Amycolicicoccus subflavus DQS3-9A1T is the type strain of a novel species in the recently described novel genus Amycolicicoccus, which was isolated from oil mud precipitated from oil produced water. The complete genome of A. subflavus DQS3-9A1T has been sequenced and is characteristic of harboring the genes for adaption to the harsh petroleum environment with salinity, high osmotic pressure, and poor nutrient levels. Firstly, it characteristically contains four types of alkane hydroxylases, including the integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153, a long-chain alkane monooxygenase (LadA) and propane monooxygenase. It also accommodates complete pathways for the response to osmotic pressure. Physiological tests proved that the strain could grow on n-alkanes ranging from C10 to C36 and propane as the sole carbon sources, with the differential induction of four kinds of alkane hydroxylase coding genes. In addition, the strain could grow in 1–12% NaCl with the putative genes responsible for osmotic stresses induced as expected. These results reveal the effective adaptation of the strain DQS3-9A1T to harsh oil environment and provide a genome platform to investigate the global regulation of different alkane metabolisms in bacteria that are crucially important for petroleum degradation. To our knowledge, this is the first report to describe the co-existence of such four types of alkane hydroxylases in a bacterial strain.The moderate halophile  For example, several types of alkane hydroxylases have been characterized to hydroxylate n-alkanes to alcohols, which are further oxidized to the corresponding aldehydes and fatty acids and finally converted to acetyl-CoA via the β-oxidation pathway. The integral-membrane non-heme di-iron monooxygenase (AlkB) alkane monooxygenase (LadA) have been reported to be involved in long-chain alkane (>C18) metabolism Dietzia strains n-alkanes is attributable to some unknown hydroxylation pathways or to the co-existence of multiple types of hydroxylases in the same strain.Microbial enhanced oil recovery (MEOR) is one of the tertiary methods purported to increase oil recovery. Degradation of hydrocarbons such as Amycolicicoccus subflavus DQS3-9A1T with the ability to degrade crude oil and grow in 1–12% (wt/vol) NaCl alkB homologous genes and a complete n-alkane metabolism pathway n-alkanes with different chain lengths. In addition, the genes required for living in environments with different salinities have been analyzed. Further transcriptional analysis suggested the possible functions of these genes. To our knowledge, this is the first report that a halophilic bacterial strain can harbor most of the known hydroxylase gene homologs that allow the strain to grow on a wide range of n-alkanes.Recently, we isolated a moderate halophile A. subflavus DQS3-9A1T was isolated from oil “mud,” which was sedimented from oil produced water at Daqing Oilfield, China A. subflavus DQS3-9A1T was performed with a combined strategy of 454 sequencing The strain http://www.pathogenomics.sfu.ca/islandviewer) http://img.jgi.doe.gov) of the Joint Genome Institute for detailed analysis and genome comparison t-test was used to evaluate the statistically significant differences of the function profiles based on cluster of orthologous groups (COG) between A. subflavus DQS3-9A1T and genomes deposited in the IMG bacteria genome database. Briefly, the data about the abundance of a COG category distributed in each genome were transformed into normal distribution data using Box-cox transformation t-test was performed using the SPSS program . Genes that could be functionally assigned according to the KEGG Orthology classification system (http://pathways.embl.de/iPath2.cgi) Protein coding genes were predicted using Glimmer 3.0 n system  were usen-alkanes, the strain DQS3-9A1T was first grown in lysogeny broth (LB) at 30°C till the middle exponential phase. The cells were then collected by centrifugation , washed 3 times with sterile phosphate-buffered saline, and finally suspended in sterile minimal salt medium 2SO4, 1 g K2HPO4, 0.2 g MgSO4, and 3 g KNO3 per liter of deionized water, pH = 7.2) to make the inoculum cell suspension. For growth tests, the cell suspension was inoculated into 100 mL of MSM in 300-mL flasks  containing 0.1% (vol/vol) MT microelements , supplemented with 0.2% liquid n-alkanes  (vol/vol) or 0.1% solid n-alkanes  (wt/vol) as the carbon sources. To examine the growth on gas alkanes , the cell suspension was inoculated into 40 mL of the above MSM containing MT microelements in 100-mL gas-tight serum bottles with 60-mL headspace of air. Then 20 mL of gas alkanes were pressurized into each bottle using syringe. Cultures with 1% (wt/vol) glucose as the sole carbon source were used for comparison. In addition, cultures containing cells without carbon source were used as controls for calculating the background growth baseline. Cell growth was detected by the increase of OD600 in the culture with time. Six duplicates  of all cultures were made and incubated in the dark at 30°C while shaking at 150 rpm. To examine the transcriptional levels of putative genes for alkane degradation, three parallel flasks of each culture in the middle exponential phase were sampled for RNA extraction and reverse transcription PCR. Cells grown in 1% glucose were collected as the control for transcriptional analysis. For the n-alkane degradation test, the cell suspension was inoculated into 100 mL of the above MSM containing MT microelements, supplemented with C14, C16, C20, C24, C28, C32, and C36 n-alkanes as the sole carbon source. Cultures containing hydrocarbons without cells were used as controls for calculating the background evaporation of the n-alkanes. At 8 days after incubation, residual n-alkanes in the culture were extracted and detected, as described previously To examine growth on 2SO4, 0.68 g KCl, 0.1 g KBr, 0.025 g H3BO3, 5.4 g MgCl2·H2O, 1.5 g CaCl2·2H2O, 0.024 g SrCl2·6H2O, 0.2 g NaHCO3, 0.04 g Na2HPO4, 0.5 g NH4Cl, and 0.002 g NaF per liter of deionized water, pH 8.0), with NaCl added at the indicated concentration when necessary. The cell growth was determined by detecting OD600 with time. To examine the transcriptional levels of putative genes for osmosensing and responding, cells were grown in the modified ASW medium till the middle exponential phase, followed by the addition of NaCl into the medium at a final concentration of 4% NaCl. Cells were then collected at 15 min, 30 min, 60 min, and 24 h after NaCl was added. Cells harvested before NaCl was added were used as the control for transcriptional analysis.To test growth with different NaCl concentrations, cells were grown in modified artificial seawater (ASW) medium g for 5 min, 4°C). Total RNA from the above samples was extracted using TRIzol Reagent (Invitrogen), treated with DNase I, and purified according to the manufacturer’s instructions. Reverse transcription was performed using 0.5 µg of total RNA with random primers and the ReverTra Ace reverse transcription kit (ToYoBo). Specific cDNA was then quantified using real-time PCR  with the SYBR Green Ex Taq Kit II (TaKaRa). The specific gene primer pairs are shown in Table S1 in t method n-alkanes and cells responding to osmotic stress, respectively. All the experiments were performed in triplicate.Immediately after the samples for real-time reverse transcription PCR were obtained from the cultures, ice-cold stop solution  was added at 10∶1 (vol:vol) ratio. Samples were then maintained on ice for 10 min before being collected using centrifugation  and two plasmids  (The complete genome of 784 bp])  that con784 bp])  and dist784 bp]) .T was significantly (P<0.001) more abundant in the genes responsible for energy production and conversion (C); lipid transport and metabolism (I); secondary metabolite biosynthesis, transport, and catabolism (Q); and transcription (K)  . In conttion (K) .T genome is also characteristic, with its low content of potential mobile genetic elements. Six and 2 GIs were predicted using the IslandPath - DIMOB and SGI-HMM T and 118 bacteria selected to represent the gene transfer of all bacterial genomes in the IMG collection T . The third type of hydroxylase found was a LadA homolog coding gene (AS9A_3890)  encoding multiple components of propane monooxygenase was also found in the genome of DQS3-9A1T   with 52%QS3-9A1T , with 90Escherichia coli, MtrA/MtrB TCS from Corynebacterium glutamicum for osmosensing, Trk-type K+ transport systems + accumulation, and glycine betaine transporter (BetP) for the uptake of compatible solutes T was isolated from oil “mud” sedimented by oil produced water with the concentration of total dissolved solids of around 8,000 mg/L, it could grow in 1–12% NaCl. A number of genes for osmosensing and regulation genes were found in the DQS3-9A1T genome , in response to K+ limitation or salt stress +/H+ antiporters (Nha) + transport systems +/H+ antiporters (Mnh) Bacteria are frequently exposed to osmotic stresses. In defense, bacteria may accumulate compatible solutes like amino acids and their derivatives, oligosaccharides, and glycosides in their cytoplasm. A number of osmoregulated receptors and transport systems have been investigated. These include KdpD/KdpE and EnvZ/OmpR two component systems (TCSs) from T strain grew with C10–C36 n-alkanes and propane as the sole carbon sources  were upregulated by C10–C36 n-alkanes and propane (C3), with the highest expression level detected at C16–C24. The transcription of the CYP153 homologous gene AS9A_2813 was mostly induced by C20 and C24 but not by C10 to C16, and it was distinct from another CYP153 coding gene AS9A_4287, which was induced by C10 to C20. The results suggested the different roles of these two CYP153 genes in the degradation of n-alkanes. The transcriptional level of putative ladA (AS9A_3890) was induced from C16 to C36, with the highest expression level at C24; this indicated its potential role in the degradation of long-chain-length n-alkanes. One of the subunits of the putative propane oxidation gene (prmC) AS9A_2157 was significantly upregulated with propane as the sole carbon source and only slightly induced by C16 and C36. The transcriptional profiles of these alkane hydroxylase genes suggested a potential “team” of multiple alkane hydroxylases in strain DQS3-9A1T during n-alkane degradation, which needs to be further researched for more biochemical evidences.The DQS3-9A1tion PCR . The traT could grow in 1–12% NaCl without significant growth differences between 1% and 4% NaCl  was added into the culture medium , which were flanked by KdpD/KdpE (AS9A_4249 and AS9A_4250) and seemingly grouped together as one operon, were induced after 30 min following exposure and decreased from 60 min to 24 h. In contrast, the expression of another Trk-type K+ transporter coding gene (AS9A_0248 and AS9A_0249) did not change with incubation time. Moreover, the transcriptional level of genes coding for Na+/K+ antiporter (Nha)  were upregulated at 30 min after exposure and were back to the control (untreated culture) level after 60 min. The gene (AS9A_1552) coding for multiple subunits of Na+/K+ antiporter (Mnh) was also upregulated at 30 min after exposure; however, another gene (AS9A_0231) coding for MnhA was induced at 60 min after exposure. These results suggested that the strain could resist osmotic stress by importing H+ and pumping out Na+, in addition to K+ uptake. Gene encoding MtrB (AS9A_3791), the histidine kinase of the MtrA/MtrB TCS system, was upregulated 30 min after exposure and reached its maximal transcriptional expression level at 60 min and 24 h after exposure. However, expression of the gene encoding MtrA (AS9A_3792), which is the transcriptional regulator of MtrA/MtrB TCS, was not induced after exposure to osmotic stress. As for the compatible solute transport and synthesis functions, genes AS9A_1011 and AS9A_2468 coding for BetP were induced at 30 min after treatment, but the putative betP gene AS9A_3514 was not changed. Among these genes, the putative betP genes AS9A_0979 and AS9A_2953 showed the highest expression level at 24 h after treatment and were likely to be important for long-term adaptation to osmotic stress. The gene coding for the Na+- and Cl–dependent choline cotransporter (AS9A_1074) was also upregulated 30 min after treatment, whereas other ABC-type compatible solute transporter encoding genes were not changed.Strain DQS3-9A1e medium . As for A. subflavus DQS3-9A1T was isolated from oil “mud” that contained sediment from oil produced water. The bacterium has a lower abundance of genes in COG categories of cell duplication (L), intracellular trafficking, secretion, and vesicular transport (U), cell wall/membrane/envelope biogenesis (M), cell motility-related genes (N), as well as genes for carbohydrate transport and metabolism (G), compared with those in bacterial genomes released. The loss of redundant genes in carbohydrate metabolism did not influence the bacterium’s life functions, which was confirmed by the fact that the strain could utilize various carbohydrates, including D-fructose and D-glucose, as the sole carbon source Polymorphum gilvum SL003B-26A1TT, including sensing and responding to environmental factors, regulating the expression of target genes, transporting harmful byproducts out of the cells, or producing protective materials. Two temporal gene regulation responses were found during the osmotic stress in strain DQS3-9A1T. First, rapid increase of the transcriptional levels of the genes coding for KdpD/KdpE TCS and Trk-like K+ transporter suggested that a rapid K+ influx by Trk-like K+ transporter under the control of KdpD/KdpE TCS and possible accumulation of K+ inside the cells was induced to resist the osmotic stress caused by the high concentration of NaCl. Meanwhile, genes encoding Mnh and Nha were also upregulated immediately after osmotic stress, suggesting an immediate efflux of Na+ and influx of K+ by multiple K+ and Na+ transporters in the early phase of adaptation.The genes involved in responding to osmotic stresses provide a clear pathway for osmotic tolerance of DQS3-9A1betP genes, AS9A_0978 and AS9A_2953, showed the highest expression levels 24 h after treatment. Moreover, upregulation of the membrane-bound histidine kinase MtrB, which leads to the induction of betPmtrA was not induced in osmotic adaptation, compared with the high expression level of mtrB 60 min after osmotic stress. This result suggested that mtrA and mtrB were transcribed by different promoters. A similar case was also observed in Mycobacterium, and 2 unique promoters were found in front of the individual genes + efflux and compatible solute transport and biosynthesis were upregulated following exposure of the DQS3-9A1T strain to osmotic stress, e.g., mnh operons, Na+/H+ antiporters, and Trk family K+ transporters  in DQS3-9A1n-alkanes), which are substrates that are relatively easy to use in comparison with other petroleum components. Alkane degradation is a complex process that includes the synthesis of glycolipids and/or lipopeptides as biosurfactants n-alkane degrading microorganisms have a higher abundance of genes for lipid metabolism T, which had a significantly higher than average abundance of these genes when compared to all known whole genomes in the IMG collection.For many bacteria living in oil reservoirs, hydrocarbons may be the main carbon resources. The main components of crude oil are aliphatic hydrocarbons  could be induced by n-alkanes ranging from C10 to C36, suggesting their potential functions in degradation of medium- and long-chain n-alkanes. Both CYP153 genes clustered in an operon-like structure, with the difference being that AS9A_4287 was possibly under the regulation of AraC while AS9A_2813 was under the regulation of TetR , phenol 2-monooxygenase (for phenol and toluene degradation), and catechol 1,2-dioxygenase and catechol 2,3-dioxygenase . It is interesting to note that Polymorphum gilvum SL003B-26A1T, isolated from a similar environment as that for DQS3-9A1T, displays an opposite gene content in that it actually contains many genes and complete pathways for aromatic compound degradation but relatively few genes for alkane degradation Although genes related to A. subfalvus DQS3-9A1T has an efficient alkane metabolism together with a functioning osmosensing system, allowing it to adapt well to the oil field environment. More specially, the strain DQS3-9A1T harbors propane monooxygenase, CYP153, AlkB and LadA  and bioremediation under saline conditions.In conclusion, the genomic, transcriptional, and physiological analyses we describe here show that File S1Amycolicicoccus subfalvus DQS3-9A1T with different n-alkanes as the sole carbon source. Figure S3, The degradation ratio of n-alkanes by Amycolicicoccus subfalvus DQS3-9A1T. Figure S4, The growth of Amycolicicoccus subfalvus DQS3-9A1T with different concentrations of NaCl. Table S1, Primers used in this study. Table S2, Comparative analysis of COG categories between Amycolicicoccus subfalvus DQS3-9A1T and other selected genomes in the IMG bacterial genome database. Table S3, Genomic island prediction by different methods. Table S4, Genes in GIs. Table S5, Comparison of the frequency of gene transfer events between DQS3-9A1T and 118 other bacteria. Table S6, Genes discussed and described in this work. Table S7, Genes involved in TCS. Table S8, Genes in compatible solutes transport and biosynthesis.Figure S1, Genomic Island (GI) prediction by different methods. Ring 1 (red) (from outside in) indicates the GIs by multiple methods; ring 2 (blue) indicates the GIs predicted using the IslandPath-DIMOB method; ring 3 (orange) indicates the GIs predicted using the SIGI-HMM method; and the black line plot indicates the G+C content. Figure S2, The growth of (DOCX)Click here for additional data file.File S2KO list.KO. The list of KEGG annotation results for construction of metabolic pathways using interactive tools like iPATH.(KO)Click here for additional data file.


This review highlights the current knowledge on B10 cells and discusses their potentialas novel therapeutic agents in autoimmunity.B cell abnormalities contribute to the development and progress of autoimmune disease.Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited tothe production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells haveother functions potentially relevant to autoimmunity. Such functions include antigen presentation toand activation of T cells, expression of co-stimulatory molecules and cytokine production. Recently,the ability of B cells to negatively regulate cellular immune responses and inflammation has beendescribed and the concept of regulatory B cells has emerged. A variety of cytokines produced byregulatory B cell subsets have been reported, with IL-10 being the most studied. In this review,this specific IL-10-producing subset of regulatory B cells has been labeled B10 cells to highlightthat the regulatory function of these rare B cells is mediated by IL-10, and to distinguish themfrom other B cell subsets that regulate immune responses through different mechanisms. B10 cells area functionally defined subset currently identified only by their competency to produce and secreteIL-10 following appropriate stimulation. Although B10 cells share surface markers with otherpreviously defined B cell subsets, currently there is no cell surface or intracellular phenotypicmarker or set of markers unique to B10 cells. The recent discovery of an effective way to expand B10cells  Traditionally, B cells have been thought to contribute to the pathogenesis of autoimmune diseasethrough antigen (Ag)-specfic autoantibody production . Nonethe3-fold to 104-fold more efficiently than nonprofessional Ag-presenting cells [+ T cellexpansion, memory formation, and cytokine production [+ T cell responses in mouse models ofautoimmune disease [H cell development may all beinfluenced by B cells during the formation of immune responses [B cells can positively regulate immune responses by producing Ag-specfic antibody and inducingoptimal T cell activation ,3. B celoduction -7. B cel disease ,9. Furth disease . The pos disease , signifi disease -15, abse disease ,17, and  disease ,19. Finaesponses .B cells can also negatively regulate cellular immune responses through their production ofimmunomodulatory cytokines. B cell-negative regulation of immune responses has been demonstrated ina variety of mouse models of autoimmunity and inflammation -30. Althin vivo both in mice and humans, where theirdevelopment shows multiple similarities.One of the most fundamental basic biology questions about B10 cells relates to the stimulidriving their development. Ag and B cell receptor (BCR) signaling are critical in early development,although additional stimuli such as CD40 ligation and Toll-like receptor (TLR) ligands appear to beinvolved in the developmental process. Figure exvivo stimulation with lipopolysaccharide (LPS) or CpG oligonucleotides, phorbol esters(phorbol-12-myristate-13-acetate (PMA)) and ionomycin for 5 hours [PRO cells). B10PROcells develop into B10 cells after maturation through CD40 ligation or exposure to LPS or CpG.B10PRO cells can be identified indirectly following ex vivo stimulation with LPSor CpG in the presence of CD40 ligation for 48 hours with the addition of PMA and ionomycin for thelast 5 hours. The IL-10+ B cells measured following this 48-hour stimulation includecells that would have been IL-10+ even with the shorter 5-hour stimulation (B10 cells),and thereby represent the sum of B10 plus B10PRO cells (B10+ B10PRO).B10 cells are a functionally defined B cell subset. There are no unique phenotypic markers forB10 cells, and these cells are currently defined only by their competency to produce and secreteIL-10 following appropriate stimulation. B10 cells share surface markers with other previouslydefined B cell subsets both in mice and humans, such as marginal zone B cells, transitional B cells,B1a B cells, and memory B cells. However, no one marker or set of markers is unique to B10 cells.For identification of B10 cells, intracellular cytoplasmic IL-10 staining is used, following  5 hours . B10 celPROcells are