A prospective, nonblinded and nonrandomized controlled trial was conducted to test the hypothesis that vitamin D supplementation would improve glycemic control in patients with type 1 diabetes mellitus who have vitamin D deficiency.Eighty patients with type 1 diabetes mellitus who had 25-hydroxyvitamin D levels less than 50 nmol/L were assigned to receive 4000 IU of vitamin D3. Calcium supplements were provided to ensure a total calcium intake of 1200 mg/d. Glycosylated hemoglobin and 25-hydroxyvitamin D levels were measured at baseline and at 12 weeks.P=.02). There was a significant difference in glycosylated hemoglobin change from baseline between the groups that achieved 25-hydroxyvitamin D levels of <35.4 nmol/L, 35.4-51 nmol/L and >51 nmol/L at 12 weeks (P=.04). There was a significant difference in 25-hydroxyvitamin D level between the groups that achieved glycosylated hemoglobin levels of <7.8, 7.8-9.9 and >9.9 at 12 weeks (P=.001). Patients were more likely to achieve lower glycosylated hemoglobin levels at 12 weeks if they had higher 25-hydroxyvitamin D levels at 12 weeks .There was a significant difference in mean (SD) glycosylated hemoglobin level (%) between the groups that achieved 25-hydroxyvitamin D levels of <35.4 nmol/L, 35.4-51 nmol/L and >51 nmol/L at 12 weeks (There was an observed effect of vitamin D supplementation on glycemic control in vitamin D-replete, type 1 diabetes mellitus patients. Further studies are needed to determine if these findings are applicable. Type 1 diabetes mellitus results from cellular-mediated autoimmune destruction of the beta cells of the pancreas.34A relationship between type 1 diabetes mellitus and vitamin D deficiency has been reported.53345We conducted a prospective, nonblinded and nonrandomized controlled trial to test the hypothesis that vitamin D supplementation would improve glycemic control in patients with type 1 diabetes who are vitamin D deficient.Patients aged more than 12 years who had type 1 diabetes mellitus were eligible.Vitamin D deficiency was defined as a 25-OHD level of less than 50 nmol/L.t test and paired t test where appropriate. The chi-square test was used for categorical data comparison. Pearson correlation coefficients between continuous variables were used as a measure of association. The data were analyzed by one-way analysis of variance (ANOVA), followed by post hoc LSD multiple comparison test to estimate the significance of difference between groups, and linear regression analysis was used as appropriate. All statistical analyses were performed using SPSS version 16.0. All P values were based on two-sided tests. The difference between groups was considered significant when P was <.05.Univariate analyses of baseline and follow-up demography and clinical laboratory endpoints were accomplished using the unpaired Table 1) were similar for each serum 25-OHD tertile (Table 2). There was also a significant difference in mean glycosylated hemoglobin level between the groups that achieved 25-hydroxyvitamin D levels of <35.4 nmol/L, 35.4-51 nmol/L and >51 nmol/L at 12 weeks (P=.02). There was a significant difference in glycosylated hemoglobin change from baseline for each tertile. The correlation of baseline and follow-up glycosylated hemoglobin levels in patients who achieved 25-hydroxyvitamin D levels of >51 nmol/L was -1.0 , P=.001, Pearson test. Mean (SD) glycosylated hemoglobin levels in patients who achieved 25-hydroxyvitamin D levels of >75 nmol/L as compared to those who achieved <75 nmol/L at 12 weeks were 7.3(1.5) vs. 9.1.Eighty patients completed the study. Baseline characteristics . There were no significant differences in the mean and the tertiles of glycosylated hemoglobin between the groups that achieved 25-hydroxyvitamin D levels of <35.4 nmol/L, 35.4-51 nmol/L and >51 nmol/L at 12 weeks in both genders (Tables 4).There was a significant difference in achieved 25-hydroxyvitamin D level between the groups that achieved glycosylated hemoglobin levels of <7.8, 7.8-9.9 and >9.9 at 12 weeks—61.9(23.4), 45.1(22.1) and 38.5(19.3), respectively, Diabetes mellitus has been recognized as a main independent risk factor for cardiovascular diseases.Insulin resistance plays a larger role in the type 1 diabetes disease process than is commonly recognized. Subsets of people with mild manifestations of the type 1 autoimmune disease process could benefit from treatments aimed at improving the insulin-resistant state.31932830The patients in our study were repleted with vitamin D3, and using up to 4000 IU of vitamin D3 to reverse states of deficiency was found to be safe.The study had some limitations. Increased hypoglycemia is a well-known complication of improved glucose control, and an important goal of therapy is to minimize this risk. Hypoglycemic episodes were not obtained by our patients. The study was neither blinded nor randomized, and the incremental doses of insulin were not analyzed.In conclusion, diabetes is one of the fastest-growing chronic diseases worldwide. Vitamin D deficiency is common, and repletion might improve glycemic control in type 1 diabetes. Vitamin D3 is inexpensive and readily available. Well-designed clinical studies are required to ascertain if improving 25-OHD levels from deficiency to sufficiency improves glycemic control in patients with type 1 diabetes.


