The role of innate immunity in general and of type I interferon(IFN-I) in particular in HTLV-1 pathogenesis is still a matter of debate.ADAR1-p150 is an Interferon Stimulated Gene (ISG) induced by IFN-I that can editviral RNAs. We therefore investigated whether it could play the role of ananti-HTLV factor.We demonstrate here that ADAR1 is also expressed in the absence ofIFN stimulation in activated primary T-lymphocytes that are the natural target ofthis virus and in HTLV-1 or HTLV-2 chronically infected T-cells. ADAR1 expressionis also increased in primary lymphocytes obtained from HTLV-1 infectedindividuals. We show that ADAR1 enhances HTLV-1 and HTLV-2 infection inT-lymphocytes and that this proviral effect is independent from its editingactivity. ADAR1 expression suppresses IFN-α inhibitory effect on HTLV-1 and HTLV-2and acts through the repression of PKR phosphorylation.invivo. The balanced expression of those proteins could determine thefate of the viral cycle in the course of infection.This study demonstrates that two interferon stimulated genes, i.e.PKR and ADAR1 have opposite effects on HTLV replication The online version of this article (doi:10.1186/s12977-014-0093-9) contains supplementary material, which is available to authorizedusers. Human T-cell leukemia virus type 1 (HTLV-1) was the first humanoncoretrovirus to be discovered . It infeInnate immunity plays a critical role in host response to a viralinfection by triggering effector mechanisms that restrict infection . EffectoThere are two ADAR1 isoforms. ADAR1-p110 is constitutively expressedand found exclusively in the nucleus, while ADAR1-p150 is an Interferon StimulatedGene (ISG) induced by IFN-I .ADAR1-pAs ADAR1-p150 expression can be induced by type I interferon, it wasanticipated that it would be an effector of type I interferon antiviral effect onviral replication. Indeed, viral genome editing by ADAR1 was described for severalviruses ,32. For ex vivo samples obtained from HTLV-2 infectedindividuals, thus demonstrating that ADAR1 editing of HTLV in vivo is a very rare phenomenon and is unlikely to play an antiviralrole [IFN-α is being used in combination to AZT to treat ATL patients,although the relationship between stimulation of innate immunity and impact on HTLVcycle are still not fully understood ,54,55. Wralrole . Though,+cells obtained from HTLV-1 infected individuals. We show that ADAR1 enhances HTLV-1and HTLV-2 infection in T-lymphocytes and that this proviral effect is independentfrom its editing activity. ADAR1 expression suppresses IFN-α inhibitory effect onHTLV-1 and HTLV-2 through repression of PKR phosphorylation. Together with ourprevious results, these studies demonstrate that two interferon stimulated genes,i.e. PKR and ADAR1 have opposite effects on HTLV replication in vivo. The balanced expression of those proteins could determine thefate of viral cycle in the course of infection.Here, we first demonstrate that ADAR1 is expressed in PHA/IL-2activated primary T-lymphocytes in the absence of IFN-I stimulation, in HTLV-1 orHTLV-2 chronically infected T-cells and in primary CD4in vivo. Given that HTLV-1infected T-lymphocytes display an activated phenotype [ADAR1 was previously shown to edit the genome of some RNA virusesand to act therefore as an antiviral factor ,34,44,45henotype , we firsThen, we compared the expression of both ADAR1 isoforms innon-infected Jurkat T-lymphocytes as well as in HTLV-1 (C91-PL and MT2) or HTLV-2(C19 and MO) infected T-lymphocytes  antigen and comparedto the p19gag negative population. Interestingly,HTLV-1 productively infected cells expressed higher levels of ADAR1 than cellsthat were also stimulated but did not express HTLV-1 antigens  editing of cellular and viralgenes ,31. Editnhibited .in vivo, at least in HTLV-2infected cells [We previously demonstrated that co-transfecting ADAR1 together withHTLV-2 or STLV-3 molecular clones in 293-T cells led to A-to-G editing although thefrequency of such editing could not be quantified . A similed cells .+T-lymphocytes and HTLV-2 infects CD8+ T-lymphocytes[ex vivo samples[+ T-cellsobtained from HTLV-1 infected individuals, thus suggesting that ADAR1 is expressedin response to HTLV infection. Interestingly, Clerzius etal have also shown that ADAR1-p110 and ADAR1-p150 expression isincreased when Jurkat cells are infected with HIV-1 [As HTLV-1 mostly infects CD4phocytes, we measphocytes, both AD samples, we haveADAR1 proviral effect was previously observed for different viruses,40,49-52et al have recentlydeveloped an assay to study the effect of hundreds of ISG products on differentviruses that belong to Flaviviridae ,Togaviridae  or Retroviridae (HIV-1) [Shoggins  (HIV-1) . This le (HIV-1) . Thus, tde novo infection through PKR activation[et al., PKR could play an antiviral role[We have previously shown that IFN-α inhibits HTLV-1 and HTLV-2tivation. We demoral role. Given tral role, itwoulIn conclusion, our results demonstrate that IFN-I promotes theexpression of two ISG that have antagonist roles on HTLV replication.2.293T and 293T-LTR-GFP cells were maintained in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% fetal bovine serum  and 100 μg/mL penicillin-streptomycin .Jurkat, Jurkat-LTR-luc, HTLV-1-  and HTLV-2-infected T-cell lines, were maintained in RPMI 1640 medium supplemented with 10% fetal bovineserum  and 100 μg/mL penicillin-streptomycin . Peripheral blood lymphocytes (PBL) were purified from bloodsamples obtained from healthy donors  orfrom HTLV-1 infected individuals. HTLV-1 infected blood samples were obtained inthe context of a Biomedical Research Program approved by the "comité de protectiondes personnes", Ile de France III, Paris  withinformed consent of all individuals. Cells maintained in RPMI 1640 mediumsupplemented with 10% fetal bovine serum  and 100 μg/mLpenicillin-streptomycin . PBL were either left quiescentor stimulated with phytohemagglutinin  and interleukin-2(IL-2 at 150 U/mL). When required, IFNα-2a (Tebu-Bio) was added for 3 days to thecells. 293T-LTR-GFP and Jurkat-LTR-luc are stably transfected with a plasmidencoding GFP (green florescent protein) or luciferase under the control of HTLV-1long terminal repeat (LTR), respectively ,64. All + B cells were purified bydepletion/negative selection using a B-cell isolation kit (Miltenyi Biotech).Purity of isolated B-cells was monitored using anti-hCD19 VioBlue (MiltenyiBiotech) and was analyzed by fluorescence-activated cell sorting .CD19B-lymphocytes were cultured in RPMI 1640 medium (Gibco BRLInvitrogen) supplemented with 10% FCS and 1% penicillin/streptomycin with 50 ng/mlIL2 (Miltenyi Biotech). They were either treated with Pansorbin 200 ng/ml (activated condition) or left untreated (non activatedcondition). 24 h and 48 h later cells were labeled with hCD19-PE, hCD3-PE andhCD69-PEvio770 (Miltenyi Biotech). ADAR antibodies (Abcam) were used afterpermeabilization/fixation using the Cytofix/Cytoperm kit (BD).+ T-cells obtained from healthydonors or HTLV-1 infected individuals were purified by depletion/negativeselection using T-cell isolation kit (Miltenyi Biotech). Purity ofCD4+ T-cells was monitored using anti-hCD4APCantibodies (Miltenyi Biotech) and was analyzed by fluorescence-activated cellsorting . CD4+T-lymphocytes were then cultured at 1.106/ml with IL-2(150 U/ml) and PHA (1 μg/ml) for 3 days and then maintained only with IL-2 for5 days. Cells were analyzed with a surface staining against hCD4-APC and hCD69-PE(Miltenyi Biotech) or intracellular staining for ADAR and p19 (Zeptometrix) afterpermeabilization/ fixation with the Cytofix/Cytoperm kit (BD). Cells were analyzedon a FACS-Canto II (BD Sciences) collecting 100.000 events. Results were analyzedusing the FlowJo software.CD4HTLV-1 proviral DNA clone (pACH) was provided by Dr L. Ratner. HTLV-2 6) or PBLs(5.105) were transfected with 2.5 μg of the HTLV-1 orthe HTLV-2-LTR-luciferase plasmids and 2.5 μg of the ADAR1 plasmid  following the manufacturer’s instructions.Twenty-four hours later, they were co-cultured with irradiated  C91-PL or C19 cells (3:1 ratio). When needed, PBLs weretreated with 50 μM AZT (Sigma) 24 hours and 3 hours before infection. Twenty-fourhours later, reporter activity was assayed . Luciferase activity was normalized by protein concentration asdetermined by the Bradford method (Bio-Rad).Jurkat (106) cells were seeded onto100 mm dishes. Twenty-four hours later, cells were transfected with 4 μg of HTLV-1(pACH) or HTLV-2 (pH6neo) molecular clones and increasing amount  of ADAR1 expression plasmid . Forty-height hourspost-transfection, 293 T cells were washed and co-cultured with reporterJurkat-LTR-Luciferase (1:3 ratio) cells for 24 hours. Reporter activities wereassayed using the luciferase reporter assay system (Promega) and normalized byprotein concentration as determined by the Bradford method (Bio-Rad).293T (3.106) were seeded onto100 mm dishes. The following day, 4 μg of pACH (HTLV-1), pH6neo (HTLV-2) or SV2Neo(control) plasmids were transfected with or without 5, 50, 500 ng of ADAR1 usingPolyFect reagent (Qiagen) following manufacturer's instructions and cells weretreated with increasing amount  of IFN-α2a (Tebu-Bio) for48 hours. Reporter activity was assayed  and normalized by protein concentration as determined by the Bradfordmethod (Bio-Rad).293T cells (3.106) or PBL (5.105) were transfected with 2 μg of the HTLV-1 or theHTLV-2-LTR-luciferase plasmids, 2 μg of the HTLV-1 (pACH) or HTLV-2 (pH6neo)molecular clones with or without 1 μg of the ADAR1 expression plasmid  following manufacturer’s instructions.Forty-eight hours later reporter activity was assayed . Luciferase activity was normalized by protein concentration asdetermined by the Bradford method (Bio-Rad).Jurkat cells  following the manufacturer’s instructions as previouslydescribed [Cell culture supernatants were collected 48 hours post-transfectionand the presence of the HTLV matrix p19escribed . In brie5 cells per well onto 6-well plates. Thefollowing day, 20 nM of PKR siRNA  orcontrol siRNA  were transfected as previously described [293T cells were seeded at a concentration of3.10escribed . Twelve 3VO4, 10 mMimidazole) in the presence of protease inhibitors and incubated with Ni2+-NTA (nitrilotriacetic acid)beads (Sigma) at +4°C under agitation. Beads were then washed extensively in Chrisbuffer. Bound proteins were eluted in Laemmli buffer and processed for immunoblotanalyses. The following antibodies were used: anti-His ,anti-OPTN , anti-PKR  with a Tax1-His-encoding plasmid (4 μg), together with an OPTN- or aPKR-encoding plasmid (4 μg). Twenty-four hours later, cells were harvested andlysed in Chris buffer  and protease inhibitors (Complete RocheApplied Science) as previously described . Cell de