reduced by approximately 90% in transgenic mice with a fixed BCR [in vivo.CD19-deficient mice  have a 70 to 80% decrease in B10+B10PROcells [/− −mice that alsoectopically express CD40L show dramatically enhanced numbers of CD1dhiCD5+ Bcells and B10 cells [+ B cells with regulatory activity by T cellimmunoglobulin domain and mucin domain protein 1 (TIM-1) ligation [in vitro and in vivo [BCR specificity, affinity and signaling are the most important currently identified factors inB10 cell development. B10 cell regulation of inflammation and autoimmunity is Ag specific ,30,36. Tixed BCR . SignaliROcells , also reROcells , also inligation  further ligation ,42, and  in vivo . The imp in vivo . Remarka in vivo . All of PRO cell development in vivo.Despite the requirement for BCR expression and function during mouse B10 cell development, B cellstimulation with mitogenic anti-IgM antibody alone does not induce cytoplasmic IL-10 expression. Thecombination of anti-IgM stimulation with CD40 ligation and LPS or CpG significantly reduces IL-10competence . BCR-gen+ T cells in B10 cell development [Ex vivo stimulation of purified spleen CD19+ B cells with IL-21induced 2.7-fold to 3.2-fold higher B10 cell frequencies, and 4.4-fold to 5.3-fold more IL-10secretion compared with stimulation with media alone. Remarkably, IL-21 induced B10 cells to produceIL-10 without the need for stimulation with phorbol esters and ionomycin. Interestingly, IL-21induced a threefold increase in IL-10+ B cells within the splenicCD1dhiCD5+ B cell subset, but did not induce IL-10+ B cells withinthe CD1dloCD5-- B cell subset. Both B10 cells and non-B10 cells expressedIL-21R at similar levels, and ex vivo B10, B10pro and CD1dhiCD5+ Bcell numbers were similar among IL-21R-deficient (IL-21R--/--), MHC-II-deficient(MHC-II--/--) and CD40-deficient (CD40--/--) mice. Nevertheless, IL-21R,MHC-II and CD40 appear to be required for B10 cell effector functions, at least in experimentalautoimmune encephalomyelitis (EAE) [A recent study revealed the importance of IL-21, major histocompatibility complex class II(MHC-II) and CD40 during cognate interactions with CD4elopment . Ex vivois (EAE) . Regulatis (EAE) .+ T cells are important for B10 cell effectorfunctions [in vitro also does not affect the frequencyof B10 cells. Although increased B10 cell frequencies in T cell-deficient mice suggest that T cellsmight actually inhibit B10 cell development, it is equally possible that the immunodeficient stateof these mice allows subclinical inflammation that induces B10 cell generation. The role of T cellsin B10 cell development in vivo is thereby complex and, although T cells are not requiredfor B10 cell development, cognate interactions between CD4+ T cells and B10 cells arerequired for B10 cell effector function.Although cognate interactions with CD4unctions , T cellsunctions . The prePRO cells acquire the ability to function like B10 cells after in vitromaturation following stimulation with LPS, but not CpG, in the presence or absence of agonisticCD40 mAb [in vivo, since B10 cells develop normally in MyD88--/-- mice [--/-- mice when their maturation or IL-10 production are measuredfollowing CD40 ligation or PMA plus ionomycin stimulation, respectively, demonstrating that B10PRO and B10 cells are present at normal frequencies in MyD88--/-- mice.Thereby, while TLR signaling is not required for B10 cell development, MyD88 expression is requiredfor LPS to induce optimal B cell IL-10 expression and secretion in vitro.B10 cells can be driven to produce IL-10 by TLR4 (LPS) or TLR9 (CpG oligonucleotides) ligands.Mouse B10CD40 mAb . TLR4 anCD40 mAb . Neverth/-- mice . SpecifiThe involvement of TLR signals in B10-cell IL-10 production was recently demonstrated . IL-10 pblimp1 and irf4 transcription factors along with decreased expression of pax5and bcl6, a significant but small fraction of B10 cells can differentiate intoantibody-secreting cells producing IgM and IgG polyreactive antibodies that are enriched forautoreactivity to single-stranded or double-stranded DNA and histones [Although certain transcription factors are involved at some point in B10 cell development, it isimportant to stress that there is no known transcription factor signature unique to B10 cells.Following a transient period of IL-10 transcription characterized by increased expression of thehistones . WhetherPRO cells and B10 cells have been recently identified in humans [PRO cells to CpG comparedwith their human counterparts. Human B10PRO cells can be driven to develop ex vivointo B10 cells with LPS or CpG stimulation, or CD40 ligation. Interestingly, BCR ligationaugmented human B cell IL-10 responses to CpG in one study [M) remains to be determined.B10n humans  and theine study . This fine study  and micene study , where BPROcells and B10 cells share phenotypic features with memory B cells (see later section on HumanB10 cell phenotype).The most critical unsolved issue relates to the nature of antigenic stimuli driving B10 celldevelopment. The identification of B10-cell BCR specificity is imperative since it will provide newinsights into their early development. The autoreactive nature of mouse B10-cell BCRs  suggestshiCD5+ B cell subset, where they represent 15 to 20% of the cells inC57BL/6 mice. This phenotypically unique CD1dhiCD5+ subset shares overlappingcell surface markers with a variety of phenotypically defined B cell subsets such as CD5+B-1a B cells, CD1dhiCD23−IgMhiCD1dhimarginal zone B cells, and CD1dhiCD23+IgMhiCD1dhiT2 marginal zone precursor B cells, which all undoubtedly contain both B10PRO cellsand B10 cells [lowIgMhi, and <10%co-express IgG or IgA, but they can differentiate into antibody-secreting cells secretingpolyreactive or Ag-specific IgM and IgG [+ compartmentand TIM-1+ B cells are enriched in the CD1dhiCD5+ compartment [+ B cells are also present in the TIM-1--compartment and TIM-1+ B cells are present in the non-CD1dhiCD5+compartment. Intracellular cytoplasmic IL-10 staining thereby remains the only current way tovisualize the entire subset of IL-10-competent B cells. Nonetheless, the isolation ofCD1dhiCD5+ B cells or other phenotypically defined B cell subsets where B10cells are enriched currently provides the best current means for isolating a viable B cellpopulation that is significantly enriched for B10 cells and can be used for adoptive transferexperiments and functional studies in mice.Although a variety of cell surface markers have been proposed ,32, ther10 cells ,30,50. M and IgG . IL-10+ partment . HoweverPRO cells are highly enriched in theCD24hiCD27+ B cell subset, with approximately 60% also expressing CD38.Similar total numbers of IL-10+ B cells have been described in theCD24hiCD38hi and CD24intCD38int B cell subsets [+ and the CD38hicompartments [-- [+ B cell subset can also expand during the course of autoimmunity andhas been proposed as a marker for disease activity [hiCD148+ phenotype of B10 cells and B10PROcells may thereby indicate their selection into the memory B cell pool during development, orthey may represent a distinct B cell subset that shares common cell surface markers with memory Bcells. Consistent with a memory phenotype, the proliferative capacity of human blood B10 cells inresponse to mitogen stimulation is higher than that for other B cells [+CD24hiCD38hi cells that are also CD27-negative [The IL-10-producing B cell subset characterized in humans normally represents <1% ofperipheral blood B cells . Periphe subsets . A separartments . Human Bartments . CD48 isartments  and CD14artments . CD27 exents [-- -56. The activity ,56. The  B cells . Human tnegative ,56; sincnegative , its abs+ T cells, IL-10 suppresses TH1 [H2 polarization [in vitro [in vivo [+ T cells. Co-culture of mouse CD1dhiCD5+B cells with CFSE-labeled naive CD4+ T cells suppresses TH17 celldifferentiation [H17 responses [H17 responses by B10 cells in vivo wasdemonstrated recently [+CD25--T cell proliferation [in vitro.B10 cells exert a variety of IL-10-dependent regulatory effects potentially involved inautoimmune disease. The anti-inflammatory effects of IL-10 are mediated by multiple mechanismsinvolving both the innate and adaptive arms of the immune system. In innate cells, these mechanismsinclude downregulation of proinflammatory cytokine production  and decrsses TH1  and enharization ,59. B10 in vitro  and INFγ[in vivo  by Ag-spntiation  and IL-1esponses . The suprecently . IL-10 pferation  and reguferation in vitro.REG) pool [REGS. These two studies suggesting that B10 cells are not involved in the generationand maintenance of the TREG pool are both in models of EAE [REG pool specifically in EAE [REG numbers through IL-10-independent mechanisms [A number of studies suggest that IL-10-producing B cells are important for the generation and/ormaintenance of the regulatory T cell (TEG) pool ,63-72. HEG) pool  and our EG) pool  do not ss of EAE ,73. In cchanisms . Human BPRO cells are present in patients withautoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, primarySjögren's syndrome, autoimmune bullous diseases, and multiple sclerosis. Interestingly,B10+B10PRO cell frequencies are expanded in some but not all cases, while mean B10+B10PRO cell frequencies are significantly higher in patients with autoimmune diseasecompared with age-matched healthy controls [+ B cells producedcytoplasmic IL-10 than did controls. A different study also demonstrated spontaneous B cell IL-10production that is higher in untreated rheumatoid arthritis, systemic sclerosis, and systemic lupuserythematosus patients than in controls [Studies of B10 cells and human autoimmune disease are limited but of outmost importance sincethey provide valuable insights relevant to the potential future therapeutic application of B10 cellsin humans. Peripheral blood B10 cells and B10controls . A diffecontrols . Blood mcontrols .hiCD38hi regulatory Bcells in human systemic lupus erythematosus [+ T cellresponses. When CD24hiCD38hi B cells were removed from the culture, higherfrequencies of CD4+IFNγ+ and CD4+TNFα+ T cellswere noted in healthy individuals but not in systemic lupus erythematosus patients; this effect waspartially IL-10 dependent. In addition, CD24hiCD38hi B cells isolated from theperipheral blood of systemic lupus erythematosus patients were refractory to CD40 ligation andproduced less IL-10 compared with their healthy counterparts. The results of this study are ratherintriguing but these findings need to be validated in view of the complexity of the culture systemused and the non-uniformity of the CD24hiCD38hi B cell subset with regards toits IL-10-dependent regulatory properties. In conclusion, B10 cells are present in the peripheralblood of autoimmune disease patients, where they appear to be expanded, whereas the functionalcapacity of human B10 cells in autoimmunity needs to be further defined.By contrast, the concept of functional impairment of B10 cells in autoimmune disease was recentlyintroduced by demonstrating functional impairment of CD24ematosus . Culturein vivo and their therapeutic potential inautoimmunity have been demonstrated in a variety of mouse