/)  assumingThe influenza cases had been admitted to five different wards  at RXH. The study sample size was limited by the low number of influenza positive samples acquired from RXH during the study period (12 April- 1 July 2011). It is noteworthy that influenza detection between these dates fell within the beginning and peak of the influenza detection season for 2011 (epidemiologic weeks 15 to 26) in the Western Cape, as determined by the South African Viral Watch, Influenza Surveillance program (Bar Graph in Transmission case definitions identified nine suspected HAI cases and five CAI laboratory-confirmed H1N1pdm cases within the observed time period (April-July 2011). HAI cases were distributed across physically separated wards E1 (n = 3), E2 (n = 2), ICU (n = 3) and B2 (n = 1) while CAI cases were distributed across E1 (n = 1), ICU (n = 3) and MREG (n = 1).The time-point post-admission at which patients were found to be H1N1pdm positive was assumed to be the earliest time-point when ILI symptoms were noticed by attending clinicians . The lenNested PCR was used to generate partial Hemagglutinin (HA) amplicons spanning HA nucleotide positions 379 to 1204 (HA-1 sequence domain). Eighteen ~800 base pair amplicons were sequenced, aligned with 105 publically available H1N1pdm sequences  and used to construct a maximum likelihood tree. . In thisThis analysis revealed four strongly supported (74–87% bootstrap support) potential RXH transmission clusters (clusters 1–4) containing 16 of the 18 Cape Town sequences  phylogenies were constructed using BEAST. By the explicit inclusion of sampling times and estimated H1N1pdm evolutionary rates during the construction of the MCC tree, we were able to estimate the dates of the nodes in the tree. The dates of the root nodes of the four RXH clusters  were used to indicate whether they could plausibly have been the consequence of HAIs. Similar to the ML phylogenetic analysis, putative HAI transmission clusters were inferred if two or more sequences clustered with 0.95 or higher posterior probability support. Based on this criterion, exactly the same four RXH potential HAI transmission clusters were identified in the MCC tree  as were Cluster 1 consists of five isolates sampled from individuals that were admitted to RXH, during a 22 day period between 12 May and 3 June 2011. Within a period of three days all of these individuals were confirmed by laboratory testing to have H1N1pdm infections Figs .The other sequences in cluster 1: 1.2 and 1.3, differed from 1.1 a-c by one and three mutations, respectively. To determine the plausibility that the most recent common ancestor (MRCA) of all five of these sequences was present in RXH we estimated the date of the root node of this cluster. The date of this node was estimated, with 95% confidence to have been between the 26 January and 6 May 2011 with 26 March being the most probable date Figs .The three identical sequences in this cluster 1.1 a–c have a mean estimated tMRCA of 7 May (range: 31 March-3 June), the upper limit of which coincides with the admission of patient 1.1 b, the presumed CAI infection in this cluster. 1.1 a and 1.1 c (the two probable HAI samples) have a mean estimated tMRCA of 24 May (range: 22 April-4 June), which is well within the period of admission of these patients implying that it is plausible that these viruses have a common hospital-derived source. While it is also plausible that patients 1.1 a and 1.1 c acquired their infections either directly or indirectly from a hospital source, due to the brief time lapse between of their diagnoses/symptoms relative to when patient 1.1b was admitted it is questionable whether these patients acquired their viruses from patient 1.1 b.Cluster 2 consists of six related sequences, three of which were sampled from RXH. Individuals 2.1 a (with a suspected CAI) and 2.1 b (with a suspected HAI) were infected with viruses that had identical HA sequences  both demCluster 3 consists of 3 unique sequences, all derived from RXH 3.1–3.3) . Patient–3.3 . PaCluster 4 had two identical RXH-derived isolates (4.1 a and 4.1 b) isolated from RXH patients that were admitted six days apart to two different wards (E1 and ICU) and were confirmed as H1N1pdm positive by laboratory-testing within 4 days of one another, neither was observed to have ILI symptoms at admission. Patient 4.1 b qualified as having a potential CAI testing H1N1pdm positive two days post admission (admitted after Patient 4.1 a). Patient 4.1 a on the other hand had a suspected HAI and tested H1N1pdm positive at four days post admission. The MCC tree supports the inference that these infection instances are unlikely to have a common hospital source as the mean estimated tMRCA of 3 June (range: 29 April-13 June), the upper limit of which coincides with the admission of patient 4.1 a, the patient in this cluster with a likely CAI.To determine the value of the insights derived from the influenza sequence analyses, we compared our interpretations to those derived from an epidemiological study conducted on a subset of our samples. Aside from an Influenza positive PCR, the epidemiological study used no other molecular techniques. Epidemiological observations was available for nine RXH patients  in 3 of the 4 clusters of interest  and are summarised in Based on the molecularly indistinguishable Influenza instance data shown in This study demonstrated the value of including molecular sequence analysis as a diagnostic tool to identify nosocomial infections and trace the origins of viruses causing these infections. We have shown how such sequence data could enrich more traditional observational epidemiological data to reveal what may appear to be individual transmission chains but may in fact be multiple independent chains.Unexpectedly, with the available data we were unable to conclusively identify patient sources of infection for any of the potential HAI instances identified . Furthermore, temporally-scaled phylogenetic analysis suggested that most instances of potential HAI in RXH—including instances where virus HA sequences were identical between different cases—likely resulted from independent introductions of Influenza A viruses into the hospital. Our inability to establish epidemiological transmission linkage of cases/viral isolates could be attributed to limited sampling in RXH during the study period which could imply that the viruses we detected originated in either asymptomatic hospital patients, visitors, or hospital staff. Transmission from asymptomatic patients is serious in the context of South Africa’s high HIV prevalence. While studies have demonstrated that HIV-infection status alone in adults does not increase risk of Influenza infection, evidence suggests that prolonged Influenza shedding periods (> 1 week) can be observed in patients exhibiting non-HIV related immunosuppression , 63. SucFurthermore, while we are also unable to confirm the vaccination status of patients, it is unlikely that they were vaccinated as the South African public sector is not provided with free access to flu vaccination. Flu vaccination via herd immunity may nevertheless have mitigated transmission from and between vaccinated adults. Approximately 75% of all influenza positive cases in South Africa during the 2011 influenza infection season were as a result of influenza A(H1N1)pdm09 virus infection . The genNevertheless, the protective effect of herd immunity would be limited as influenza vaccination compliance in the general public and HCWs in South Africa is expected to be low. This occurs despite its potential to reduce morbidity in HCWs and limit transmission to patients, which might invaluably contribute to the maintenance of the workforce during outbreaks. In a bid to increase flu vaccination compliance some health-care institutions have provided HCWs with a voluntary, free immunization program , 64. In Limitations of study include an incomplete epidemiological analysis of study patients to account for patient movements within the hospital. The epidemiological observations excluded five of the 14 RXH patients of which two were potential HAIs. Patients 1.1 c and 2.1 b developed infection symptoms within the RXH ICU at nine and 17 days post hospital admission, respectively. Influenza PCR-based testing was unable to determine whether they acquired the infection within this ward. However, access to this information would not have aided our ability to establish virus transmission linkage. Furthermore, the identification of HAI sources may ultimately require exhaustive longitudinal sampling and screening of hospital staff, visitors and patients, including review of patient records and collection of other epidemiological data on questionnaires throughout the duration of a single influenza season. Such a large-scale study would also likely benefit from analyses of larger sequence fragments , which would provide greater analytical power with respect to resolving clusters within phylogenetic trees for the identification and characterization of likely virus transmission chains within the hospital setting.S1 Fig(PNG)Click here for additional data file.S1 Table(PDF)Click here for additional data file.