Human T-cell Lymphotropic virus (HTLV) prevalence is low in the UK, but higher in individuals originating from endemic countries. Anti-HTLV screening of all blood donations was introduced in 2002 to reduce the risk of transfusion transmission. The UK HTLV Cohort (UHC) was established for longterm follow up of people affected by HTLV. Recruitment by clinicians at blood centres and specialist clinics began in July 2003. Participants are asked to complete a baseline self completion questionnaire (SCQ) about their health and HTLV risk factors, flagged in central registries for cancer or death, and followed up about their health and well being every 2-3 years. Recruiting clinicians report participant’s clinical state at diagnosis. By March 2013, a total of 161 HTLV positive individuals had consented to take part: 89 of these were blood donors. Most were female (77%), of non-white ethnicity (64%) with a mean age of 49 years. Most (83%) positive participants were asymptomatic for HTLV at registration and eight (4%) participants died during follow-up. Baseline questionnaires were received from 160 (85%). Follow up was done in 2003, 2007 and 2010, the fourth is underway. Once complete, approximately 1,000 person years of follow up will be analyzed, factors associated with symptoms will be investigated and an assessment of the progression of HTLV related disease among the UHC will be presented. Information from the UHC, obtained from a decade of follow up, will provide a unique perspective since it is the only cohort of its kind in Europe.


Infections with human retroviruses, others than the HIV virus, are not very well known in Romania, despite the fact that screenings for HTLV I and II are constantly performed in blood donation centers. The authors draw attention on the possible severe consequences that may occur in chronic infections with these viruses by presenting the case of a 35 year-old male, who was known to be infected with HTLV I virus for several years and who consequently developed adult cell leukemia.The authors also plead for the development of an algorithm for the detection, surveillance, monitoring and treatment of HTLV infections, throughout interdisciplinary cooperation between infectious disease specialists, hematologists, dermatologists.The authors strongly recommend the development of means to promote prevention by: securing blood transfusions; forbidding breastfeeding in women who tested positive for HTLV; using condoms in order to prevent sexual transmission in discordant couples.


Human T cell lymphotropic virus type-1 (HTLV-1) is the etiologic agent of Adult T cell Leukemia and Lymphoma (ATLL) and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus prevalent worldwide with foci of high prevalence. However, effective vaccines or drugs against HTLV-1 infection are not available. Because of the severity of the diseases and the absence of a satisfactory treatment, there is indeed an urgent need for an efficient vaccine against HTLV-1 in endemic regions. HTLV-1 is associated with persistent and lifelong infections and a low incidence of lymphomas within its hosts, and can be spread through the contact with bodily fluids containing infected cells, most often from mother to offspring through breast milk. These previous observations suggest that HTLV-1 has common features in infection and protective immune mechanisms with Bovine Leukemia virus (BLV). Imm une protection of newborns by vaccination is difficult to achieve since there is not enough time to mount an immune response before exposure to the virus. Interestingly, previous study revealed that antibodies from the maternal colostrum protect from BLV infection. On the basis of the above reports, we have designed a vaccination strategy mediating transfer of neutralizing antibodies from the mother to the offspring during pregnancy and / or lactation. In the present study, we investigated the effectiveness of intranasal vaccine using HTLV-1 Env protein as an antigen in combination with a synthetic double-stranded RNA analog for the induction of specific antibody in serum and breast milk in mother mice. Full-length Env protein was synthesized using the wheat germ cell-free protein production system. Intranasal administration for mother mice with the Env protein and the synthetic double-stranded RNA adjuvant, polyriboinosinic-polyribocytidylic acid [poly(I:C)] generated significant Env-specific systemic and mucosal immune responses as evidenced by the prominent induction of serum and milk antibodies. Our results provide the first evidence that intranasal co-administration of HTLV-1 Env protein with the poly(I:C) adjuvant augments the viral-specific immunity against HTLV-1.


Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety. In both the germline (endogenous retroviruses) and in somatic cells , retroviruses can integrate and persist in humans for years. Certain retroviruses are major pathogens, while others have played important roles in genome evolution ,2. The lin vivo. Transmission between individuals occurs through infected breast milk, blood or semen [HTLV-1 and the closely related HTLV-2 both preferentially infect T cells or semen . Efficieor semen . Membranor semen .+ T cells [+ cells [in vitro that this preference may be due to differential proliferation of infected cells rather than a differential tropism [Following cell entry, the HTLV-1 viral RNA genome undergoes reverse transcription, and a single copy  of the r T cells , while H[+ cells . There i tropism .ex vivo [in vivo, at least at some times and in some cells. How Tax silencing and re-expression are regulated is not understood.Although expression of HTLV-1 genes provides a proliferative advantage to the infected T-cell clone, it also leads to viral epitope display on host HLA class I molecules, making the infected cell a target of the host cytotoxic lymphocyte (CTL) response. In chronic infection, viral gene products from the HTLV-1 positive strand (including Tax) are rarely detected in PBMCs directly ex vivo ,15, suggex vivo ,17 impliin vivo and is detected in PBMCs directly ex vivo [In contrast to Tax, expression of HBZ from the minus strand is maintained at a low level  ex vivo . HBZ app ex vivo . Althoug ex vivo . HBZ is  ex vivo . Thus th ex vivo . The surin vivo, which increases the success of therapy [The retroviral DNA integrated in the host genome is transmitted to daughter cells at mitosis. This makes retrovirus-based gene delivery systems an attractive method to stably introduce new genetic information into host cells, for instance to correct gene defects. Substantial progress has been made in developing retrovirus-based gene therapy vectors, especially for primary immunodeficiency disorders. In cases where HLA-matched donor bone marrow is not available, patient-derived autologous hematopoietic stem cells (HSC) can be isolated, transduced and transplanted back into the patient . Disease therapy .Gene therapy vectors based on the gammaretrovirus murine leukaemia virus (MLV) have been used in a number of gene therapy trials following the first trials in SCID-X1 ,25. The Recently, HIV-based self-inactivating vectors have been used in a number of trials ,28,29,30Ligation-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR) have been the most frequently used systems to determine retroviral provirus or vector integration site. Both techniques involve ligation of a linker DNA cassette to fragmented genomic DNA, which enables PCR amplification between known sequences in the viral LTR and the linker. High-throughput sequencing (for instance Illumina technology) is then used to sequence the host integration site DNA between the LTR and the flanking restriction site ,35. LAM-i.e., sharing the genomic integration site) has a shear site in its genome at a unique distance from the viral integration site. This makes it possible to count the sequenced sister cells of each clone. A small correction is needed for very abundant clones, because identical shear sites can occur by chance [Recent modifications to both LM-PCR and LAM-PCR have addressed these issues. A modified protocol for nrLAM-PCR has eliminated the need for restriction enzymes by ligating single stranded linker oligonucleotides to linear single-stranded amplified vector-genome junctions . nrLAM-Py chance ,40. Furty chance . Finallyy chance .Retroviral integration is not random at the primary sequence level. MLV and derived gene therapy vectors share weakly conserved, apparently palindromic target sequences with symmetry around a 4 bp target sequence that is duplicated during integration ,44. ThisThe conserved DNA sequence does not completely describe the observed retroviral integration site profile: for example, MLV integration sites are strongly clustered in the genome. Recent high-throughput sequencing of MLV integration sites (>1 million sites sequenced per study) demonstrated that at least half of the sequenced MLV integration sites could be assigned