In Vietnam the blackwater fever syndrome (BWF) has been associated with malaria infection, quinine ingestion and G6PD deficiency. The G6PD variants within the Vietnamese Kinh contributing to the disease risk in this population, and more generally to haemoglobinuria, are currently unknown.Eighty-two haemoglobinuria patients and 524 healthy controls were screened for G6PD deficiency using either the methylene blue reduction test, the G-6-PDH kit or the micro-methaemoglobin reduction test. The G6PD gene variants were screened using SSCP combined with DNA sequencing in 82 patients with haemoglobinuria, and in 59 healthy controls found to be G6PD deficient.G6PD variants were identified in the Vietnamese population, of which two are novel (Vietnam1 [Glu3Lys] and Vietnam2 [Phe66Cys]). G6PD Viangchan [Val291Met], common throughout south-east Asia, accounted for 77% of the variants detected and was significantly associated with haemoglobinuria within G6PD-deficient ethnic Kinh Vietnamese .This study confirmed that G6PD deficiency is strongly associated with haemoglobinuria . Six G6PD mutations, including novel mutations, in the Vietnamese Kinh population are reported and the contribution of G6PD mutations to the development of haemoglobinuria are investigated.The primary frequency of several  Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is one of the most common enzymatic disorders of red blood cells in humans and has a varied clinical presentation . The WorG6PD gene, which is located at chromosome Xq28. The G6PD gene exhibits remarkable polymorphism in human populations and G6PD is known to have over 400 variants. These variants are distinguished by their electrophoresis and biochemical characteristics and some variants are not associated with significantly reduced enzyme activity in erythrocytes . 35/82 haemoglobinuric patients had a further methaemoglobin reduction test at follow up . From the follow up test 23 were confirmed as G6PD deficient. Therefore in this study 48/82 haemoglobinuric patients are defined as "probable G6PD deficient" and 23/82 are defined as "definite G6PD deficient". Plasma was removed from the remaining blood sample and the blood cell pellet was stored at -20°C.The third group was comprised of 82 patients with haemoglobinuria admitted to the Hospital for Tropical Diseases (HTD) HCMC between 1993 and 1996 . All patDNA was extracted from frozen blood cell pellets or whole blood by the Nucleon genomic DNA extraction Kit .G6PD gene . PCR cycling conditions were as follows; 95°C for 5 min, annealing temperature . PCR products that were sequenced directly where purified using the QIAquick PCR Purification kit . Plasmid DNA templates were sequenced using the M13 forward and reverse primers, whereas PCR templates were sequenced using the specific primers that had initially generated the PCR fragment (Table G6PD in GenBank (accession No. X55448) to identify the mutations.Two or three samples for each unusual band pattern detected on an SSCP gel were prepared for sequencing to confirm the location of the sequence change. The PCR products were gel purified using a Geneclean kit BIO 101, USA) and then cloned using the TOPO TA Cloning kit for Sequencing . Cloned plasmids for sequencing were extracted from 01, USA aFisher's exact test was used for all comparisons and was performed within the STATA software package.Phenotypic screening for G6PD deficiency in healthy individuals from two ethnic groups, the Vietnamese Kinh and S'tieng, showed that the overall prevalence of the G6PD deficient phenotype in the southern Vietnamese population is relatively high at 11.3% (59/524). The difference in the prevalence of G6PD deficiency between the two Vietnamese ethnicities was not significant; 8.7% (23/266) in the Kinh and 14% (36/258) in the S'tieng (p = 0.07). Of the 23 G6PD deficient Kinh, 17 were male, five were female and one unknown (sex not recorded). Of the 36 G6PD deficient S'tieng, 28 were male and eight were female. In Vietnamese Kinh patients with haemoglobinuria, the frequency of "probable G6PD deficiency" was 58.5% (48/82). Twenty-eight percent (23/82) of haemoglobinuric patients had "definite G6PD deficiency." Of the 82 haemoglobinuric patients, five were female and 75 were male. All patients with probable G6PD deficiency were male (N = 48).G6PD gene was screened for mutations in healthy individuals (of both Kinh and S'tieng ethnic groups) who were G6PD deficient, and in all haemoglobinuric patients (N = 82). Across the three groups eight polymorphisms were identified within the G6PD gene (Table 32Arg), Coimbra "Shunde" , Chinese-5 , and Viangchan Jammu  and nt13714C>T [3 Lys); Vietnam2 ; and Vietnam3  (Table To identify the molecular cause of G6PD deficiency in this population the nts N = 8. Across nts N = 8. Across G6PD gene and 28.8% (17/59) have a synonymous polymorphism only. 33.9% (20/59) of the healthy G6PD deficient group have detectable non-synonymous G6PD variants. In the haemoglobinuria group, 25.6% (21/82) of patients harbour non-synonymous polymorphisms in the G6PD gene. Within the haemoglobinuria group of those patients with measured G6PD deficiency 39.6% (19/48) harboured non-synonymous G6PD polymorphisms, and 14/48 had a synonymous polymorphism (9/48 of these patients harboured both synonymous and non-synonymous G6PD polymorphisms). In contrast the patients without measured G6PD deficiency harboured significantly fewer non-synonymous G6PD polymorphisms (2/34 (6%), P = 0.001) but a similar number of synonymous polymorphisms (10/34 (29%), P = 1.0).In the healthy G6PD deficient group (both Kinh and Stieng) 37.3% (22/59) have no detectable mutation in the 437Tyr). 