models of human autoimmune disease.The important regulatory effects of B10 cells + T cells [--/-- B cells normalizes EAE severity inμMT mice [H1-mediated immuneresponses persist and mice do not recover from the disease.EAE is an established model of multiple sclerosis induced by immunization with myelin peptides(such as myelin oligodendrocyte glycoprotein) leading to demyelination mediated by auto-Ag-specificCD4 T cells ,77. B ce T cells . However T cells . NonetheμMT mice . DiseasehiCD5+ B cells purifiedfrom IL-10/− −mice does not affect EAE severity. B10 cell effectorfunctions in EAE require IL-21 along with cognate interactions with CD4+ T cells sincethe adoptive transfer of CD1dhiCD5+ B cells into CD19--/-- micefrom IL-21R--/--, MHC-II--/-- or CD40--/-- mice prior to theinduction of EAE does not alter disease course [REG depletion enhances late-phase disease.Therefore, in EAE, depending on the stage of the disease, different regulatory mechanisms areinvolved in limiting inflammatory responses, with B10 cells regulating disease initiation andTREGS being involved predominantly in the regulation of late-phase disease.The EAE model demonstrates the complexity of regulatory mechanisms mediated by different cellsubsets during different stages of the disease. When B cells from wildtype mice are depleted by CD20mAb treatment 7 days before EAE induction, there is an increased influx or expansion ofencephalitogenic T cells within the central nervous system and exacerbation of disease symptoms . This efe course . Once distat3) activation rather than by altering polarized THcell responses. The adoptive transfer of these mesenteric lymph node B cells also suppressesinflammatory bowel disease through a mechanism that correlates with an increase in TREGsubsets [--/-- mice (where B10 cells are absent) than in wildtype mice [hiCD5+B cells producing IL-10. B10 cells therefore emerge during chronic inflammation in mousemodels of inflammatory bowel disease, where they suppress the progression of inflammatory responsesand ameliorate disease manifestations.IL-10-producing B cells regulate intestinal inflammation in inflammatory bowel disease . Early ssubsets . Oral adype mice , and theCIA is a model for human rheumatoid arthritis that develops in susceptible mouse strainsimmunized with heterologous type II collagen emulsified in complete Freund's adjuvant ,81. CIA H1 responses, prevents arthritis development, and is effective in amelioratingestablished disease. The adoptive transfer of CD21hiCD23+IgM+ Bcells from DBA/1 mice in the remission phase prevents CIA and reduces disease severity through IL-10secretion [ex vivoexpanded CD1dhiCD5+ B cells in collagen-immunized mice delays arthritisonset and reduces disease severity, accompanied by a substantial reduction in the number ofTH17 cells [hiCD5+ B cells with naive CD4+ Tcells suppresses TH17 cell differentiation in vitro, and co-culture ofCD1dhiCD5+ B cells with TH17 cells results in decreasedproliferation responses in vitro. Furthermore, the adoptive transfer of TH17cells triggers CIA in IL-17--/-- DBA mice; however, when TH17 cells areco-transferred with CD1dhiCD5+ B cells, the onset of CIA is significantlydelayed. Finally, in a different study, administration of apoptotic thymocytes along with ovalbuminpeptide and complete Freund's adjuvant to mice carrying an ovalbumin-specific rearranged T cellreceptor transgene (DO11.10 mice) up to 1 month before the onset of CIA resulted in an increase inovalbumin-specific IL-10 secretion and is protective for severe joint inflammation and bonedestruction [in vitroby increasing secretion of IL-10, and inhibition of IL-10 in vivo reversed thebeneficial effects of apoptotic cell treatment [Activation of arthritogenic splenocytes with Ag and agonistic anti-CD40 mAb induces a B cellpopulation that produces high levels of IL-10 and low levels of IFNγ . The adoecretion ; a signi17 cells . Co-culttruction . Activatreatment .hiCD5+B220+ B cellsubset, which is enriched in B10 cells, is increased 2.5-fold during the disease course, whereasCD19--/-- NZB/W mice lack this CD1dhiCD5+ regulatory B cell subset [--/-- NZB/W recipients following the adoptive transfer ofsplenic CD1dhiCD5+ B cells from wildtype NZB/W mice [B cell-negative regulatory effects are important in NZB/W mice, a spontaneous lupus model, sincemature B cell depletion initiated in 4-week-old NZB/W F1 mice hastens disease onset, which parallelsdepletion of B10 cells . B10 cell subset . FinallyB/W mice . StudieshiCD5+ and CD21hiCD23hi B cells, were not enrichedin IL-10 transcription. This observation suggests fundamental differences in the pathogenesis andimmune dysregulation in the NZB/W lupus model compared with the MRL.Fas(lpr) model.In the MRL.Fas(lpr) mouse lupus model, B cell-derived IL-10 does not regulate spontaneousautoimmunity . B cell-in vitro can maintain tolerance and transfer protection fromtype 1 diabetes in NOD mice [--/-- B cells do not confer protectionfrom type 1 diabetes or the severe insulitis in NOD recipients. The therapeutic effect of adoptivelytransferred activated NOD B cells correlates with TH2 polarization. The limited dataabove suggest that B10 cells may be protective in preventing establishment of type 1 diabetes in NODmice.Studies on B10 cells and mouse models of diabetes are limited to the nonobese diabetic (NOD)mouse, a spontaneous model of type 1 diabetes in which autoimmune destruction of theinsulin-producing pancreatic β cells is primarily T cell mediated . AlthougNOD mice ,93. The PRO cells or theselective expansion of B10 cells. Since there are no identified surface molecules specific fornon-B10/B10PRO cells, it is currently impossible to selectively target and deplete matureB cells while sparing B10/B10PRO cells. B10 cell expansion appears to be a more viableapproach since some of the stimuli driving their development have been identified. B10 cells can beexpanded for therapeutic purposes either in vivo or ex vivo. Expansion of B10cells in vivo by means of agonistic CD40 antibody has shown benefit in CIA [in vivo carries additional risks since thecurrently identified stimuli driving B10 cell development are rather nonspecific and, ifadministered systemically, will trigger responses in a variety of immune cells. For example, thesystemic administration of agonistic CD40 antibodies in humans has been associated with seriousadverse effects such as cytokine release syndrome [PROcells is not currently possible, and in vivo B10 cell expansion by nonspecific agentssuch as agonistic CD40 antibody is potentially associated with serious off-target effects.Harvesting the anti-inflammatory properties of B10 cells can provide a new approach to thetreatment of autoimmunity. Manipulation of this subset for treating autoimmune disease is possibleby either selective depletion of mature B cells while sparing B10/B10t in CIA . Howeversyndrome . In summex vivo appears more preferable than in vivo B10 cellexpansion by nonspecific agents because it offers a potential therapy without the risk ofundesirable nonspecific off-target effects. However, ex vivo B10 cell expansion introducesnew challenges related to the method of expansion, to the magnitude of expansion and to the time ittakes to generate B10 numbers that will be sufficient for therapeutic use. The method of ex vivoB10 cell expansion can be the source of safety concerns when it comes to human applications.Large numbers of regulatory B cells have been successfully generated in mice by means of geneticmanipulation of immature B cells through lentiviral transfection [ex vivo, concerns remain aboutadministering infusions of lentivirus-infected B cells to humans . Safety concerns thereby limit the use of infectious agents in manipulating human cells,which could render this approach inappropriate for use in humans.Expanding B10 cells sfection . These cex vivo B10 cell expansion is very important since the number of cellsinfused during adoptive transfer experiments is critical. In humans, the most convenient potentialsource of B10/B10PRO cells prior to ex vivo expansion is obviously peripheralblood. Since B10/B10PRO cells are rare in peripheral blood and there are limitations onthe volume that can be drawn at any given time, a method of expanding B10 cells by severalmillion-fold is needed. Furthermore, since this method will be used for treatment of active disease,the time it will take to expand these cells ex vivo is also of great significance; ideally,this process should not take more than 1 or 2 weeks. There is accumulating hope that such anapproach will soon be available for human cells since mouse B10 cell ex vivo expansion canbe accomplished within 9 days by means of combined CD154, B-lymphocyte stimulator, IL-4 and IL-21stimulation [ex vivo expanded B10 cells arevery effective in limiting inflammatory responses in EAE. This approach appears promising since itprovides an effective way of generating large numbers of B10 cells without the use of infectiousagents. The development of a similar system for expanding human B10 cells is of outmostimportance.The magnitude of mulation . After tin vitromethod to efficiently expand mouse B10 cells provides an invaluable tool for studying the basicbiology of B10 cells as well as manipulating them for therapeutic purposes. The development of asimilar method for human cells will open new opportunities for studying the basic biology of humanB10 cells and a promising novel approach in treating human autoimmune disease, potentially withoutundesirable off-target effects.The phenotypic and functional characterization of B10 cells is an important advance for theregulatory B cell field. Numerous additional functionally defined subsets of regulatory B cells willprobably be identified in the future. B10 cells share phenotypic markers with a variety ofpreviously defined subsets, but their only unique phenotypic marker is intracellular IL-10production. Although certain transcription factors are involved at different points in B10 celldevelopment, there is currently no transcription factor signature unique to B10 cells. BCR-relatedsignals are most critical in B10 cell development and the finding of B10-cell BCR auto-reactivitysuggests that autoantigens may be of particular importance. The recent discovery of an H: T-helper; TIM-1: T cell immunoglobulin domain andmucin domain protein 1; TLR: Toll-like receptor; TNF: tumor necrosis factor; TREG:regulatory T cell.Ag: antigen; B10 cells: IL-10-producing regulatory B cells; BCR: B cell receptor; CIA:collagen-induced arthritis; EAE: experimental autoimmune encephalomyelitis; IFN: interferon; IL:interleukin; LPS: lipolysaccharide; mAb: monoclonal antibody; MHC: major histocompatibility complex;MyD88: myeloid differentiation primary response gene 88; NOD: nonobese diabetic; PMA:phorbol-12-myristate-13-acetate; TThe authors declare that they have no competing interests.Arthritis Research & Therapy Volume 15Supplement 1, 2013: B cells in autoimmune diseases: Part 2. The supplement was proposed by thejournal and content was developed in consultation with the Editors-in-Chief. Articles have beenindependently prepared by the authors and have undergone the journal's standard peer review process.Publication of the supplement was supported by Medimmune.This article has been published as part of


YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36 Traversing the plasma membrane of the host cell is a significant challenge for many viruses during the infection cycle, and the efficiency of detachment from the host cell and subsequent release can have implications in pathogenesis. Vaccinia virus exits cells through the loss of an outer membrane but remains attached via viral envelope proteins that mediate adhesion between the cell and virus. Here we report that actin filament nucleation by the viral protein A36 promotes the disengagement of virus attachment. Viruses unable to locally induce actin nucleation displayed significantly reduced release and particles were found trapped in small pits at the plasma membrane. Mutations in luminal viral proteins that disrupt attachment identified an alternative route to virus release, bypassing the requirement for actin nucleation. Our results suggest that untethering virus attachment to the cell surface is a rate-limiting step during exocytic release of vaccinia virus. We have elucidated that the force of actin nucleation is the primary mechanism that operates to relieve these interactions. P189S, A34K151E and a C-terminal deletion in A33) Crossing the membrane of host cells, either during entry or escape, is a major obstacle facing prospective viral pathogens during infection. Enveloped viruses are released from cells by either the acquisition or loss of an outer membrane and both strategies pose unique challenges to the final separation of pathogen from host. Where viruses gain a membrane, for example influenza virus and human immunodeficiency virus, a tight association must be formed between assembling viral complexes and the internal surface of the cell membrane that is loaded with viral envelope proteins Y112, A36Y132 respectively) Y112 and A36Y132, which generates binding sites for the SH2 domains of Nck and Grb2 adaptor proteins that combine to stabilise N-WASP, a potent activator of the Arp2/3 complex The viral transmembrane protein A36 is currently the only Abl substrate that has been implicated in VACV morphogenesis, although its role in Abl-mediated release is unknown Here we show that introducing both Y112F and Y132F mutations into A36 leads to a severe decrease in the production of EEV. In the absence of virus-induced actin nucleation, virus particles remained trapped at the plasma membrane in small invaginations.When parental VACV was examined using three-dimensional structured illumination microscopy (3D-SIM), it was found that upon reaching the surface, A36 redistributed to a discrete region beneath CEV, reducing contact between extracellular virus and the host cell. This redistribution was dependent on actin-based motility but could also be phenocopied by a mutation in the luminal domain of the envelope protein B5. In our model, following the fusion of IEV at the plasma membrane, CEV remain tethered to the cell in tight membrane pits through interactions between viral proteins. Tethering can be relieved either by mutations in the luminal domains of viral proteins that disrupt these interactions, or by the force supplied by localised actin nucleation.Y112 and A36Y132 on the release of EEV. These residues are critical to the canonical pathway whereby VACV induces actin nucleation by recruitment and activation of the Arp2/3 complex. Plaque assays performed under semi-solid overlay are used to quantitate replication dynamics and cell-to-cell transmission. When performed under a liquid overlay, these are termed ‘comet’ assays and the efficiency of EEV release can be qualitatively assessed by the formation of satellite plaques that disperse from primary plaques due to the action of convection currents Y112F, A36Y132F and A36YdF ( = A36Y112F/Y132F) to assess cell-to-cell spread  seen with A36YdF and a significant inhibition seen when Y112 was mutated  in EEV was observed in A34K151E/A36YdF virus compared to A34K151E (K151E) YdF mutation potently supresses EEV release. Conversely, in a B5P189S background, which is strongly deficient in actin-based motility owing to a failure of kinase activation, the A36YdF allele has no effect on EEV release Deletion of genes encoding envelope-specific proteins can increase  or decrease  production of EEV, often due to earlier defects in morphogenesis ckground . EEV reln A36YdF . There wn A36YdF . In contA34K151E . TherefoYdF released similar levels of EEV , a potent inhibitor of actin polymerisation that caps the fast growing ends of actin filaments. Previous studies have shown that Cyt D significantly reduces WR EEV while having little effect on morphogenesis or formation of CEV K151E , no motile virus particles were associated with F-actin. In cells infected with A36Y112F (n = 9), six cells had at least one example of virus motility associated with transient F-actin accumulation over the course of two minutes. This was in contrast to actin tails that were associated with parental WR virus, which were robust and longer lived . The majority of exiting enveloped viruses were found loosely associated with the plasma membrane in WR infected cells  alleles or transgenes. Initially, cells expressing Lifeact fused to cerulean fluorescent protein  were infected with A36-YFP/B5-mRFP virus. Lifeact is a 17 amino acid peptide that binds to filamentous actin YdF-YFP, while distinguishing between extracellular and intracellular enveloped virus with an antibody label  and the surface of the cell (labelled with A36-YFP). Analysis of individual z-planes in a 3D reconstruction revealed contact between the virus and cell was limited to a few planes in A36-YFP, but in A36YdF-YFP A36 colocalised with exposed B5 over the majority of a CEV particle  leads to a far stronger reduction in EEV (10-fold) than treatment with imatinib (3-fold). Secondly, imatinib inhibits EEV release regardless of the integrity of A36 Y112 and Y132 residues. Therefore, while Abl family kinases may regulate EEV release through phosphorylation of A36, they do so redundantly with Src-family kinases, as we have previously proposed for actin-based motility Inhibiting the action of Abl-family kinases with the specific inhibitor imatinib leads to a reduction in EEV release, implicating these kinases in the regulation of this process. Despite our previous work demonstrating that A36 was a direct Abl substrate YdF mutation into high EEV release backgrounds. These included strains carrying mutations in the envelope proteins B5 (B5P189S) and A34 (A34K151E). The B5P189S virus exhibits high levels of EEV release irrespective of the status of A36 Y112 and Y132 residues K151E EEV release was potently suppressed by the A36YdF mutation. These data show that in strains that activate actin-based motility (WR and A34K151E) the YdF mutation suppresses EEV release, but has no effect in cases where actin-based motility is absent (B5P189S-infected or Nck null cells) K151E and B5P189S result in high levels of EEV, only the latter bypasses the requirement for actin-based motility to facilitate release. A34K151E appears to exhibit a faster rate of enveloped virus production, but EEV release is nonetheless regulated in the same manner as the parental WR strain P189S a high rate of EEV release is achieved at a cost to morphogenesis, hence the small plaque phenotype. Collectively, these findings indicate that two pathways can liberate EEV from the surface of cells: localised actin filament nucleation or disruption of viral protein-to-host membrane interactions. The exact nature of these interactions is unclear as few experiments have been performed that are able to distinguish between interactions occurring in cis and trans in the viral envelope et al.et alP189S mutation on the surface of EEV also disrupts super-repulsion actin-based motility cis interaction in the membrane between these proteins and raising the possibility that A33 might mask and/or regulate the availability of the adjacent SCR4 To better characterise the mechanism by which actin nucleation promotes virus release, we examined the effects of recombining the A36P189S and A33 C-terminal truncations lead to increased EEV release but also a reduction in plaque size K151 allele, display reduced mortality compared to those infected with WR  in vivo spread and transmission.What is the role of EEV release during pathogenesis? The literature does not offer a clear consensus as to whether strains that release more EEV are more or less virulent. Mutations isolated in a WR background such as B5YdF are reduced in size, attesting to the role of these residues in enhancing cell-to-cell spread , murine embryonic fibroblasts (NIH3T3) and Nck-null NIH3T3 cells were maintained in Dulbecco's Modified Eagle's Medium  supplemented with 5% foetal bovine serum (FBS), 292 µg/ml L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin (DMEM-FPSG) at 37°C and 5% COYdF, A36Y112F, A36Y132F and B5-YFP have been described previously YdF-YFP, A36-YFP/B5-mRFP, B5P189S, B5P189S/A36YdF, A34K151E and A34K151E/A36YdF, A36YdF/B5-YFP and A36Y112F/B5-YFP were prepared as described previously YdF-YFP, B5P189S and A34K151E, or infected with A36YdF or A36Y112F and transfected with the relevant vector for production of B5P189S/A36YdF, A34K151E/A36YdF, A36YdF/B5-YFP and A36Y112F/B5-YFP. For the other fluorescent viruses, BSC-1 cells were infected with A36-YFP and transfected with the relevant vector for production of A36-YFP/B5-mRFP, B5P189S/A36-YFP and A34K151E/A36-YFP, or infected with B5P189S or A34K151E and transfected with the A36YdF-YFP vector to produce B5P189S/A36YdF-YFP and A34K151E/A36YdF-YFP, respectively. At 24 hours post infection (hpi) cells were scraped and recombinant viruses purified by three rounds of plaque purification. Insertion at the correct locus and correct sequence was confirmed by PCR and sequencing.Vaccinia virus strain Western Reserve (WR) and the mutant viruses A362 for 1 h, the cells were washed twice and overlaid with either 1.5% carboxymethyl cellulose (CMC) in minimal essential medium (MEM) containing 2.5% FBS, 292 µg/ml L-glutamine, 100 units/ml penicillin and 100 µg/mL streptomycin for plaque assays, or DMEM-FPSG for comet assays. For experiments with imatinib treatment, imatinib mesylate (ChemiTek) at a final concentration of 10 µM or 0.01% of the carrier, DMSO, was included in the overlay. Cells were incubated for 3 days and then the overlay removed and cells stained with 1% crystal violet in methanol for visualisation.BSC-1 or NIH3T3 cells were seeded in six-well plates and grown to confluence. The virus strains were diluted in serum free DMEM (SFM) and approximately 25 PFU was added to each well. After incubation at 37°C in 5% COYdF, A36Y112F or A36Y132F in triplicate at a multiplicity of infection (MOI) of 5 for 1 h. The inoculum was removed, and the cell monolayer was washed three times with PBS. Cells and supernatant were collected at various times post-infection , freeze-thawed three times and