to tight clusters that covered less than 2% of the genome ,50.MLV preferentially integrates near transcriptional start sites (TSS) and gene regulatory elements such as promoters and enhancers ,50,51,52versus random integration can be extremely high  [in vitro and in vivo infection [Like MLV, HTLV-1 integration near TSS is also favoured. This bias is greatest at approximately 1Kb from the TSS, and diminishes sharply within 100 bp from the TSS , perhapsd HDAC6) , althougnfection ,56,57,58In vitro, we observed preferential HTLV-1 integration in a human T cell line (Jurkat) within 10 Kb of epigenetic marks associated with active promoters or enhancers [HTLV-1 preferentially integrates in areas containing active epigenetic marks (~twofold higher than random frequency of integration). nhancers ,47. Thernhancers   proteins  ,71,72. BAs has been demonstrated for other retroviruses, it is likely that the HTLV-1 integrase is guided to preferred locations in the genome by interactions between the integrase and host proteins. Identifying the putative partners of the HTLV-1 integrase is a subject of current research.in vivo through mitotic division to become clonal populations. Cells containing certain integration sites can undergo selective expansion in vivo, resulting in abundant clonal populations [Following initial integration, infected or transduced cells persist for years ulations ,75.Strongyloides stercoralis [We have sequenced HTLV-1 integration sites from PBMCs in individuals with chronic HTLV-1 infection, both asymptomatic individuals and those with the inflammatory disease HAM/TSP or the HTLV-1 associated malignancy ATLL, and in people co-infected with HTLV-1 and rcoralis ,56,57,58rcoralis .+ clones appear to make up a disproportionately high number of the most highly expanded clones [Strongyloides, but the total number of clones in the blood was not significantly different from asymptomatic individuals [There are of the order of 10,000 integration sites in a typical HTLV-1 infected person . This imd clones . The disd clones . The infd clones . The gred clones . Highly ividuals . In ATL,ividuals . In nearividuals . The conRetroviral gene therapy trials have also shown differential expansion of clonal populations, and in some cases this has led to malignancy. The first two clinical trials of gammaretroviral gene therapy for X-linked severe combined immunodeficiency (SCID-X1) in France and the UK showed effective reconstitution of the patient’s immune system ,25. HoweIntegration sites near certain growth-promoting genes, including LMO2, were associated with leukaemia in the SCID-X1 trials ,4,75,81.In both gene therapy and exogenous retroviral infections such as HTLV-1, key questions include the following. What is the role of the host genome flanking the integration site in regulating viral or transgene expression? How does this influence cell proliferation or targeting by the host response? Conversely, what is the effect of the introduced viral promoter on the expression of host genes? And finally, what other factors contribute to the skewing of the integration site clone profile?de novo infection and error-prone reverse transcription. Differential expression between integration site clones, therefore, must be predominantly influenced by the host genomic environment of the integration site.There is little variation in the sequence of HTLV-1, either between clones in an infected person or between different infected individuals , comparein vitro, such as regions marked by high levels of epigenetic marks associated with active regulatory elements and sites in close proximity to certain transcription factor binding sites, are less frequent in vivo than in vitro—although still more frequent than random [in vivo are enriched in large genomic regions (1Mb) with high levels of inhibitory marks, compared to in vitro integration and in silico random sites. It is likely that active integration sites are counter-selected, for instance due to expression of Tax, during the early response to HTLV-1 infection. Studies of acute HTLV-1 infection in humans are rare. However, in a closely related deltaretrovirus, bovine leukaemia virus (BLV), active sites near promoters or genes favoured during initial integration were strongly selected against in the acute phase of the host response [The expression of HTLV-1 viral genes exposes the infected cell to attack by the host immune response. Preferred integration sites n random ,55. We hresponse . These tresponse , making ex vivo at either the mRNA or protein level. Upon in vitro culture the majority (~60%) of cells begin to express Tax within 24 h, although what regulates this release from repression is incompletely understood. However, clones that remained Tax-negative even after overnight in vitro culture have a higher clonal abundance in vivo. Clones carrying proviruses integrated in the same transcriptional orientation as an upstream flanking gene were more likely to be Tax- silenced in vitro, implying that transcriptional interference may silence Tax expression [tax gene [HTLV-1 clones that persist into the chronic phase of HTLV-1 infection proliferate to form clonal populations, while remaining a target of a chronic CTL response. Virtually all infected patients have detectable Tax-specific CTLs , and Taxpression . Tax exppression   is associated with silencing, for example the inactive X chromosome [Integration sites throughout the acrocentric chromosomes  are also significantly over-represented om sites . This doromosome : thus, iin vivo, clone abundance is also correlated with the frequency of proviruses inserted in genes and actively transcribed areas of the genome during HTLV-1 chronic infection [in vivo and Tax-targeted CTLs are common, it is possible that integration in an active genomic region may instead favour increased expression of HBZ. Integration in transcriptionally active genomic sites were also found to be more frequent in clones from a Southern Japanese asymptomatic cohort compared to clones of a similar abundance in Japanese HAM/TSP patients. This observation may be explained by a requirement for greater clonal proliferation in AC to reach a given absolute abundance, in the face of a stronger host response [Although Tax silencing is associated with survival and abundance nfection ,86. Highnfection . This obresponse ,77.in vivo [The transgenes expressed in gene therapy vectors do not have the transactivating effects of the viral proteins, and do not drive unregulated proliferation. Analogous to HTLV-1, however, gene therapy transgene expression may support expansion of transduced cells, and integration site environment may alter transgene expression. Transgene expression may confer a selective advantage on the transduced cell, which is desirable to maintain the transduced population in vivo . For insThe particular transgene expressed may contribute to the risk of adverse events. MLV-based retroviral vectors were used in both SCID-X1 and ADA-SCID trials, yet in contrast to the SCID-X1 trials no recipients in the ADA-SCID trials developed clonal dominance or leukaemia ,89,90. Ti.e., an antigen “foreign” to the host immune system. Residual retroviral epitopes may provide immunogenic epitopes  to stimulate the host response [In gene therapy, except in diseases involving a severely compromised immune system, such as both SCID-X1 and ADA-SCID, the immune response may also target and restrict transduced cells to expressing a transgene, which encodes a neoantigen, response . Indeed,response . Recent response ). As in in vivo. A study comparing HIV-1 latency in five different primary T cell clones or T cell lines found no common predictors of latency across all lines. However, proviruses integrated in the same chromosomal region of the same cell type shared the same pattern of expression (either expressed or silent/inducible), implying an effect of genome location on expression status [At present there is little direct evidence that the characteristics of the integration site of gene therapy vectors alter the expression of the transduced gene. During the Frankfurt CGD trial of two patients, although successful engraftment led to an initial functional recovery, transgene expression became silenced by promoter methylation . Understn status . The actn status , and virn status .Insertional mutagenesis by retroviruses, as well as by MLV- and lentiviral-based vectors, has been extensively used to identify oncogenes . Theex vivo from SCID-X1 patients transduced with an IL2GR gammaretroviral vector showed only minor vector-mediated expression changes but no changes in cellular growth rate or mutation rate [c-myb promoter, demonstrating the ability of integrated retroviruses to influence distant genes [While retroviral vector integration in certain sites can clearly be hazardous, there is little evidence of more widespread disturbance of gene expression due to the integrated retroviral gene therapy vectors. Analysis of single T cell clones ion rate ,103. In ion rate ,103. Thent genes .It is interesting that no abnormal clonal expansion has been observed to date in lentiviral vector clinical trials in adrenoleukodystrophy , Wiskotin vivo mouse models [Many lentiviral systems use a self-inactivating vector, in which the promoter within the U3 region of the viral LTR is deleted in both LTRs of the inserted provirus. Transgene expression is driven by an internal promoter . Strong e models . Physiole models ,110. Game models  and usede models .It is clear that insertional mutagenesis contributes to leukaemogenesis caused by gammaretroviral vectors. In contrast, it has been widely believed that the transactivating effects of Tax and HBZ are responsible for leukaemogenesis in HTLV-1 infection . Howeverin vivo  and those that are more frequently observed because of intrinsic initial vector targeting