42.1% (45/107) of G6PD deficient individuals in the Vietnamese population had this polymorphism and 29.3% (24/82) in the haemoglobinuria group. The second most common polymorphism found in this study was the Viangchan variant; 36% (13/36) of healthy G6PD deficient S'tieng, 9% (2/23) of healthy G6PD deficient Kinh and 22% (18/82) of Kinh with haemoglobinuria (35.4% with G6PD deficiency and 2.9% without).The most common polymorphism found in this study was the synonymous polymorphism nt13714C>T  was significantly higher in G6PD deficient Kinh with haemoglobinuria than in G6PD-deficient healthy Kinh . The Chinese-5 mutation has a low frequency in this population and the frequency was not significantly different between G6PD deficient Kinh with haemoglobinuria and G6PD deficient healthy Kinh . Two variants, Vietnam1 and Gaohe Gaozhou, were only seen in the G6PD deficient Kinh with haemoglobinuria and not in the G6PD deficient healthy Kinh. In contrast, G6PD Vietnam2 was only seen in the G6PD deficient healthy Kinh group.Table The frequency of G6PD deficiency in Southeast Asia is highly variable . Within et al has reported that nine variants are present in China, nine in Malaysia, five in Thailand and four in Myanmar [Studies have shown that the distribution of G6PD variants also vary with geographical area and/or ethnic group. Matsuoka  Myanmar . In Viet Myanmar . In this Myanmar , Cambodi Myanmar ,24, Thai Myanmar ,26 and M Myanmar  are G6PD Myanmar ,28,29. G Myanmar ,30 and M Myanmar .In Vietnam, the G6PD Viangchan is the most common variant. A previous study in Lam Dong province also reported that G6PD Viangchan is the dominant G6PD variant in the Vietnamese population . It can et al [Studies in Southeast Asia report that G6PD Viangchan is in linkage disequilibrium with the silent polymorphism nt13714C>T ,26,27. Iet al  also fouBWF has been associated with recent or active malaria, and with the ingestion of oxidant drugs such as primaquine. 25.6% of the patients with haemoglobinuria had confirmed malaria. In this Vietnamese Kinh cohort, the association between G6PD deficiency and haemoglobinuria was strong. The association between G6PD deficiency and haemolysis has been shown in at least four clinical syndromes, oxidative stress-induced haemolysis, favism, neonatal jaundice and chronic non-spherocytic haemolytic anaemia .Of the G6PD variants, only G6PD Viangchan was shown to be associated with haemoglobinuria in the Kinh group. The G6PD Viangchan variant is one of the class 2 G6PD variants known to be associated with chronic haemolytic anaemia . The frThe haemoglobinuric patients investigated in this study of G6PD variation were hospitalized between 1993 and 1996. Since the early 2000s it has been rare for the Hospital for Tropical Diseases in HCMC to admit patients with haemoglobinuria associated with malaria. This is may be related to a change in the treatment policy for malaria in Vietnam. In 1990 the first intervention trial investigating the use of artemisinin derivatives for the treatment of malaria in southern Vietnam was published . FollowiG6PD mutations in Vietnam. G6PD Viangchan, a common south-east Asian variant, was found to be the most prevalent and two novel non-synonymous variants (Vietnam1 and Vietnam2) and one silent polymorphism (Vietnam3) were identified in this Vietnamese population. G6PD deficiency is a major risk factor for haemoglobinuria in Vietnamese Kinh and within the G6PD deficient population G6PD Viangchan was significantly associated with haemoglobinuria.This study reports the primary frequency of several G6PD: Glucose-6-phosphate dehydrogenase; BWF: Blackwater fever; SSCP: Single strand conformation polymorphism.The authors declare that they have no competing interests.NTH carried out the molecular genetics study, collected healthy samples, carried out the G6PD test using the G-6-PDH kit, performed the analysis and drafted the manuscript. JC participated in the design of the study. TTHC carried out the micro-methaemoglobin reduction test, recruited the patients and collected clinical data. TTH and ND were involved in recruiting the patients and collecting clinical data. JF conceived of the study, participated in its design and edited the manuscript. SJD participated in the data analysis, drafted and edited the manuscript


In plants, auxin distribution and tissue patterning are coordinated via a feedback loop involving the auxin-regulated cell polarity factor ICR1 and the secretory machinery. icr1 mutant embryos and plants display an a array of severe developmental aberrations that are caused by compromised differential auxin distribution. ICR1 functions at the plasma membrane where it is required for exocytosis but does not recycle together with PINs. ICR1 expression is quickly induced by auxin but is suppressed at the positions of stable auxin maxima in the hypophysis and later in the embryonic and mature root meristems. Our results imply that ICR1 is part of an auxin regulated positive feedback loop realized by a unique integration of auxin-dependent transcriptional regulation into ROP-mediated modulation of cell polarity. Thus, ICR1 forms an auxin-modulated link between cell polarity, exocytosis, and auxin transport-dependent tissue patterning.Development in multicellular organisms depends on the ability of individual cells to coordinate their behavior by means of small signaling molecules to form correctly patterned tissues. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells connects cell polarity and tissue patterning and thus is required for many aspects of plant development. Direction of auxin flow is determined by polar subcellular localization of PIN auxin efflux transporters. Dynamic PIN polar localization results from the constitutive endocytic cycling to and from the plasma membrane, but it is not well understood how this mechanism connects to regulators of cell polarity. The Rho family small GTPases ROPs/RACs are master regulators of cell polarity, however their role in regulating polar protein trafficking and polar auxin transport has not been established. Here, by analysis of mutants and transgenic plants, we show that the ROP interactor and polarity regulator scaffold protein ICR1 is required for recruitment of PIN proteins to the polar domains at the plasma membrane.  The coordination of different cells during pattern formation is a fundamental process in the development of multicellular organisms. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells demonstrates the importance of cell polarity for tissue patterning. The direction of auxin flow is determined by polar subcellular localization of auxin transport proteins called PINs, which facilitate auxin efflux. At the same time, an auxin-mediated positive feedback mechanism reinforces the polar distribution of PINs. However, the molecular mechanisms that underlie polar PIN localization are not well understood. In eukaryotic cells, the Rho family of small GTPases function as central regulators of cell polarity. We show that a Rho-interacting protein from plants, called ICR1, is required for recruitment via the secretory system of PIN proteins to polar domains in the cell membrane. As a result, ICR1 is required for directional auxin transport and distribution and thereby for proper pattern formation. In addition, both the expression and subcellular localization of ICR1 are regulated by auxin, suggesting that ICR1 could function in a positive feedback loop that reinforces auxin distribution. Thus, ICR1 forms an auxin-modulated link between cell polarity, protein secretion, and auxin-dependent tissue patterning. Polar auxin transport and resulting asymmetric auxin distribution within tissues (auxin maxima and gradients) are essential for proper development of the embryo, the root, and the shoot, differentiation and regeneration of vascular tissues, and for tropic responses In this work we show that ICR1 is an essential component of the auxin transport machinery functioning in exocytosis and as a part of an auxin modulated feedback loop. Thus, ICR1 links between Rho-regulated cell polarity and auxin associated pattern formation.Col-0 and icr1 mutant plants using the auxin sensitive reporters DR5::GUS and DR5rev::GFPDR5-visualized auxin maximum in the quiescent center (QC) and proximal columella cells is required for root meristem maintenance and correct tissue patterning icr1 roots the auxin maximum was displaced towards the distal tier of root cap and was reduced   reduced . Concomi reduced . In olde reduced . These ricr1icr1 embryos and roots, it was plausible that columella cells may be specified in young roots but would later disappear, concomitant with the proximal shift of the auxin maximum. To examine the specification of the columella cells, the existence of starch granules was examined in WT and icr1 roots at 2, 4, and 6 DAG  of the icr1 embryos showed defects in stereotypic cell divisions of the basal embryo pole at the globular stage as well as abnormal divisions in the suspensor, including the uppermost cell, which forms hypophysis  WOX5 and SCR in embryos was examined  and SCR (SCARECRAW), that define the stem cell niche examined  and S6. icr1 mutants resulted from compromised auxin transport rather than the genetic framework that regulates root development, we compared the expression of WOX5, SCR, and SHR in WT and icr1 roots. This analysis showed that like in embryos, all the three markers were expressed in the icr1 roots , which is located 254 base pairs (bp) upstream of the initiation AUG codon of ICR1 , indicatd to GFP . In globd to GFP . Similard to GFP . The abspICR≫GFP-ICR1 and pICR1≫GUS reporters following growth of seedling on medium containing the polar auxin transport inhibitor NPA  and followed by addition of auxin (NAA) to the growth medium were studied. Confirming the prediction, ICR1 expression was lower when seedlings were grown on NPA and was induced as early as 4–6 h following treatment with NAA  showed that ICR1 expression is quickly induced following 30 min of incubation in auxin (NAA) . The expwith NAA . As showAA  was expressed under regulation of the ICR1 promoter. To reduce the possibility of differences in expression due to position effects, expression was carried out using the LhG4/pOp transcription/transactivation system pICR1 promoter activator lines to express GFP-rop6CA, GUS, or GFP-ICR1 . These results reconfirmed that ICR1 is likely not involved in the Rab5-mediated endocytosis accumulation of PIN proteins following BFA treatments and indicated that ICR1 affects the recycling of PIN proteins back to the plasma membrane.The effects of BFA on endocytic recycling are reversible. When BFA is washed from the medium, the BFA bodies disappear and PIN proteins regain their localization in the plasma membrane and icr1 . HoweverT plants . The dificr1 mutant plants was carried out. SecGFP is a secreted form of GFP that is fused to the chitinase signal peptide at its N-terminus and to HDEL, ER-retention signal, at its C-terminus and has been used in analysis of protein trafficking icr1 background. Qualitative and quantitative fluorescence imaging analyses showed that the GFP fluorescence in icr1 roots was significantly stronger  than in WT roots  to high (pH 8.5) pH medium. Quantitative analysis showed significant  increase in GFP fluorescence of WT roots, while no increase was observed in icr1 roots  integration of a previously described constitutive endocytic recycling that involves clathrin-dependent endocytosis membrane . Thus, iicr1 are consistent with a previous finding that