the virus concentration determined by plaque assay of 10-fold serial dilutions in triplicate on BSC-1 cells. Cells were incubated for 3 days and then the overlay removed and cells stained with 1% crystal violet in methanol for visualisation.BSC-1 cells were seeded into 12-well dishes, and confluent monolayers were infected with either WR, A3612-well dishes were seeded with BSC-1 or NIH3T3 cells and incubated with VACV strains (in triplicate) at an MOI of 0.1 for 1 h. Cells were then washed twice with PBS and DMEM-FPSG was added. For inhibitor treatments, imatinib mesylate at a final concentration of 10 µM, Cytochalasin D (Sigma-Aldrich) at a final concentration of 0.1 µg/ml or 0.01% of the carrier, DMSO, was added to the overlay media. The supernatants were collected at 16 hpi. To quantify the infectious EEV, plaque assays of 10-fold serial dilutions of the supernatant were performed on BSC-1 cells as described above. After 3 days cells were stained with methanol/1% crystal violet and plaques enumerated. All EEV assays were performed on at least three separate occasions.Confluent monolayers of BSC-1 cells were infected at an MOI of 5 and processed for TEM at 9 hpi. Briefly, cells were rinsed twice with PBS and fixed with 1.5% glutaraldehyde in Na-cacodylate buffer (0.1 M and 0.1 M sucrose) at pH 7.4 for 1 h. The samples were subsequently washed with PBS and postfixed with 1% osmium tetroxide in 0.1 M Na-cacodylate at pH 7.4 for 1 h. The cells were next dehydrated in a graded ethanol series. After dehydration, samples were embedded in Epon and after hardening of the embedding medium, the plastic of the multiwell dishes was removed using liquid nitrogen. Sections of 120 nm under various angles were cut with a diamond knife, stained first with uranyl acetate, subsequently with lead citrate, and examined in a Jeol 2100  at 200 kV.Y112 and A36YdF and fixed 8 hpi with 3% paraformaldehyde (PFA) in cytoskeletal buffer (CB)  for 10 minutes at room temperature. Cells were blocked in blocking buffer  for 20 minutes then incubated for 40 minutes with 19C2 rat anti-B5 primary antibody (1/300) BSC-1 cells were grown to 80% confluency on glass coverslips and infected with WR, A36For 3D-SIM, BSC-1 cells grown to 80% confluency on glass coverslips were infected with the fluorescently-tagged viruses at an MOI of 1. Transfection with plasmids expressing Lifeact-cerulean 2, Sigma-Aldrich). Cells were infected with B5-YFP, A36YdF/B5-YFP or A36Y112F/B5-YFP and transfected with pE/L Lifeact-mRFP  HeLa cells at 80% confluency were grown on Ibidi glass bottom μ-Dishes coated in fibronectin  software. To determine A36 distribution in 3D-SIM images, for each virus type 100 virus particles selected from five representative cells were classified as having polarised , intermediate  or circular  A36 distribution see . ClassifVideo S1Live cell microscopy of VACV B5-YFP-induced actin nucleation. HeLa cells were infected with B5R-YFP (green), transfected with pE/L Lifeact-mRFP (red) and imaged for 80 seconds at 6–9 hours post infection. A virus particle is seen colocalising with F-actin tail of approximately 3–4 µm. See (MOV)Click here for additional data file.Video S2Y112F/B5-YFP-induced actin nucleation. HeLa cells were infected with A36Y112F/B5-YFP (green), transfected with pE/L Lifeact-mRFP (red) and imaged for 80 seconds at 6–9 hours post infection. A virus particle is seen undergoing rapid, presumably microtubule-dependent motility, before pausing and nucleating F-actin. See Live cell microscopy of VACV A36(MOV)Click here for additional data file.Video S3YdF/B5-YFP that appears in one frame to colocalise with F-actin. HeLa cells were infected with A36YdF/B5-YFP (green), transfected with pE/L Lifeact-mRFP (red) and imaged for 80 seconds at 6–9 hours post infection. Colocalisation between a virus particle and F-actin is observed transiently at the beginning of the movie but is not associated with virus motility. See Live cell microscopy of VACV A36(MOV)Click here for additional data file.


Furthermore, we observed efficient virus-like particle production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of spherical progeny, which may contribute to the rapid spread of the pandemic virus.In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains. This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet, despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/California/04/09 , to be predominantly spherical by immunufluorescence and electron microscopy analysis in human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain. Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1), a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between influenza morphology and efficient cell-to-cell spread  Moreover, strains containing Eurasian origin NA with increased enzymatic activity released more viral particles, thereby proposing a mechanism by which the NA segment may be contributing to enhanced viral transmission of the pH1N1 virus.Both the NA and M segments encode viral proteins with key roles in the influenza assembly, budding and release processes, which are required for efficient viral transmission. NA encodes the type II transmembrane protein, neuraminidase, which is found as a tetramer on the surface of the host-derived lipid envelope. It functions during the final stage of viral budding where it cleaves sialic acid containing receptors to allow for the release of progeny virions from infected cell surfaces in vivo using the guinea pig model The M segment of influenza encodes two viral proteins, matrix protein (M1) and the M2 ion channel. While M2 is essential for uncoating of the virus during entry, M1 is known to be the key component for both assembly and budding Specific M1 residues are also known to affect virion morphology. Influenza virions can range from 100 nm spheres to filamentous particles reaching several micrometers in length in vitro. These data suggest that a few mutations present in pH1N1 contribute to morphological change and efficient transmission of influenza viruses.In this study, we characterized the morphology of pH1N1 isolates, and found that they are predominantly spherical in our cultured cells. This was in contrast to the filamentous phenotype we observed with the closely related, but poorly transmissible, TR swine viruses. In addition, we found that, unlike other strains, pH1N1 M1 by itself can efficiently induce virus-like particles from transfected cells. To evaluate the contribution of M1 mutations in virus morphology, we rescued various pH1N1 virus containing mutations at M1 residues different between pH1N1 and TR. Our results revealed that pH1N1 M1 residues 30, 207 and 209 are involved in regulation of virus morphology and enhanced viral spread st century. Although multiple factors are likely to be involved in the emergence of the pH1N1, efficient virion production is among the requirements for the outbreak. Because efficient virion production has previously been shown to correlate with spherical rather than filamentous particle formation, we first determined the morphology of pH1N1 and TR viruses. Initially, we investigated the presence or absence of filamentous protrusions at the surface of human lung epithelial A549 cells infected with the viruses by immunufluorescence (IF) analysis. A549 cells were infected with either the TR strain A/Wisconsin/87/2005 , or a pH1N1 virus A/California/04/2009 . At 18 h, the cells were fixed and processed for visualization of viral surface proteins using anti-H1N1 mouse serum. Noticeably, cells infected with AWisc05 exhibited long filamentous structures on their surface  by itself in cultured cells. Previous studies have shown that expression of influenza NA, M2, or HA alone, but not M1, is sufficient for efficient VLP production 35S-labeled 293T cells transfected with plasmids expressing wild-type or mutant ACal-04/09 M1, or with plasmids expressing avian or human virus M1. In agreement with previous reports, we found trace amounts of M1 protein released into the supernatant of 293T cells transfected with A/chicken/Nanchang/3-120/01 , A/WSN/33 , and A/Aichi/2/68  M1 plasmids , yet disagrees with an investigation which reported the pH1N1 strain as predominantly filamentous when analyzed using MDCK or primary human airway epithelial cells In this study, we determined the role of the pH1N1 M1 protein in virus morphology and growth By introducing TR M1 residues into pH1N1 M1, we identified three residues at 30, 207 and 209, which affected virion morphology and virus growth. Single mutations at these residues, reduced plaque size, growth rate and overall virus production . pH1N1 mIt is unclear at this stage how pH1N1 M1 mutations at 30, 207 and 209 affect virus morphology. Influenza virus assembly and budding is a complex, multi-step process involving interactions between viral and cellular proteins. Many viral protein interactions are known to affect virus assembly and morphology, such as M1-HA, M1-NA, M1-M2, and M1-vRNP interactions Recent cryo-electron tomography data indicate that M1 forms a helical net under the viral membrane. The pitch of the helical turn of the M1 protein differs between spherical and filamentous virions, suggesting that M1-M1 interaction and its structure determine virion morphology Human lung epithelial (A549), human embryonic kidney (293T), and Madin-Darby canine kidney (MDCK) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8% fetal calf serum (FCS). Porcine kidney (LLC-PK1) cells were obtained from American Type Culture Collection and were cultured in Medium 199 supplemented with 8% FCS. Normal human bronchial epithelial (NHBE) cells obtained from Lonza were maintained in bronchial epithelial cell basal medium (BEBM) with the supplements provided. Triple reassortant swine isolates A/Wisconsin/87/2005 (H1N1), A/Iowa/01/2006 (H1N1), A/Iowa/02/2006 (H1N1), A/Ohio/02/2007 (H1N1) and A/Minnesota/03/2008 (H1N1) cDNAs encoding Nan, Aichi and ACal-04/09 M1 protein were synthesized by RT-PCR using SuperScript III One-step RT-PCR Platinum Taq HiFi (Invitrogen) from total RNAs extracted from virus-infected cells, and subcloned into pCAGGS. Mutations in the M1 gene were created using the QuikChange II site-directed mutagenesis kit . cDNAs used to rescue ACal-04/09 were synthesized from RNAs provided by R. Webster and R. Webby  using RT-PCR kit and subcloned into the pPolI vector given to us by Y. Kawaoka . Amino acid substitutions at positions 30, 142, 207, and 209 were introduced into the pPolI-M plasmid by site-directed mutagenesis and used for rescue of the ACal-04/09 M1 mutant viruses.Viruses were rescued using the 12-plasmid rescue system developed by Neumann and Kawaoka A549 or LLC-PK1 cells were infected with the viruses at MOI 1 and incubated with DMEM containing 0.15% BSA for 18 h. For detection of viral surface proteins by IF, cells fixed with 4% paraformaldehyde were incubated with anti-H1N1 mouse serum supplied by D. Topham  followed by goat anti-mouse IgG conjugated with Texas Red (Invitrogen). Images were taken using an Olympus inverted microscope. For scanning electron microscopy, cells