preferences. Common integration sites observed in an X-linked ALD lentiviral vector clinical trial and in transduced human hematopoietic stem progenitors cells engrafted into immunodeficient mice both showed broad (megabase) regions where lentiviral integration frequency was enriched. This seems likely to reflect an underlying lentiviral preference for these genomic regions [+ hematopoietic stem cells, which have histone marks reflecting open chromatin at that location [in vivo selection [in vivo.It is important to distinguish between sites that are more common because of preferential expansion or survival  regions . It is p regions ,61. Simi regions ,116,117.election . The genelection . Howeverin vivo. In T lymphocytes, for example, the specificity of the T cell antigen receptor (TCR) of the infected clone also differs between clones. In chronic co-infection with HTLV-1 and the helminth Strongyloides stercoralis, and in patients with bacterial infection in infective dermatitis associated with HTLV-1 (IDH), the most abundant clones are not enriched in transcriptionally active genomic integration sites [Clones of retrovirus-infected cells can differ in factors other than the integration site that influence survival and proliferation on sites . It is pIn addition, the conditioning regimen may play an important role in expansion and the risk of adverse events. One difference between the ADA-SCID and SCID-X1 trials was the lack of conditioning in the latter . SCID-X1+ cells are transduced, only some will be long-lived hematopoietic stem cells, which will contribute to long-term generation of cells in vivo.Stochastic effects are also likely to play a role in determining which gene therapy integration site clones form the long-term transduced repertoire. For instance, if a heterogeneous population of CD34in vivo. In HTLV-1, a key driver of clonal abundance is the expression level of the genes including Tax and HBZ, and the balance between proliferation and immune system targeting. Expansion of gene therapy vector transduced cells can be influenced by insertional mutation, or advantages conferred by certain transgenes, or by the engraftment potential of the cell. In both HTLV-1 infection and in gene therapy, not all clonal expansion is pathogenic, and progression to pathogenesis, such as leukaemia, is the result of a complex integration of factors.Long-term clonal integration site composition in both chronic HTLV-1 infection and gene therapy depends on both the initial profile of viral integration and on selective survival and expansion of certain integration sites in vitro and causes disease. It had been widely accepted that oligoclonal proliferation of HTLV–1 infected cells plays a central part in the pathogenesis of both the inflammatory diseases  and the malignant disease (ATL) caused by HTLV-1. But recent data on clonal abundance and HTLV-1 proviral expression strongly indicate that oligoclonal proliferation per se does not contribute to the pathogenesis of either type of disease, even if continuous or intermittent HTLV-1 gene expression is required for viral persistence in vivo [The recent work on HTLV-1 integration has overturned several preconceptions of how the virus behaves  in vivo ,114. Addi.e., insertional mutagenesis) is also gaining attention, analogous to the effects seen with gene therapy vectors and other retroviruses (The focus in the HTLV-1 field to date has been on the role of viral proteins such as Tax and HBZ in driving clonal proliferation and influencing oncogenic progression. The recent observations have revealed the extent of the interaction between host genome and virus in driving viral gene expression, which may be favourable or unfavourable for the virus. The role of the provirus in influencing neighbouring host genes (oviruses . It is aThe adverse events in the early gene therapy trials in SCID led to extensive studies to determine the genotoxic safely of retroviral vectors, and the insights from these studies have considerably reduced the side effects of gene therapy. Both benign clonal expansion and malignant transformation in gene therapy are the result of multiple, often interacting, factors, including insertional mutagenesis. The viral integrase determines preferential integration sites; some locations, such as active promoters and enhancers, may increase the chance that integrated sequences alter host gene expression levels. Physiologically appropriate promoters have been used to reduce the risk of vector-mediated host gene expression changes. In addition, the transgene expressed and the conditioning regimen may also support cell survival or proliferation, in turn favouring successful long-term engraftment.Investigation into the role of the host immune response in limiting clone expansion due to increased expression, as observed in HTLV-1, is becoming more relevant as gene therapy moves into patients with intact immune systems. The host may mount an immune response both to novel or previously unexpressed transgenes that form a neoantigen. More work is also needed on how the host genomic environment of gene therapy vectors influences transgene expression, as in HTLV-1 infection, to optimize the pattern and intensity of transgene expression.High-throughput integration site analysis allows integration site profiles to be monitored during therapy. LM-PCR was recently used in HIV-1 infection to show that clonal proliferation contributes to the maintenance of the reservoir of HIV-1 during anti-retroviral therapy . In HTLVOur understanding of clonal expansion and malignancy in both HTLV-1 and gene therapy has been instrumental in altering our view of pathogenic mechanisms of HTLV-1 associated diseases, is informing application of novel ATL treatments, and has led to safer gene therapy in the clinic.


Vibrio parahaemolyticus inactivation on sterile shelled fresh shrimp were obtained using response surface methodology: NaCl concentration to electrolysis 1.5 g/L, treatment pressure 400 MPa, treatment time 10 min. Under the optimal conditions mentioned above, AEW dramatically enhanced the efficiency of HHP for inactivating V. parahaemolyticus and Listeria monocytogenes on artificially contaminated shelled fresh shrimp, and the log reductions were up to 6.08 and 5.71 log10 CFU/g respectively, while the common HHP could only inactivate the two pathogens up to 4.74 and 4.31 log10 CFU/g respectively. Meanwhile, scanning electron microscopy (SEM) showed the same phenomenon. For the naturally contaminated shelled fresh shrimp, AEW-HHP could also significantly reduce the micro flora when examined using plate count and PCR-DGGE. There were also no significant changes, histologically, in the muscle tissues of shrimps undergoing the AEW-HHP treatment. In summary, using AEW as a new transmitting medium for HHP processing is an innovative non thermal technology for improving the food safety of shrimp and other aquatic products.Acidic electrolyzed water (AEW), a novel non-thermal sterilization technology, is widely used in the food industry. In this study, we firstly investigated the effect of AEW as a new pressure transmitting medium for high hydrostatic pressure (AEW-HHP) processing on microorganisms inactivation on shelled fresh shrimp. The optimal conditions of AEW-HHP for  Shrimp is one of the most popular and important seafood worldwide because of its delicacy and abundant nutrients  has been regarded as an effective sanitizer and widely used in the food industries of many countries . A response surface (RS) model was applied to quantify the influence of AEW on high hydrostatic pressure processing for reducing pathogenic bacteria on shelled fresh shrimp. And the preservation effect of this new technology was evaluated by the reduction of indigenous microflora and the change of muscle fiber of shelled fresh shrimp. This novel sterilization and preservation technology could provide a powerful tool for eliminating pathogenic microorganisms and improving the seafood quality and safety.V. parahaemolyticus strains  and a two-strain cocktail of L. monocytogenes  were used in the study. Each strain for V. parahaemolyticus (stored in 25% glycerol at -80°C) was separately cultured in tryptic soy broth  plus 3% NaCl and incubated at 37°C for 18 h. Other strains of L. monocytogenes were grown in TSB at 37°C for 18–20 h. Enriched cultures were pooled into a sterile centrifuge tube and centrifuged at 3000 g, 25°C for 10 min . Pelleted cells were re-suspended in sterile peptone water  to obtain a multi-strain cocktail of ~ 9 log10 CFU/mL of V. parahaemolyticus and L. monocytogenes, respectively.A four-strain cocktail of Litopenaeus vannamei) were purchased alive from a local market in Shanghai, PR China before each experiment and transported to the laboratory in plastic bags containing pond water sparged with oxygen to keep the shrimps viable. At the laboratory the shrimps were rinsed with running tap water, a protocol described in GB/T 4789.7-2008  for 30 min, and then transferred to plastic plates for 30 min to allow for bacterial attachment. The above protocol was carried out at room temperature (23 ± 2°C). The inoculated shrimps were used for the subsequent treatments.Fresh shrimps  was used in the present study. The come-up rate was approximately 240 MPa/min and the pressure release time was almost immediate. Samples were packaged in sterile plastic bags and treated under the desired levels 200, 300, or 400 MPa, for treatment time 5, 10, or 15 min. Common water or AEW was used as the transmitting medium for HHP.V. parahaemolyticus, L. monocytogenes and indigenous microflora on shrimp samples. TCBS ager for V. parahaemolyticus, PALCAM agar  for L. monocytogenes and Trypticase Soy Agar  for indigenous microflora was used, respectively. The detection limit of the direct-plating was approximately 2 log10 CFU/g. Colonies