ICR1 can interact with both ROPs and AtSEC3A exocyst subunit at the plasma membrane icr1 mutant background  gene, which encodes an RPT2a 26S proteasome subunit, share a similar phenotype with icr1. Like icr1, the root meristem of hlr collapses, the QC is lost, and expression pattern of several markers including SCR and SHR is altered icr1, however, additional tiers of columella cells, compared to WT, were observed in 6-d-old hlr seedling, while in icr1 plants, of similar age, the specification of the columella is significantly reduced (icr1 expression pattern were observed when pICR1≫GFP-ICR1 plants were treated with a proteasome inhibitor (unpublished data). Thus, it is currently unclear whether the proteasome is involved in regulation of ICR1 expression.Auxin modulates its polar efflux from cells by inhibiting PIN endocytosis  reduced . No diffBased on computational modeling, it has been proposed that the PIN-mediated auxin fluxes in the root are sufficient for maintaining a stable auxin maximum Constitutive active ROP-induced cell deformation has been associated with reorganization of actin and MT cytoskeleton, as well as compromised vesicle uptake at the plasma membrane ROPs orchestrate cell polarization by regulating cytoskeleton organization through proteins that include RIC1 (ROP Interacting CRIB containing 1), RIC3 and RIC4 ICR1 promoter was cloned by amplifying a 2,050 bp fragment upstream of the initiation ATG codon from genomic DNA with the oligonucleotide primers SYP1502 (CCGGTACCTTTGATTTCGTGTTGAGG) and SYP1503 (CGTGTCGACCCTCCTACAGAAGGTTGG) and cloned into pGEM (Promega). The resulting plasmid (pSY1500) was digested with Kpn1 and Sal1, and the resulting fragment was subcloned into pLhG4-Bj36 upstream of the synthetic transcriptional factor gene LhG4pICR1-LhG4 fragment was subcloned into the plant binary vector pART27 to yield pSY1502.The ICR1, including the entire 5′-UTR of the gene, exons, introns, and the 3′-UTR, was cloned into the plant binary vector pMLBART as follows: the ICR1 5′-UTR was amplified from genomic DNA by PCR using oligonucleotide primers SYP321 (CAGCCATGGGACGTCGACATTTGATCAGC) and SYP322 (ATTATATCCTCAACACGAAATCAAACCATGGCTG), digested with NcoI and subcloned upstream of GFP in pGFP-MRC ICR1 gene starting from the initiating ATG codon was amplified by PCR from genomic DNA using primers SYP323 (CTAGAGCTCATGCCAAGACCAAGGTTACG) and SYP324 (GCAGGTACCGTTTAACGGGTTTCTCCATTTACGA), digested with SacI and KpnI and subcloned into pSY350 downstream of GFP to yield pSY351. pSY351 was in turn digested with SalI and KpnI, and the resulting fragment containing genomic-ICR1-3′UTRpICR1-5′UTR′-GFP-ICR1 was subcloned into pOPTATABJ36 to yield pSY352. pSY352 was in turn digested with NotI, and the resulting fragment was subcloned into the plant binary vector pMLBART to yield pSY353.A 2,339 bp genomic fragment of 6-GFP-rop6CAHis was prepared as previously described 6-GFP-rop6CAHis was subcloned into p10Op-Bj36 6-GFP-rop6CA-OCSOp-His terminator was subcloned into the plant binary vector pMLBART promoter::LhG4 activator lines with the respective pOp::reporter lines.The pSY812 plasmid containing TCAAAATGCCAAGACCAAGA and SY1583: TTGGAATGATTGGAATCAGAAG was carried out by q-PCR using an ABI Prism 7700 StepOnePlus™ Instrument . Study samples were run in triplicate on 8-well optical PCR strips (Applied Biosystems) in a final volume of 10 µl. Primers were designed using Roche Universal Probe Library (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp). The PCR cycles were run as follows: 10 min initial denaturation at 95°C, followed by 40 subsequent cycles of 15 s denaturation at 95°C, and 1 min annealing and elongation at 60°C. The specificity of the unique amplification product was determined by a melting curve analysis according to the manufacturer's instructions . Relative quantities of RNA were calculated by the ddCt method (Applied Biosystems Incorporated (2001), User Bulletin #2: ABI PRISM 7700 Sequence Detection System, http://www.appliedbiosystems.com). cDNA dilution series were prepared to calculate amplification efficiency coefficient. The relative levels of RNA were calculated according to the amplification efficiency coefficient and normalized against an UBQ21 gene standard http://medgen.ugent.be/jvdesomp/genorm/) and was calculated as M≤0.7. The analysis was repeated with three independent biological replicates.Total RNA was isolated with “SV Total RNA isolation” according to the manufacturer's instructions . cDNA first strand synthesis was carried out using Super ScriptII reverse transcriptase . Quantification with the oligonucleotide primer set SY1582: Columbia-0 (Col-0) and mutant Arabidopsis plants were sown on soil  and left for 2 d at 4°C. Then, plants were transferred to a growth chamber and were grown under long-day conditions (16 h light/8 h dark cycles) at 22°C. The light intensity was 100 µE m−2 s−1. For seedling analysis, seeds were surface sterilized and sown on plates containing 0.5× Murashige & Skoog (0.5× MS) salt mixture (Duchefa) titrated to pH 5.5 with MES and KOH, 1% sucrose, and 0.8% plant agar (Duchefa) and left for 2 d at 4°C. Then plates were transferred to the growth chamber and grown under long-day conditions (16 h light/8 h dark cycles) at 22°C for an indicated period. The light intensity was 100 µE·m−2·s−1. For auxin induction experiments, seedlings were germinated on plates (as above) for 5 d, then transferred to liquid medium (0.5× MS), and grown for 2 additional days before application of either NPA (10 µM) or NAA (10 µM).Seeds of WT Two to 3-d-old seedlings were submerged in 25, 50, or 90 µM BFA (prepared from a 100 mM stock solution in DMSO) and 4 µM FM4-64 (prepared from a 1 mM stock solution in DMSO) diluted in 0.5× MS