were fixed with 2.5% glutaraldehyde and visualized under a Zeiss Auriga supra 40VP Field Emmison microscope. For the analysis of virions, supernatant from virus-infected A549 cells was spun through a 100 K Amicon filter unit (Millipore) for 18 min at 3,500 rpm. A 10 µl volume of concentrated virus was absorbed to carbon-coated 200 square mesh nickel grids for 3 min, washed once with PBS(+), and negatively stained for 1 min with filter sterilized 2% phosphotungstic acid. Transmission electron micrographs were obtained at ×200,000.MDCKs in 6-well culture plates were infected with the viruses at MOI 0.01 and incubated in 2 ml DMEM containing 0.15% BSA and 2 ug/ml TPCK-treated trypsin. At various times points, 200 µl of the culture supernatant was harvested and replaced with an equal volume of fresh media. Virus titers were determined in MDCK cells as described by Reed and Muench 35S-Met/Cys in DMEM lacking methionine and cystine. Cell culture supernatants were cleared of cell debris by low speed centrifugation, layered onto 20% sucrose cushions and centrifuged at 35,000 rpm for 2 h in an SW41 rotor (Beckman). Pellets containing virus-like particles were resuspended in laemmli buffer (BioRad) and analyzed by SDS-PAGE. Cell lysates were harvested in RIPA buffer , clarified, and separated by SDS-PAGE. Proteins were transferred to a PVDF membrane and subject to Western Blot analysis with anti-M1 GA2B monoclonal antibody (Sigma). Band intensities were quantified with BioRad Quantity One software and the amount of M1 detected in culture supernatant was normalized to M1 expressed in cell lysates.293T cells transfected with pCAGGS-M1 vectors were labeled for 24 h with g. Half ml aliquots of supernatant were mixed with 3.5 ml 70% sucrose in low salt buffer (LSB) (50 mM Tris HCl (pH 7.5) 25 mM KCl and 5 mM MgCl2), overlaid with 5.5 ml 55% sucrose and 2 ml 10% sucrose in LSB and ultracentrifuged at 38,000 rpm for 18 h at 4°C in an SW41 rotor. One-ml fractions were collected from the top of each gradient to which 0.25 ml 100% trichloroacetic acid was added for protein precipitation. After 10 min incubation on ice, fractions were spun at top speed for 5 min. Protein pellets were then washed twice with cold acetone, heated to 95°C for 5 min and resuspended in NuPAGE sample buffer (Invitrogen) for SDS-PAGE and Western Blot analysis with anti-M1 GA2B antibody. Band intensities within each fraction were quantified using BioRad Quantity One software.Flotation analysis on 293 T cells transfected with pCAGGS-M1 plasmids was performed as described by Sanderson et al. Isolation of lipid rafts from 293 T cells transfected with pCAGGS-M1 plasmids was performed as described by Carrasco et al.


Host genetic variations may contribute to disease susceptibility of influenza. IL-1A and IL-1B are important inflammatory cytokines that mediate the inflammation and initiate the immune response against virus infection. In this study, we investigated the relationship between single-nucleotide polymorphisms (SNPs) of Interleukin-1A (IL-1A) and Interleukin-1B (IL-1B) and the susceptibility to 2009 pandemic A/H1N1 influenza (A(H1N1)pdm09). 167 patients whom were confirmed with A(H1N1)pdm09 and 192 healthy controls were included in this study. Four SNPs  in IL1A gene and three SNPs  in IL1B gene were genotyped by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry platform, and the associations of the genetic variants of IL-1 with susceptibility to A(H1N1)pdm09 were then assessed.The polymorphisms of rs17561 in IL1A gene and rs1143627 in IL1B gene were found to be associated with susceptibility to A(H1N1)pdm09 with P values of 0.003  and 0.002 , respectively. However, no significant difference in allelic frequency was observed for other SNPs between cases and controls.This study provides a new insight into pathogenesis of A(H1N1)pdm09, suggesting that genetic variants of IL-1A and IL-1B may exert a substantial impact on the susceptibility of A(H1N1)pdm09 virus infection. The global pandemic caused by the novel AH1N1)pdm09 was first declared in Mexico and the United States in April 2009, inducing severe morbidity and mortality in a subset of the population [N1pdm09 wIt has been suggested that the host genetic factors may affect the susceptibility and progression of microbial infections . For exaDuring influenza virus infection, IL-1B has been demonstrated to mediate acute pulmonary inflammatory pathology . Pro-IL-In this study, we examined 7 SNPs in IL1 gene and evaluated their association with A(H1N1)pdm09 susceptibility. The results indicated that rs17561 polymorphism in IL1A gene and rs1143627 polymorphism in IL1B gene, two novel SNPs of IL-1 gene, contributed to the susceptibility to A(H1N1)pdm09.167 patients with A(H1N1)pdm09 were recruited in Shenzhen Third People’s Hospital, China during 2009 influenza pandemic period. 167 patients  were included in this study, and the average age of patients is 22.37 (SD = 11.95). The diagnosis of A(H1N1)pdm09 was followed the guideline released by the Ministry of Health of the People’s Republic of China. 192 Healthy donors (HD) were from Shenzhen, China during the same period, and no occurrence of A(H1N1)pdm09 was found in the HD during 1 year’s follow-up. 192 health individuals  were recruited as controls, and the average age of healthy individuals is 24.53 (SD = 10.29). All subjects were Chinese Han people whom have received flu vaccine previously. Venous blood was drawn from all subjects after an overnight fasting. Blood corpuscles were stored at −80°C for DNA purification. The study was approved by the Institutional Review Board of Shenzhen Third People’s Hospital, and informed consent was obtained from each participant. There is no significant difference for the average age and gender distribution between cases and controls (P >0.05).Genomic DNA was obtained from peripheral blood samples, using the QIAamp DNA Blood Mini kit  according to the manufacturer’s instruction. Isolated DNA was stored at −80°C before usage. SNPs were selected according to methods described previously , with fohttp://www.ncbi.nlm.nih.gov/projects/SNP/). We then used a PWM_SCAN Algorithm [2 = 0.8 and default settings for all other parameters. We also selected several other SNPs in the IL-1 gene region that were reported to be associated with disease severity [To search for the SNPs with alleles that alter the putative transcription factor binding sites, we scanned the promoter region of the gene in TRANSFAC database . For thelgorithm  to scan lgorithm . To searlgorithm , and choseverity -23. The SNPs’ genotyping was based on analysis of primer extension products generated from previously amplified genomic DNA using a chip-based MALDI-TOF mass spectrometry platform . The amplification PCR primer pairs and the extend primers were designed by MassARRAY assay designer software v3.1, in combination with manual adjustment , followed by a final extension done at 72°C for 3 min. After desalting extension products for MALDI-TOF mass spectrometry analysis by the addition of 16 μL water and 6 mg of clean resin to each sample. Data analysis was performed by using MassARRAY typer software 4.0.χ2 test was used to compare allele and genotype distribution in cases and controls. Odds ratios (ORs) and their 95% confidence intervals (CI) were calculated with Miettinen’s method. Each SNP was calculated under four alternative models , as previously described [The frequencies of allele and genotype were determined by direct counting. All SNPs investigated in this study were tested for Hardy-Weinberg equilibrium. The Pearson escribed ,24-26. Bhttp://www.ncbi.nlm.nih.gov/SNP/; CYPallele nomenclature: http://www.cypalleles.ki.se). After bioinformatic analysis and prediction for SNPs that may influence gene expression or protein structure and function, a total of 7 SNPs, including 4 in the IL1A gene , and 3 in the IL1B gene , were selected finally. The locations of these SNPs in IL-1 were shown in Figure To choose relevant SNPs for IL1A and IL1B gene, we considered about 100 known and rare variants, which are publicly available in database pdm09. We examined 7 SNPs in IL1A and IL1B genes and evaluated the association between SNPs of IL-1 and A(H1N1)pdm09 susceptibility. We found that rs17561 polymorphism in IL1A gene and rs1143627 polymorphism in IL1B gene contributed to the susceptibility to A(H1N1)pdm09.In humans, IL-1 exists in two forms, IL-1A (encoded by IL1A gene) and IL-1B (encoded by IL1B gene), and both forms of IL-1 locate on chromosome 2 . IL-1A aIn fact, IL1A with rs17561 allele T has been suggested to be associated with immunopathogenesis of several diseases, including nasal polyposis , chronicDuring influenza virus infection, the immune response was triggered by the influenza virus ion channel M2 which was essential for virus entry and replication, leading to the assembly of inflammasome in macrophages and dendritic cells (DCs) . Then, tHere, we found a significantly high frequency of IL-1B rs1143627 allele C in A/H1N1 patients, suggesting the hosts with allele C were more susceptible to this virus infection. Importantly,the frequency of the rs1143627 allele C is quite rich in Han Chinese . Thus, elucidation of the underlying mechanism of the polymorphism of rs1143627 is of great importance for in-depth understanding of the susceptibility of influenza in Chinese population. Rs1143627 polymorphism involved the variation of IL-1B −31 allele C to T and is located on the TATA box of IL1B gene promoter region, which has been demonstrated to be a critical motif partially determining IL-1B transcriptional efficiency . FurtherThus, it is possible that IL1B SNP rs1143627 C allele has a lower binding affinity to c/EBPβ and reduced levels of IL-1B expression, which may therefore weaken the inflammatory response of Th-17 and Th-1 cells in humans during influenza infection. A cohort of study with a larger population size is needed to further confirm the association of IL1A rs17561 allele T and IL1B rs1143627 allele C with an increased risk for the susceptibility to A(H1N1)pdm09. Functional studies will also be needed to further investigate the role and mechanism of genetic variants of IL-1 on the impact of susceptibility to A(H1N1)pdm09.IL1A rs17561 allele T and IL1B rs1143627 allele C may confer an increased risk for the susceptibility to A(H1N1)pdm09. Thus, this study provides a new insight into the immunopathogenesis of influenza A virus infection in humans.SNPs: Single-nucleotide polymorphisms; IL: Interleukin; A(H1N1)pdm09: 2009 pandemic A/H1N1 influenza; MALDI-TOF: Matrix-assisted laser desorption/ionization time-of-flight; OR: Odds ratio; CI: Confidence interval.The authors declare that they have no conflict of interests.YL, SL GZ and GN collected the samples, performed the experiments and analyzed the data. ZM, DM and CC analyzed the data. XC, BZ and GZ oversaw the research and wrote the manuscript. All authors read and approved the final manuscript.Y Liu, MD, PhD, Director, S Li, BS, G Zhang, MD, PhD, G Nie, PhD, Z Meng, PhD, D Mao, BS, C Chen, BS, X Chen, MD, PhD, B Zhou, MD, PhD, Dean, G. Zeng, PhD.