were counted after the plates were incubated at 37°C for 24 h. Three replicates at each sampling time were done.The bacterial enumeration method used in this study was the plating counting method according to previous studies  was used to design the treatment conditions  was used to describe the relationship and interactions between three technological variables Table  on the ins Table , for regns Table , and theANOVA) and the corresponding post-hoc contrasts were performed to compare the results of the different experimental conditions. The statistical significance and goodness of fit of the model were evaluated using the determination coefficients (R2), the P-values from the Fisher F-test and the Lack of Fit test. The accuracy of the RS model describing the inactivation of V. parahaemolyticus was evaluated by the following criteria: the accuracy factor , bias factor  and the root-mean-squares error  and the Bf (Equation 2) as proposed by Ross . The PCR program was performed according to Muyzer et al.  using 20 μL of the purified and concentrated PCR product. The PCR products were applied to 8% (m/v) polyacrylamide gels in 1 × TAE buffer which contained a 40–55% urea-formamide denaturing gradient. Electrophoresis was conducted at 100 V for 10 h under a constant temperature (60°C). The DGGE gels were stained with SYBR green I and visualized under UV light . Pi was calculated as Equation (5).Pi is the importance probability of the bands in a gel lane, ni is the height of a peak and N is the sum of all the peak heights of the bands in the densitometric profile . Shannon index was calculated by DGGE banding pattern analysis. Shannon index of bacterial diversity, The histological analysis was performed according to Liu D. et al.  with somAn image analysis of each picture was made according to the methods of Díaz-Tenorio et al. . Blank aSD). Statistical analysis was performed by using SPSS statistical package 17.0 . One way analysis of variance was conducted to compare the effects under different conditions. The least significant difference (LSD) test was used to determine differences at α = 0.05.All values were expresses as the mean ± standard deviation  were designed by BBD. Table V. parahaemolyticus on shelled fresh shrimp under different conditions. The actual values of Log reductions ranged from 2.72 to 6.03 log10 CFU/g.The initial load on shelled fresh shrimp was approximately 7.1 logR was response value (log10 CFU/g); X1, X2, and X3 represent variables, including NaCl concentration to electrolysis, treatment pressure and treatment time, respectively. The results of the ANOVA for quadratic model indicated the goodness of fit of the regression equation: the R2 and Adj. R2 were 0.994 and 0.986, respectively. The value of the Adj.R2 indicated a high degree of correlation between the observed and predicted values, which suggested that only 1.6% of the total variation cannot be explained by the current model  indicated that the differences between different treatments were highly significant. The model is adequate since the Lack of Fit test (p > 0.05) was not significant  as the concentration of NaCl and pressure increased. The change of NaCl concentration played a similar role with treatment time in V. parahaemolyticus inactivation, and the bacteria were less sensitive to both NaCl concentration and time than pressure . The Af value was 1.03, revealing a merely 3% difference between the observations and predictions. The calculated Bf was 1.01, which lies in the acceptable range of 0.9–1.05 proposed by Ross . All results showed that AEW-HHP was more effective than HHP for inactivation of V. parahaemolyticus and L. monocytogenes, with a maximum reduction of about 6.08 and 5.71 log10 CFU/g at 400 MPa, respectively for AEW-HHP .Efficacy of AEW-HHP in reducing indigenous microflora on shelled fresh shrimp is reported in Figure H′ of untreated shrimp samples was 2.883, and this value changed to 2.418 by HHP treatment. Surprisingly, the Shannon index H′ was decreased to 2.064 when AEW-HHP was applied to treat the shrimp samples. Moreover, the average similarity coefficient of DGGE fingerprints of microbial communities treated with AEW-HHP (0.567) was lower than that with HHP (0.716). Therefore, these results directly demonstrated that AEW-HHP had a stronger efficacy on reducing the diversity of microbial communities in shelled fresh shrimp.PCR-DGGE method was carried out to investigate the effect of HHP and AEW-HHP on the diversity of microbial communities in shrimp. The results of PCR-DGGE are shown in Figure p > 0.05).Figure An effective sterilization and preservation technology is a prerequisite for food safety and quality in the food industry (Heinz and Buckow, V. parahaemolyticus inactivation on shelled fresh shrimp were obtained by using a response surface methodology. The results of the model showed that 1.5 g/L was the optimal NaCl concentration to produce the AEW, and the optimal treatment pressure and treatment time were determined as 400 MPa and 10 min respectively. Under the optimized treatment conditions, this method could effectively reduce V. parahaemolyticus on the shelled fresh shrimp by 6.08 log10 CFU/g. The response surface model developed in this study gave strong evidence that AEW combined with HHP could effectively eliminate V. parahaemolyticus in seafood, and also this model could be applied in the future for assessing the infection risk reduction of V. parahaemolyticus (Espinosa et al., The optimal conditions of AEW-HHP for L. monocytogenes and indigenous microflora on shelled fresh shrimp were investigated in this study. Due to the fact that the optimal conditions for V. parahaemolyticus inactivation may not be appropriate for other microorganisms, we evaluated the reduction of L. monocytogenes and indigenous microflora on shrimp samples at 200, 300, and 400 MPa (Figures V. parahaemolyticus inactivation from 4.74 up to 6.08 log10 CFU/g. For L. monocytogenes, AEW-HHP could reduce 5.71 log10 CFU/g, while HHP could only reduce 4.31 log10 CFU/g. A similar result was observed for indigenous microflora on shelled fresh shrimp, with AEW-HHP showing an additional reduction of 1.37 log10 CFU/g. The fact that the effect of AEW-HHP was remarkably higher than that of HHP might be explained by the synergy between bactericidal techniques. As an effective sterilizing agent, AEW not only was widely used to eliminate a variety of pathogens in food products (Xie et al., In order to further verify the sterilization performance of this AEW-HHP method for other bacteria,  Figures . CompareV. parahaemolyticus and L. monocytogenes shrimp samples treated by AEW-HHP in comparison with HHP treatment. Although SEM is not a quantitative technique, it provided more comprehensive information and clearly displayed the survival situation of bacteria on shrimp after AEW-HHP treatment.The microbial populations on shrimp were further observed by SEM Figure . SEM hasBesides traditional methods, molecular techniques were also employed in this study to evaluate the efficiency of AEW-HHP. DGGE fingerprints directly demonstrated that AEW-HHP had a stronger efficacy on reducing the diversity of microbial communities on shrimp than conventional HHP Figure . PreviouTo summarize, various results of this study have demonstrated that AEW could be used as a new transmitting medium for improvement of sterilization and preservation performance of HHP processing. The AEW-HHP method could serve as a potential sterilization and preservation technology to reduce the risk of microbial infections and ensure the aquatic products safety. Hence, the AEW-HHP approach developed in this study could provide an effective tool for improving the seafood safety and protecting public health.YZ and YP designed and drafted the work, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy, and final approval of the version to be published. SD, ZZ, LX, and YL did the work of acquisition, analysis and interpretation of data for the work, and revising it critically for important intellectual content, and integrity of any part of the work are appropriately investigated and resolved, and final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HTLV-1/2 and HIV likely share the same transmission routes and thus in Europe HTLV occurs mainly among IDUs, interests in clinical implications of HTLV-1/2 co-infection on HIV disease progression have risen. So far the available evidence suggests a protective role of HTLV-2 and adverse effect of HTLV-1 on HIV infection. In Europe, the overall prevalence of HTLV infection is up to 18% among IDUs and less than 1% among blood donors. However, there is no data regarding the overall prevalence of HTLV-1/2 in Eastern-European IDU population where the HIV epidemic mainly lies. We aimed to determine the prevalence of HTLV among IDUs in Estonia and compare it with the rate in healthy volunteers.The study included 345 Caucasian IDUs and 145 healthy volunteers from Estonia. The presence of HTLV-1/2 was determined by nested PCR in 5’ long-terminal repeat region; positive controls were used in every PCR run.The analysed IDUs resembled IDUs of HIV epidemic in Estonia: the IDU population was mainly male (79%) with median age of 30 years (interquartile range [IQR] 25-34), and had prolonged duration of illegal drug usage . The rate of co-infections was high – 50% were HIV+, 88% hepatitis C positive, 67% hepatitis B positive. 64% of IDUs reported receptive needle sharing in the past and 18% at least once a month during last six months. None of the IDUs carried HTLV-1 but there was a case of HTLV-2 . Healthy volunteers were HTLV-1 and HTLV-2 PCR negative.This is the first study investigating the prevalence of HTLV-1/2 among high risk population and healthy volunteers in Estonia. Our results suggest that HTLV is a rare co-infection among IDUs in Estonia.