liquid medium for 1–2 h at room temperature. For control treatments, seedlings were submerged in 0.09% DMSO solution containing 4 µM FM4-64. For BFA treatments of embryos, heart and torpedo stage embryos were dissected from ovules under stereo-microscope and immediately transferred into 0.5× MS liquid media supplemented with 1% sucrose. Dissected embryos were treated with 50 µM BFA and 5 µM FM4-64 on glass slides for 1–2 h in dark and observed with a 63× water-dipping (cover slide-free) objective using the Confocal Laser Scanning Microscope (CLSM).Seedlings were in 50 µg/ml PI in water solution submerged on microscope slides.Plasmolysis was carried out by incubating detached leaves in 0.8 M NaCl for 5 min β-Glucoronidase (GUS) staining was carried out as previously described Clearing of Arabidopsis embryos was performed as previously described Seedlings of indicated age were transferred into Lugol (IKI—0.2% w/v iodine and 2% potassium iodine) and incubated for 3 min. The Lugol-stained seedlings were then briefly washed with water and mounted on the slide with a clearing solution .2 in diameter, with or without 10 µM NAA were applied onto roots of vertically grown 7-d-old pICR1≫genomicGFP-ICR1 seedlings 5 mm above the root-tip. GFP-ICR1 expression was observed 24 h after the treatment.Small solid medium  particles, approximately 1 mmNomarsky/DIC imaging was performed with an Axioplan-2 Imaging microscope  equipped with an Axio-Cam, cooled charge-coupled device (CCD) camera by using either 40× or 100× oil immersion objectives with numerical aperture (NA) values of 1.3 or a 63× water immersion objective with NA value of 1.2. Low magnification imaging was carried out with Olympus MVX10 fluorescence stereomicroscope. CLSM imaging was performed with Leica TCS-SL CLSM with 20× multi-immersion, 63× water, and 63× water dipping (cover slide-free) objectives with NAs of 0.7, 1.2, and 0.9, respectively.GFP was visualized by excitation with an argon laser at 488 nm. Emission was detected with a spectral detector set between 505 and 530 nm. YFP was visualized by excitation with an Argon laser at 514 nm and spectral detector set between 525 and 560 nm for detection of emission. FM4-64 was visualized by excitation with an argon laser at 514 nm. Emission was detected with a spectral detector set between 530 and 560 nm. PI was visualized by excitation at 514 nm. The emission was detected with the spectral detector set between 600 and 650 nm.GFP-PIN2 and GFP-PIN2 icr1 seedlings were treated with 50 µM BFA for 1 h and then washed with 0.5× MS medium for 2 h. For calculation of epidermal cells with BFA bodies, at least 15 roots were used for each line and treatment. The experiment was repeated three times. Statistical analysis was performed with Student's t test.Four-day-old 35S::secGFP in the Col-0 background was crossed to icr1. T2 progeny homozygous for both icr1 and secGFP were selected. Seedlings were grown on 0.5× MS plates (pH 5.5) supplemented with 1% sucrose for the indicated time. Images of WT and icr1 root tips were taken with Leica LCS LSCM under identical conditions using a 10× objective, fully opened pinhole (600 µm) and emission/excitations of GFP as described above. The mean signal intensity was measured on an area spanning up to 500 µm from the root tip, using Image-J. DIC images of the same root were used for the adjustment of the measured area. At least 20 seedlings of each line were used in each experimental repeat. Statistical analysis was performed with Student's t test.Analysis of secretion was performed as previously described For comparison of secGFP fluorescence at external pH values of 5.5 and 8.5, seedlings were grown for 5 d on 0.5× MS plates pH 5.5, supplemented with 1% sucrose, and in turn transferred for 3 h to 0.5× MS liquid medium titrated to pH 5.5 or 8.5 with MES or KOH and supplemented with 1% sucrose.Figure S1Abnormal development of early icr1 embryos. (A–F) Col-0, (G–L) 90% of icr1 progeny with normal development at early embryo development, (M–P) 10% of icr1 progeny with early basal defects. (A and G) 1-cell stage, (B and H) 4-cell stage,  8-cell stage,  16-cell stage,  early globular stage, and  late globular. h, hypophysis; lc, lens-shaped cell; llc, large lower cell. Arrowheads in (M and N) mark abnormal division in hypophysis and suspensor; vertical brackets in (O and P) mark unshaped basal region. Bars correspond to 10 µm for all images.(1.88 MB TIF)Click here for additional data file.Figure S2Abnormal development of late icr1 embryos. (A–D) Triangular stage, (A) Col-0, and (B–D) icr1. Arrowheads in (B) indicate abnormal divisions in suspensor and columella initials; arrowhead in (C and D) marks abnormal division in protoderm. (D) Enlargement of the arrow-highlighted section in (C). (E–K) Early/mid-heart stage, (E and J) Col-0, (F–I and K) icr1. Arrowheads in (G–I) mark abnormal divisions in protoderm. Arrowhead in (J) indicates a normal periclinal division in a WT columella. Arrowheads in (K) point to abnormal division planes in columella initials of icr1 embryos. (L–S) A bent cotyledon stage,  Col-0, and  icr1. (N and O) Enlargement of the root meristem (RM). The arrowhead in (O) marks abnormal division in columella. Vertical bars in (L and M) indicate enlarged region shown in (P and Q). pd, protoderm; pc, procambium; col, columella initials; QC, quiescent center; v, vascular tissue; ep, epidermis; c, cortex; en, endodermis. Bars correspond to 20 µm (A–M), 10 µm (N–Q), and 50 µm (R and S).(3.72 MB TIF)Click here for additional data file.Figure S3icr1 siliques display greater developmental variability. Stacked-bar charts show the developmental stages of embryos collected from 12 representative siliques. (A) Col-0 and (B) icr1 siliques of various ages. Each color marks a single silique. The high mixing of colors in icr1 mutant indicates that the synchronization of embryo development within a single silique is compromised. Note that the embryo-lethal icr1 embryos were excluded.