Influenza virus infection activates NF-κB and is a general prerequisite for a productive influenza virus infection. On the other hand, non-structural protein 1 (NS1) suppresses this viral activated NF-κB, presumably to prevent expression of NF-κB mediated anti-viral response. NS1 proteins of influenza A viruses are divided into two groups, known as allele A and allele B. The possible functional relevance of this NS1 division to viral pathogenicity is lacking.in situ PLA.The ability of NS1 protein from two avian influenza subtypes, H6N8 and H4N6, to inhibit NF-κB promoter activation was assessed. Further, efforts were made to characterize the genetic basis of this inhibition. We found that allele A NS1 proteins of H6N8 and H4N6 are significantly better in preventing dsRNA induced NF-κB promoter activation compared to allele B of corresponding subtypes, in a species independent manner. Furthermore, the ability to suppress NF-κB promoter activation was mapped to the effector domain while the RNA binding domain alone was unable to suppress this activation. Chimeric NS1 proteins containing either RNA binding domain of allele A and effector domain of allele B or vice versa, were equally potent in preventing NF-κB promoter activation compared to their wt. NS1 protein of allele A and B from both subtypes expressed efficiently as detected by Western blotting and predominantly localized in the nucleus in both A549 and MiLu cells as shown by Here, we present another aspect of NS1 protein in inhibiting dsRNA induced NF-κB activation in an allele dependent manner. This suggests a possible correlation with the virus's pathogenic potential. Within hours of host-pathogen interaction, the type 1 interferons (IFNs), an essential arm of innate immune response, are induced to initiate a range of antiviral processes. The binding of dsRNA, produced as a viral by-product  to helicases or toll-like receptors (TLR), initiates a series of events culminating in the activation of two kinase complexes: TANK-binding kinase 1-inhibitor of kappa B-kinase ε (TBK1-IKK-ε) and IKK-α/β/γ . NF-κB cet al., [It has been demonstrated that influenza virus infection activates the NF-κB and exhibit higher levels of replication in cells where NF-κB is pre-activated, suggesting that a NF-κB signalling pathway is a general prerequisite for a productive influenza virus infection . Kumar eet al., , made fuet al., .Based on their amino acid sequences, NS1 proteins of influenza A viruses are divided into two groups, termed as allele A and allele B. There is little information available for the possible functional relevance of this difference to viral pathogenicity. However, recently we observed that allele A and B NS1s differ in their abilities to inhibit IFN-β promoter activation . It has ® T7 Quick Coupled Transcription/Translation System, Promega) with 35S radiolabeling.The NS1 genes from four viruses belonging to different subtypes (H6N8 and H4N6), each containing both allele A and B were cloned in a mammalian expression vector pcDNA3.1+ (Invitrogen). These isolates were named H6N8-A  H6N8-B , H4N6-A  and H4N6-B . The chimeric NS1s were constructed as follow. The RNA binding domain (amino acids 1-73) of H6N8-A and effector domain (amino acids 74-230) of H6N8-B constitute H6N8 chiNS1 A/B and the RNA binding domain (amino acids 1-73) of H6N8-B and the effector domain (amino acids 74-230) of the H6N8-A constitute the H6N8 chiNS1 B/A. The RNA binding domains (H6N8-A-RNA and H6N8-B-RNA) and effector domains (H6N8-A-ED and H6N8-B-ED) of each allele were also cloned in pcDNA3.1+. The inserts in all the clones were confirmed by sequencing and expression ability was checked by TNT  and either NS1 expression plasmid or empty pcDNA3.1+ were transfected with FuGENE6 (Roche) at a ratio of 1:3 per well. Twenty-four hours post-transfection, the cultures were stimulated by adding 10 μg/ml poly I:C (dsRNA) (Invivogen). Twenty-four hours post-stimulation, the cells were lysed using ONE-Glo™ Luciferase Assay System (Promega) and the lysates were subjected to Wallac Victor2™ 1420 multilabel counter  for luminescence. The plasmid pTA-Luc was transfected as a negative control for the estimation of background level of reporter gene activity and subtracted from all the values. The empty pcDNA3.1+ plasmid was transfected as a negative control and designated as mock treated. Each transfection experiment was repeated at least three times.A549 cells, a type II alveolar epithelial cell line from human adenocarcinoma , and mink lung cells (MiLu) were maintained as described . The dayin situ proximity ligation assay (PLA) was performed with the Duolink in situ PLA kit (Olink Biosciences). Western blotting was applied to check the total expression of NS1 protein, as we reported before [To check the expression of NS1 protein in the cells, d before .The NS1 protein of influenza A viruses mediates resistance to the eukaryotic immune system for the efficient replication of viruses even in the presence of a functional anti-viral system. This property of NS1 has been well characterized in different studies ,9. In thIn an initial experiment, 24 well plate was co-transfected with pNF-κB-Luc along with either NS1 expression plasmid for H6N8-A, H6N8-B, H4N6-A, H4N6-B or empty vector (mock treated) or left untreated (pNF-κB cont.). Relative luciferase activity was calculated 24 hours post-dsRNA stimulation. The A549 cells transfected with pNF-κB-luc and stimulated with dsRNA showed high activation of NF-κB promoter and was set to 100%. With the addition of NS1 from either of subtype or allele clearly diminished the NF-κB promoter activation Figure . AlthougThe results of the reporter assay proposed that NS1 proteins are involved in differential blocking of signal transduction pathways that lead to activation of the NF-κB promoters. Next, hypothesizing that these roles might be species-specific, we further examined this pathway in mink lung cells (MiLu). A corresponding picture appeared to that of A549 cells, where both allele A were consistently better in blocking the induction of NF-κB promoter compared to allele B NS1 protein of both subtypes. Allele B has shown an inhibition of 46% in H6N8 and 37% in H4N6 subtype of influenza virus and allele A has shown an inhibition of 84% and 80% in H6N8 and H4N6 compared to pNF-κB control, respectively Figure .in situ expression of NS1 protein from both subtypes and both alleles, in situ PLA, a highly sensitive technique, was applied. The same experimental set-up was followed as that of Western blotting and instead of lysing the cells, 12 hours post-NS1 transfection cells were fixed and processed for PLA. As expected and consistent with Western blotting, a high expression of NS1 was observed for both subtypes and for both alleles. Moreover, at this time point, NS1 predominantly accumulated in the nucleus in both A549 cells and MiLu cells  were constructed  may have biological relevance, which warrant further investigation. Moreover, allele A NS1 proteins differ with 71 aa (~31%) from allele B NS1 protein of both H6N8 and H4N6 subtypes. These amino acid differences most likely reflect differences in the respective protein function and would be interesting to study in terms of the biological significance in the context of viral infections.To further prove this notion, two chimeric NS1s were constructed containing either RNA binding domain of allele A and effector domain of allele B (H6N8 chiNS1 A/B) and vice versa (H6N8 chiNS1 B/A). Cell transfection was performed in A549 cells as outlined before with the difference that chimeric NS1 from both allele A and B were used. Interestingly, NF-κB promoter inhibition was reversed in both chimeric NS1 and was comparable to their wild type (wt) Figure . This abIn conclusion, the results of this study demonstrated that NS1 proteins from avian influenza A viruses (H6N8 and H4N6 subtypes) have the ability to inhibit NF-κB promoter activation and suggest a possible correlation with the virus's pathogenic potential. Mapping of the NS1 protein domains involved in inhibition of NF-κB promoter showed that NS1 devoid of RNA binding domain still exhibited property of NF-κB promoter inhibition. Conversely, RNA binding domain alone loses this inhibitory activity. However, for full elucidation, further experiments involving fusion of dimerization domain to that of RNA binding domain are required. Moreover, the full length NS1 protein is functionally interactive and can efficiently block NF-κB promoter activation regardless of RNA binding domain . Apart from NF-κB signaling, allele A and B NS1 protein from both subtypes were expressed predominantly in nucleus but also in cytoplasm in transfected human A549 and MiLu cells. On the basis of this work, we presented another dimension of NS1 protein in inhibiting dsRNA induced NF-κB activation that this character of NS1 is allele dependent.The authors declare that they have no competing interests.Conceived and designed the experiments: MM, SZ, MB. Performed the experiments: MM, SZ. Analyzed the data: MM, SZ, MB. Wrote the paper: MM, SZ, MB. All the authors have read and approved the final manuscript.