Arabidopsis thaliana, however understanding of this process is far less elucidated in monocot crops. Up to now, only four barley genes implicated in BR biosynthesis have been identified. Two of them, HvDWARF and HvBRD, encode BR-6-oxidases catalyzing biosynthesis of castasterone, but their relation is not yet understood. In the present study, the identification of the HvDWARF genomic sequence, its mutational and functional analysis and characterization of new mutants are reported. Various types of mutations located in different positions within functional domains were identified and characterized. Analysis of their impact on phenotype of the mutants was performed. The identified homozygous mutants show reduced height of various degree and disrupted skotomorphogenesis. Mutational analysis of the HvDWARF gene with the “reverse genetics” approach allowed for its detailed functional analysis at the level of protein functional domains. The HvDWARF gene function and mutants’ phenotypes were also validated by measurement of endogenous BR concentration. These results allowed a new insight into the BR biosynthesis in barley.Brassinosteroids (BRs) are plant steroid hormones, regulating a broad range of physiological processes. The largest amount of data related with BR biosynthesis has been gathered in  Arabidopsis thaliana, resulting in the identification of genes participating in BR synthesis [Brassinosteroids (BRs) are plant steroid hormones playing an important role in regulation of diverse physiological processes, vegetative growth, reproduction and response to environmental stresses . The ynthesis ,6, signaynthesis ,8) and dynthesis ,10.OsDWARF encoding BR-6-oxidase (CYP85A) is a single-copy gene and it was demonstrated that the encoded enzyme does not catalyze the synthesis of brassinolide, and therefore in this species BR synthesis is terminated at castasterone [DWF4 and DET2 involved in early steps of BR synthesis [Brd1 (Brassinosteroid-deficient dwarf1) encoding BR-6-oxidase [In Arabidopsis, the biosynthesis of BRs comprises several steps leading from episterol to brassinolide and its major part is divided into two, interconnected parallel pathways: early and late C-6 oxidation ,12. Howeasterone . Recentlynthesis ,18 and B-oxidase . It has -oxidase  that sim-oxidase , pea [21-oxidase .HvDWARF gene, encoding brassinosteroid C6-oxidase involved in BR biosynthesis, was validated in this research by measurement of endogenous BR concentration in allelic mutants of the gene. The function of the gene has been characterized through identification of the semi-dwarf barley mutants, carrying missense mutations within conserved fragments of the coding sequence. These alleles were identified in two mutant forms, 522DK and 527DK, derived from variety “Delisa”. However, these analyses were conducted based on “forward genetics” methodology, physiological screening of the isolated semi-dwarf mutants and the analysis of the HvDWARF coding sequence only, without quantification of endogenous BR biosynthesis intermediates [In barley, four genes implicated in BR biosynthesis have been identified ,24. Funcmediates .HvDWARF with the TILLING  strategy [The objective of this research was to identify and analyze functionally genomic sequence of strategy . This ststrategy . AnalyseHvDWARF gene, with mutations located in various parts of the encoded CYP450 domain in order to determine their influence on plant phenotype and site-specific effect of mutations. In the presented paper, the molecular analysis and the characterization of the new alleles are described, along with the physiological analysis of the identified mutants. Nomenclature of the newly identified alleles (brd1 (brassinosteroid deficient1)) conforms to the nomenclature suggested for barley mutants by Franckowiak and Lundqvist [brd1) identified in the homologous genes from rice [The aim of the performed TILLING analysis was to identify a series of new alleles of the undqvist  and corrrom rice ,29 and mrom rice .HvDWARF was deposited in GenBank database (HQ619227). Unlike rice OsDWARF (OJ1519_A12.12) (GenBank: AC097276) and maize ZmBrd1 (GRMZM2G103773 in www.maizegdb.org) homologs, which contain nine exons and eight introns, the barley HvDWARF gene is composed of eight exons and seven introns. Third exon of the barley gene seems to correspond to the combined exons third and fourth of the rice and maize homologs, which suggests that during the evolution from the common ancestor the third intron of barley HvDWARF gene was lost, since the length of the homologous proteins is very similar in rice, maize and barley  with identity of the proteins reaching 80% and Expect value E = 0.0.The identified genomic sequence of HvDWARF gene was identified. Upon the analysis of 701 bp-long fragment of the gene in more than 3000 M2 plants 2,153,472 nucleotides were screened. Thirteen mutations have been identified and this number includes missense, silent ones as well as the mutations located in the introns of the gene. No nonsense mutation was identified. The missense mutations and the ones located in the introns are listed in As the result of the TILLING approach, a series of alleles in the brd1-c, carries a single-nucleotide missense mutation G3050T within the 5th exon , Oryza sativa (rice), Zea mays (maize) and Hordeum vulgare (barley), as visualized in BOXSHADE (The substituted amino-acid residue (Arg-347) is fully conserved at corresponding positions among homologous proteins from BOXSHADE .The identified amino-acid substitution leads to rearrangement of the secondary spatial domains: loss of short β-sheet domain (present in wild type (WT) version of the protein at the positions 412–414) and shortening of α-helical domain by three positions in WT version of the protein, this domain occupies region 425–441, whereas in mutant version it spans the protein fragment 428–441 .brd1-d, carries the C3365T transition located in the 6th intron of the gene (HvDWARF mRNA splicing process was determined. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) reactions were performed on the basis of cDNA produced in reverse-transcription from mRNA extracted from the variety “Sebastian” and the mutant brd1-d. Reverse transcriptions were performed in three replicates for each genotype; in each case, RNA isolates were pre-treated with double volume (5 µL)  of (1 U/µL) RNase-free DNase in order to ensure complete elimination of any potential contamination with genomic DNA. Primers used in this experiment were designed to cover the transcript fragment containing exons surrounding the intron in which the mutation was identified. Upon agarose electrophoresis, the RT-PCR product amplified on the basis of cDNA of the “Sebastian” variety was ~570 bp long. However, the RT-PCR product amplified on cDNA of the homozygous mutant brd1-d was single-band and ~650 bp long  were extracted from the agarose gel and sequenced. Sequencing results confirmed the difference in the RT-PCR products’ length, which is in accordance with the length (79 bp) of the intron 6, in which the mutation was identified. Therefore, it was concluded, that the mutation leads to the altered splicing of the transcript encoded by the brd1-d allele and retention of the 6th intron. The full-length cDNA of the brd1-d allele from the homozygous mutant and full-length cDNA from the “Sebastian” variety were cloned and sequenced. The sequencing analysis indicated that the identified mutation causing retention of the 79-bp-long intron within the brd1-d transcript was the only difference between the mutant and the WT “Sebastian” version of the transcript. Out of the mutations identified in various introns of the HvDWARF gene, the brd1-d allele was the only one resulting in splicing perturbation . The predominant fragment of the P450 cytochrome domain (spanning protein region 21–472) remains unaltered in the mutant version of the HvDWARF protein  were retained in the next generations of homozygous mutants (confirmed by the HvDWARF gene re-sequencing). No phenotype segregation was observed in the consecutive generations of both genotypes, indicating that their phenotypes are caused by the identified mutations. Allelism tests of the identified mutants and the mutants carrying the brd1-a and brd1-b alleles were also conducted. For each complementation  test 15 F1 plants were analysed. In each allelism test, the F1 plants showed mutant phenotype. Genetic analyses indicated that the newly identified homozygous mutants brd1-c and brd1-d are allelic to each other, and to the brd1-a (522DK) and brd1-b (527DK) mutants, validating that their phenotype is caused by mutation of the same gene—HvDWARF. Detailed characteristics of the identified mutants are given in For each of the identified homozygous mutants Mbrd1-c allele showed a relatively mild, semi-dwarf phenotype, the plant stature was similar to the mutants identified previously in the HvDWARF gene  [brd1-c allele, a significant reduction in fertility was observed. The mutant plants produce spikes of the similar appearance as those of the parent variety, however only few grains per spike were fully developed. The reduction in fertility observed in the homozygous mutant brd1-c was correlated with its semi-dwarf phenotype and with the identified mutation, constituting an aspect of the phenotype of the homozygous mutant form. Microscopic analysis of anthers and pollen grains of this mutant with respect to the “Sebastian” variety indicated that the pollen grains of the brd1-c mutant are infertile. However, it should be emphasized, that heterozygous brd1-c mutants showed improved fertility; therefore, we postulate that it is an allele-specific effect.Homozygous mutant with the  brd1-b)  , with proportionally shortened length and decreased width of the spikes (ca. 40% of the spike length of the parent variety) and proportionally reduced length of leaves  and width was observed between the identified mutants and the parent variety. There was no reduction in the number of tillers of the mutant plants of both genotypes when compared with the parent variety.The homozygous mutant with the f leaves . The mutIn order to characterize physiologically the identified mutants, the etiolation test and leaf-segment unrolling test were performed with the parent variety “Sebastian” as a reference. The identified mutants showed a lack of enhanced elongation of seedlings during the growth in darkness, and mutant seedlings grown in the darkness were of similar length to those grown in the light, which is a feature specific for BR mutants. The mutant seedlings