(0.22 MB TIF)Click here for additional data file.Figure S4Abnormal DR5 response in icr1 embryos.DR5rev::ER-GFP expression in WT and icr1 embryos. (A–E and L) Col-0, (F–K) icr1. (A and F) Early globular stage, (B and G) triangular, (C and H) early/mid-heart, and  late heart stage. Panels (K) and (L) are enlargements of the RM of (D) and (I), respectively. Arrows mark the auxin accumulation foci in developing cotyledons. Arrowheads in  indicate position of QC. Arrowheads in (E and J) point to the downward movement of auxin through provascular tissue. (A–J) Maximum projection Z-stack of multiple confocal sections; (K and L) single confocal scans throughout the center of RM.  are fluorescence/DIC overlay images. (E) and (J) are fluorescent images. GFP fluorescence is shown in green. Bars correspond to 20 µm.(2.37 MB TIF)Click here for additional data file.Figure S5Abnormal expression pattern of pWOX5::ER-GFP in icr1 embryos.WOX5 expression was first detected in globular stage embryos. In WT embryos expression is seen in the lens shape and the upper suspensor cells. In icr1 globular and heart stage embryos expression is spread to cell neighboring the lens-shape cell (arrowhead). In adult icr1 embryos strong WOX5 expression is seen in the lower part of the hypocotyl (arrowhead) and in the cotyledons. In contrast in adult WT embryo WOX5 expression is detected in the QC and the cotyledons. However, the expression in the cotyledon is weak and its detection required using high detector sensitivity. Bars correspond to 10 µm globular embryos, 20 µm heart stage embryos, and 50 µm adult embryos.(2.19 MB TIF)Click here for additional data file.Figure S6The expression pattern of SCR is altered in icr1 embryos. Expression pattern of SCR was determined with pSCR::YFP-His2b reporter seen as yellow nuclei. In WT globular embryos SCR is expressed in the hypophysis, ground meristem, and provascular cells. The inset is a projection stack of multiple confocal scans. At the heart stage expression expanded to the QC cells. In adult embryos expression was detected in the QC and future endodermis of the embryonic roots and in the hypocotyls. Note the clear differences in the expression pattern between the hypocotyl and the embryonic root (noted by the rectangular and curved brackets). In bent cotyledon embryos the expression was confined to the QC and endodermis/cortex stem cells of the embryonic root. Low levels of expression were detected in the cotyledons. Abnormal expression pattern of SCR was seen in globular stage icr1 embryos with early developmental aberrations (arrowhead). In heart stage icr1 embryos expression was spread to additional cells (arrowheads). Similar to WT, in mature icr1 embryos SCR expression was detected in the embryonic root and hypocotyls. However, unlike WT embryos no clear distinction in expression pattern could be made between the embryonic root and the hypocotyls. In bent cotyledons icr1 embryos SCR expression was confined to the embryonic root but was more spread compared to WT embryos (arrowhead). Bars correspond to 10 µm globular embryos, 20 µm heart stage embryos, and 50 µm adult embryos.(1.78 MB TIF)Click here for additional data file.Figure S7The differentiation of vascular tissues in leaves of icr1 is compromised. Cross-sections across a rosette leave's central vascular strand. The reduced sized of the vascular strand in icr1 is apparent. Note also the altered mesophyll cell shape and large air spaces in the icr1 leaf. Bars correspond to 20 µm.(1.82 MB TIF)Click here for additional data file.Figure S8High-resolution images demonstrating the altered localization of PIN1 and PIN2 in icr1 roots. Arrowheads denote the orientation of PIN localization in cells. Bars are 20 µm.(1.72 MB TIF)Click here for additional data file.Figure S9pPIN1-driven GFP-PIN1 localization in WT and icr1 roots. GFP-PIN1 expression in 3 DAG primary root of Col-0 (A to C) and icr1 (D to F). (A and D) Overlay between DIC and GFP fluorescence, (B and E) GFP, and (C and F) PIN1-GFP expressing roots stained with 5 µM FM4-64. Insets in (C and F) are close-up views of the RM. (G–I) pPIN1::GFP-PIN1 expression in root hairs and epidermis. (G) Col-0 (H and I) icr1. Arrowheads in (H) indicate ectopic expression in epidermis and root hairs. Arrowhead in (I) marks accumulation of GFP-PIN1 in internal bodies.  Maximum projection Z-stack of multiple confocal sections.  single confocal scans.  are fluorescence/DIC overlay images.  are fluorescent images. (C and F) are GFP/FM4-64 overlay images. GFP fluorescence is shown in green and FM4-64 in red. Bars correspond to 20 µm (A to F) and 50 µm (G to I).(2.42 MB TIF)Click here for additional data file.Figure S10Localization of pPIN2-driven GFP-PIN2 in icr1 roots. (A) WT and icr1 roots expressing pPIN2::GFP-PIN2 and stained with FM4-64 at 4 DAG. White boxes indicate the enlarged regions shown on the right panels. Arrows in WT mark apical localization of GFP-PIN2 in the epidermis and basal in the cortex. Note apicalization of GFP-PIN2 in both epidermis and cortex in icr1 roots (arrows). (B) BFA treatments of WT and icr1 seedlings resulted in co-localization of FM4-64 and GFP-PIN2 in BFA compartments (arrowheads). Bars correspond to 20 µm.(3.97 MB TIF)Click here for additional data file.Figure S11The altered pPIN1-driven GFP-PIN1 localization in icr1 embryos.  Col-0,  icr1 embryos showing mild basal defects, (M and N) icr1 embryos with strong basal defects.  