of both genotypes grown in darkness and light were significantly shorter than the seedlings of the parent variety grown in corresponding conditions . This inHvDWARF gene (encoding the enzyme participating in BR biosynthesis) and the mutants are sensitive to the exogenous application of 24-Epi-brassinolide (24-EBL), the leaf segment unrolling test was performed. The leaf blades of the brd1-c and brd1-d mutants showed gradual unrolling correspondingly to the increase in the concentration of 24-EBL in the incubation solution, up to complete unrolling in response to the highest 24-EBL concentration. The lack of response in control treatment (water) was observed. Both mutants proved to be sensitive to exogenous application of BR. The leaf segments excised from mutant seedlings of both genotypes displayed a linear correlation between degree of unrolling and the hormone concentration, whereas leaf segments of the parent variety showed a decrease in unrolling at the highest concentration of 24-EBL (10−5 M), which may suggest a superfluous concentration of exogenous hormone (To confirm that the phenotypes of both mutants are attributed to the mutations identified in the  hormone .brd1-c and brd1-d) were treated with 24-EBL solutions of various concentrations  during the first three weeks of growth. Both mutants showed positive response to the treatment. Mutant plants with the brd1-c allele were of the same size as plants of the parent variety “Sebastian” (reference) when treated with both 10−6 and 10−5 M solutions of 24-EBL, however plants of the second mutant reached 90% of the parent variety size when treated with the highest concentration of 24-EBL after the three-weeks treatment.In order to prove that the observed phenotype of the identified mutants is BR-specific, a phenotype-rescue assay was performed, in which the mutant plants of both genotypes (−5 M) and gibberellic acid  during early stage of seedling development as an additional test in the phenotype-rescue assay, in which the mutant plants of both genotypes (brd1-c and brd1-d) were treated with 24-EBL solutions of various concentrations during the first three weeks of growth under light condition. Auxin and gibberellic acid slightly increased the growth rate of plants, however, these hormones did not revert the mutant phenotypes. On the contrary, BR not only significantly stimulated growth of the mutants, but also reverted their phenotypes. At the end of this three-week experiment, plants of both mutant genotypes treated with high concentrations of 24-EBL resembled plants of the “Sebastian” variety  in all three genotypes irrespective of tissue analyzed or stage of seedling development  was measured in the identified homozygous brd1-c and brd1-d mutants, and in the brd1-a and brd1-b mutants carrying missense mutations in the gene. The parent cultivar “Sebastian”, in which the brd1-c and brd1-d mutants were induced, and the parent cultivar “Delisa”, in which the brd1-a and brd1-b mutants were isolated, were used as reference genotypes in this assay. It should be emphasized that enzymatic role of the HvDWARF enzyme in castasterone biosynthesis has never been validated in this kind of assay. Results of this research confirmed the function of the HvDWARF enzyme and are congruent with the molecular, genetic and physiological data. The brd1-a and brd1-b mutants from variety “Delisa” contain similar, significantly lower content of castasterone in comparison with the parent cultivar. Similarly, the homozygous brd1-c and brd1-d mutants showed reduced concentration of endogenous castasterone in comparison with the parent cultivar “Sebastian”, however in the case of brd1-d  castasterone concentration was the lowest  [brd1-c allele, the alterations in the arrangement of secondary spatial domains were also reported in the semi-dwarf mutants brd1-a and brd1-b [The identified mutations result in different alterations of the predicted sequence of encoded polypeptide, are located in different parts of the cytochrome P450 domain, and therefore cause short stature of different degree. The ectively . The ami5 (SRS5)  what mayd brd1-b  what refbrd1-d allele is situated within the polypyrimidine tract of the 6th intron of the gene. The polypyrimidine tract in mRNA promotes assembly of the protein complex performing RNA splicing. The region is usually 15–20 bp long and located about 5–40 base pairs before the 3′ end of the intron to be spliced [The mutation identified in the  spliced . The ideHvDWARF gene, brd1-a and brd1-b, transcription level of the gene is very similar between mutants carrying the brd1-c and brd1-d alleles and the parental variety. Despite profound differences in the phenotypes of brd1-c and brd1-d mutants transcription level of the gene remains unaltered. It has been reported that the HvDWARF gene does not seem to compensate reduced level of BR biosynthesis, caused by mutations in its sequence, by elevated level of transcription [brd1-c and brd1-d alleles.However, similarly to what has been shown for the first two alleles of the cription  and it sbrd1-d mutant identified in this study is, according to our knowledge, the first in which the dwarf phenotype and BR-deficiency are caused by abnormal splicing of the BR-biosynthesis gene transcript. Its physiological and biochemical analysis allowed a new insight into the process of BR biosynthesis in barley at the stage of castasterone biosynthesis.Development of multiple alleles of the gene with application of the “reverse genetics” strategy, like TILLING, allows detailed functional analysis of the gene and encoded protein by induction of point mutations and verification of an effect of amino acid substitutions in various domains of a protein on phenotype of a mutant. This insight is a valuable complementation and validation of information about conservation and role of individual amino acid residues, which could be inferred from alignment of homologous proteins and analysis of conservation performed with the use of bioinformatics prediction tools. Several amino acid substitutions identified in this study did not result in any changes of plant phenotype, even though the substituted amino acid residues were conserved among homologous proteins from various plant species. Taking into account a type of substitution, this kind of results constitutes an ultimate validation of a role of the individual amino acid residues. The homozygous uzu1.a mutant, which carries a missense mutation in the HvBRI1 gene encoding BR receptor. This mutant shows a heat-sensitive reduction in plant height, in contrast to allelic mutants harboring amino acid substitutions in other domains of the BR receptor [HvDWARF gene, with mutations located in various fragments of the gene, and resulting in plant growth reduction of various degree may serve as a tool of basic research. Indeed, the mutants presented in this paper will be a plant material of an experiment, which has just been commenced. Along with their parent cultivars the mutants will be exposed to drought, upon which various aspects of plant physiology and reaction to the stress condition will be analyzed, including efficiency of photosynthesis and CO2 assimilation, functioning of enzymatic and non-enzymatic antioxidant system and hormonal homeostasis. Participation of BRs in regulation of these processes has been suggested, therefore it may be assumed that the BR deficiency reported in the presented mutants should affect these processes, however differentially reduced size of the mutant plants may result in reduced water requirement, and therefore counterbalance this effect improving stress tolerance.Potentially, identification of new alleles of the genes involved in BR metabolism may be of interest for agronomic purposes. BR-deficient or insensitive mutants of various species (models and crops) show growth reduction of various extent. Especially in cereal crops, including barley, mutant-based breeding strategies, aimed at fine-tuning of the brassinosteroid biosynthesis and signaling pathways could improve plant phenotypes ,24. Morebrd1 (brassinosteroid-dependent1), contains 193-bp deletion in the OsDWARF gene, accompanied by short 5-bp insertion between exons 4th and 5th. As a result of this mutation, the shift in open reading frame occurs, leading to a disruption of the protein in the fragment from Asp-300 to Tyr-470, which renders the protein nonfunctional. The mutations caused severe dwarfism, extremely short leaf sheaths and short, curled and frizzled leaf blades. The mutant plants rarely produce short panicles with small, usually sterile seeds. Reduction in tiller numbers was also observed. The mutant also shows disturbances in skotomorphogenesis [Mutations with the profound effect on the homologous OsDWARF protein structure were identified also in rice ,29. The OsDWARF gene, brd1-1, has a 113-bp deletion from 5′ part of intron 6th to 5′ fragment of exon 7th. This deletion introduces a premature stop codon just downstream the deletion and causes formation of the truncated version of the protein. The mutant plants are severely dwarfed, reaching ca. 10% of the height of parent variety. The mutant develops the malformed erect leaves with stiff leaf blades and significantly shortened leaf sheaths, produces short panicles and shows abnormalities in skotomorphogenesis [The other allele of the ogenesis . ZmBrd1 gene was characterized in maize. The single-nucleotide substitution introduces premature stop codon and profound truncation of the protein (mutant form is 165 aa long). The mutation causes a severe dwarf phenotype with marginal internode elongation, crinkled leaves and sterility of the brd1-m1 mutant plants. The maize mutant form resembles the phenotype of the rice brd1 mutants both in terms of plant height reduction and sterility [Mutant defective in the BR biosynthesis with mutation in the homologous terility .brd1 and brd1-1 mutants and the barley brd1-d mutant presented in this paper, it should be pointed out that in terms of both plant height reduction and grain production the barley mutant shows less severe phenotype . Although several changes of phenotypic features are shared by the rice brd1-d results from the fact that rice and maize genomes contain only one copy of the gene encoding BR-6-oxidase [brd1-d allele, which profoundly influences the structure of the HvDWARF protein, in this research shed more light on the BR biosynthesis in barley. In this mutant significant distortion of the predicted protein sequence and structure did not cause complete inhibition of castasterone biosynthesis—the content of endogenous castasterone was reduced by ~60% with respect to the “Sebastian” variety. However, in the severe rice mutants brd1-1 and brd1-2 castasterone was not detected [brd1 mutant when compared with the wild type [brd1-d and relatively moderate (when compared with the rice brd mutants) reduction in castasterone biosynthesis indicate that the difference may result from partial compensation of the disturbance in CS biosynthesis by the paralogous gene—HvBRD. It was quite surprising to observe that barley mutants carrying nonsense mutations at various positions of the HvBRD gene  retain relatively mild, semi-dwarf phenotype. In these mutants endogenous castasterone content was reduced by ~80% with respect to the reference variety [brd mutants). In this case perturbation in CS biosynthesis, caused by the nonsense mutations in the HvBRD gene, seems to be compensated by the other paralog—HvDWARF. All this indicates that in barley at the step of CS biosynthesis the two genes, HvDWARF and HvBRD may mutually, and, at least partially, compensate disturbances in their activity, which makes barley exceptional among other monocots—rice and maize—in which this step of BR biosynthesis is catalyzed by enzyme encoded by the single gene.It is tempting to postulate that the difference in phenotypes between the severely dwarf rice and maize mutants, and reported in this paper barley mutant -oxidase ,28,29, t-oxidase ,24. Bothbrd1-d mutant with respect to both the “Sebastian” variety and the brd1-c mutant  stimulates growth at the rate sufficient for reversion of the brd1-d mutant phenotype, whereas in the brd1-c mutant the corresponding phenotype reversion was induced by lower concentration (10−6 M) of 24-Epi-Brassinolide  TILLING population (HorTILLUS) developed at the Department of Genetics, University of Silesia was employed during the identification of the new alleles of the HvDWARF gene. The population has been developed by a mutagenic treatment of grain of the barley variety “Sebastian” consecutively with two chemical mutagens: sodium azide (1.5 mM/3 h) and N-methyl-N-nitrosourea (MNU) (0.5 mM/3 h). A six-hour inter-incubation period between mutagenic treatments was applied. To establish M2 generation only one grain from each M1 plant was sown. For establishing the TILLING platform, DNA were extracted from leaves of more than 10,000 individual M2 plants and arrayed in eight-plant pools. The barley (HvDWARF genomic sequence was identified based on mRNA of the gene (GenBank: DQ832258) and based on comparison with mRNA and genomic sequence of the homologous rice gene OsDWARF (GenBank: AC097276). Four primer pairs were designed using the Jellyfish software  and applied for amplification of fragments of the HvDWARF genomic sequence. The primer sequences and PCR profiles are available in HvDWARF gene fragment assigned for the TILLING analysis encompassed gene part in the range 2843–3543 bp, comprising exons 4–7 and was flanked by short sequences of intron 3rd and 7th. It was delineated to cover the gene fragment encoding the most conserved region of the HvDWARF protein. Sequences of primers applied in the TILLING analysis and PCR profile are available in The HvDWARF genomic sequence were verified using the Spidey software [brd1-d) with respect to the donor site, branch point, polypyrimidine tract and acceptor site of the intron. The conservation of amino-acid residues was determined using ClustalW [Position and length of exons, introns and UTR sequences within the identified software  . Reverse transcription was performed with RevertAid Kit , with double volume (5 µL) of (1 U/µL) RNase-free DNase  for RNA isolate treatment. HvDWARF transcription level was performed according to the procedure described previously [® 480 II analyzer . Relative transcription level of the HvDWARF gene in the identified mutants and the parental variety “Sebastian” (reference) was analyzed applying the LightCycler® 480 SW software (version 1.5) (Roche). The analysis of the Real-Time QPCR data was conducted according to the method by Schmittgen and Livak [Analysis of eviously  using thnd Livak . t-test and ANOVA. In the etiolation test values (seedling lengths) analyzed for each genotype were compared between the growth conditions (darkness vs. light). In the leaf-blade segment unrolling assay, values obtained for a given incubation condition (control and various concentrations of 24-EBL) were compared between genotypes. During analysis of the transcription profile of the HvDWARF gene values obtained for each of the developmental stages were compared between genotypes. In the analysis of the concentration of endogenous castasterone, value obtained for each mutant was compared with its parental variety. The mean values were subjected to the statistical analysis using the Student’s t test (p < 0.05).Physiological tests were performed in three biological replications. Etiolation test was performed according to the protocol by Gruszka and coworkers . In thisv/v) cold methanol. After centrifugation, the supernatant was enriched in deuterium-labeled brassinosteroids  and passed through a Strata X column . The bound fraction was eluted with acetonitrile and evaporated to dryness. Brassinosteroids were determined by ultra-high performance liquid chromatography with tandem mass spectrometry.Measurement of the endogenous BR biosynthesis intermediate—castasterone was performed according to a modified protocol described in ,40. All brd1-c and brd1-d, carrying mutations in the HvDWARF gene, were identified within the TILLING population (HorTILLUS) developed at the Department of Genetics, University of Silesia, Katowice, Poland. All barley mutants used in this study were identified at the Department of Genetics, University of Silesia and are deposited in the germplasm collection of this Institution.The barley mutants HvDWARF gene with insight into a role of individual amino acid residues through identification of the series of mutations in different fragments of the Cytochrome P450 domain. This approach also led to identification and characterization of the first barley mutant deficient in BR biosynthesis, whose phenotype is caused by perturbation in the splicing process. The various alterations of the HvDWARF protein sequences in these genotypes are reflected by differences in the severity of plant height reduction. Based on the performed genetic, molecular and physiological analyses and phenotype comparison, it is postulated that in barley at the step of CS biosynthesis the two genes, HvDWARF and  HvBRD may mutually, and at least partially, compensate disturbances in their activity, which makes barley exceptional among other monocots.Application of the TILLING approach allowed the detailed functional analysis of the


Cryptosporidium spp. and Giardia duodenalis are important causes of diarrheal diseases in humans and animals worldwide, and there is an increased interest in the role of animals in the mechanical transmission of these protozoa. To examine the role of yaks in this process, we examined the occurrence and genotypes of Cryptosporidium and G. duodenalis in yaks in western China.Cryptosporidium spp. and G. duodenalis were 4.0 % (22/545) and 6.0 % (16/545), respectively. Mixed infections of Cryptosporidium and G. duodenalis were also detected in four specimens. The prevalence of both protozoa differed significantly between some age groups, with higher rates of infection in animals < 1 year old. Sequence analysis of the small subunit rRNA (SSU rRNA) gene of the Cryptosporidium isolates identified the species as C. parvum (n = 12), C. bovis (n = 6), C. ryanae (n = 3), and C. ubiquitum (n = 1). Genotyping based on 60-kDa glycoprotein (gp60) gene from five C. parvum isolates identified all as IId with three isolates identified as IIdA15G1, one as IIdA18G1, and one as IIdA19G1. One C. ubiquitum isolate was identified as subtype VIIa. Amongst the G. duodenalis isolates, 16 were identified as assemblage E at the SSU rRNA gene. Four novel glutamate dehydrogenase (gdh) subtypes and two triosephosphate isomerase (tpi) subtypes were found amongst the G. duodenalis assemblage E isolates.A total of 545 fecal specimens were collected from yaks from nine different counties in the central western region of China. The prevalence for C. parvum subtype IIdA15G1, IIdA18G1, and IIdA19G1 isolates further confirms the dominance of the C. parvum IId subtypes in China. These findings also indicate that yaks may be a source of zoonotic Cryptosporidium infection, and this is the first report of G. duodenalis in yaks. The data presented here provides the basis for further genotyping or subtyping studies of G. duodenalis in yaks.The presence of The online version of this article (doi:10.1186/s12866-015-0446-0) contains supplementary material, which is available to authorized users. Cryptosporidium and Giardia are common parasitic protists that mainly cause enteric disease in humans and animals, including livestock, companion animals, and wildlife . The prevalence of G. duodenalis at the different collected sites ranged from 0–5.9 %, with the highest prevalence in Henan County ; however, significant difference was observed between the prevalence in different age groups , with the higher prevalence (5.1 %) observed in animals < 1 year old than animals > 1 year old (0.7 %).A total of 16 specimens showed positive amplification of the SSU rRNA gene, all belonging to the assemblage E. The overall prevalence for G. duodenalis assemblage E isolates was determined by amplification and sequencing of the tpi, gdh, and β-giardin (bg) genes, with 11 tpi, six gdh, and three bg gene sequences obtained (Table n = 7) was the most common subtype at the tpi gene. At the gdh gene, four subtypes of assemblage E sequences have not been reported previously.The genetic diversity of the ed Table . Subtypetpi and gdh sequences with reference sequences were performed. The phylogenetic analysis of tpi sequences in this study with the reference subtypes AI, AII, subtypes E from cattle, goat and sheep demonstrated that subtypes E1 [GenBank: KP334141] and E4 [GenBank: KP334144] clustered with reference subtypes E, whereas subtypes E2 [GenBank: KP334142] and E3 [GenBank: KP334143] formed one separate cluster in assemblage E   . The sequence accuracy was confirmed by two-directional sequencing, and sequences were identified by alignment with reference sequences downloaded from GenBank (χ2 test was used to compare the Cryptosporidium and G. duodenalis infection rates, and differences were considered significant when p < 0.05.The