mid-globular stage,  triangular stage, (I–L) heart stage. Arrowheads in  indicate the orientation of GFP-PIN1 localization, basal in procambial cells, and apical in protoderm. Arrows in (M and N) indicate the loss of PIN1-GFP expression in the basal region of icr1 embryos with strong basal defects. Insets in (I and K) are enlargements of a developing cotyledon.  Maximum projection Z-stack of multiple confocal sections.  Single confocal scans throughout the center of embryo.  are fluorescence/DIC overlay images.  are fluorescent images. GFP fluorescence is shown in green. Bars correspond to 20 µm.(2.87 MB TIF)Click here for additional data file.Figure S12A model summarizing PIN1 localization, auxin distribution, and patterning in WT and icr1 embryos. Cell outlines of WT (A) and icr1 embryos (B and C). (A) Polar membrane localization of PIN1 mediates directional auxin flux and appearance of auxin maxima in embryonic root meristem and future cotyledon tips during the development. (B) Reduced PIN1 polarity results in weak auxin flux and gradually disrupts formation of auxin maxima in icr1 embryos with late patterning defects. (C) In icr1 embryos with early patterning defects PIN1 membrane localization and polarity are strongly affected, likely leading to non-polar auxin distribution (crossed arrows).(1.24 MB TIF)Click here for additional data file.Figure S13The pICR1::LhG4 and the 5′-UTR-GFP-ICR1genomicpOp::ICR1 constructs. (A) The pICR1 driven effector/reporter lines. The LhG4/pOp system allows expression of different reporters from the same effector, thereby reducing positional effects on gene expression CA, and GUS were expressed using the same pICR1 effector lines. (B) Schematic representations of the pICR1 promoter. (C) The GFP-ICRgenomic construct.(0.30 MB TIF)Click here for additional data file.Figure S14ICR1 expression is induced by auxin.pICR1≫GUS expression following growth on NPA or induction by NAA for 6 h. Arrows denote GUS expression in pericycle cells following NAA treatments. Bars correspond to 50 µm.(2.96 MB TIF)Click here for additional data file.Figure S15Complementation of root growth in icr1 mutants by GFP-ICR1. Expression of GFP-ICR1 was driven by the ICR1 promoter using transcription/transactivation system (see stem see . Bars co(0.61 MB TIF)Click here for additional data file.Figure S16Plasma membrane localization of GFP-ICR1 detected following plasmolysis. PI-stained GFP-ICR1 expressing leaf epidermis pavement cells before and after plasmolysis. The bottom large panel is a magnification of the overlay panel after plasmolysis. Red arrowheads denote the cell wall and the white arrowheads denote the detached plasma membrane. The fluorescent green patches detected after plasmolysis indicate that some of the GFP-ICR1 was not attached to the plasma membrane. Bars correspond to 20 µm.(5.46 MB TIF)Click here for additional data file.Figure S17Effect of the apoplastic pH on secGFP fluorescence and its localization in WT and icr1 roots. (A) Mean fluorescence of WT and icr1 roots at 5 DAG that were transferred and incubated for 3 h in liquid MS medium titrated to either pH 5.5 or pH 8.5. Error bars correspond to SE, n≥20, ** significant differences in fluorescence of WT roots were detected between pH 8.5 and 5.5 . Fluorescence differences in icr1 between pH 8.5 and 5.5 were insignificant (p≥0.72). (B) A WT root incubated in MS medium titrated to pH 8.5 stained with PI (red). Arrows denote the apoplastic localization of secGFP. (C) An icr1 root incubated in MS medium tittered to pH 8.5 and stained with PI (red). Note that the GFP fluorescence remained intracellular. Bars correspond to 10 µm.(0.46 MB TIF)Click here for additional data file.Table S1The frequency of icr1−/− embryos exhibiting patterning defects at indicated developmental stages. * Embryos with strong basal defects were excluded from analysis. 1 Embryos were analyzed at 1-cell, 2-cell, 4-cell, 8-cell (octant), and 16-cell (dermatogen) stages.(0.04 MB DOC)Click here for additional data file.Video S1Expression of pICR1≫GFP-ICR1 at the root tip. The movie highlights the absence of ICR1 expression at and around the QC.(10.12 MB AVI)Click here for additional data file.


The rapidly expanding availability of de novo sequencing technologies can greatly facilitate efforts to monitor the relatively high mutation rates of influenza A viruses and the detection of quasispecies. Both the mutation rates and the lineages of influenza A viruses are likely to play an important role in the natural history of these viruses and the emergence of phenotypically and antigenically distinct strains.We evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs , and two tissue cultured H3N2 swine influenza viruses. Two waterfowl cloacal swabs were included in the analysis. Full-length sequences of all segments were obtained with 20 to 787-X coverage for the ten viruses and one cloacal swab. The second cloacal swab yielded 15 influenza reads of ∼230 bases, sufficient for bioinformatic inference of mixed infections or quasispecies. Genomic subpopulations or quasispecies of viruses were identified in four egg grown avian influenza isolates and one cell cultured swine virus. A bald eagle isolate and the second cloacal swab showed evidence of mixed infections with two (H1 and H2) and three  HA subtypes, respectively. Multiple sequence differences were identified between cloacal swab and the virus recovered using embryonated chicken eggs.We describe a new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs and add to the understanding of the ecology of influenza A virus populations. Orthomyxoviridae with a genome spanning 13.5-kilobases and consisting of eight single stranded RNA segments. The individual RNA segments range in length from 890–2341 nucleotides and encode 11 proteins Influenza A virus is an enveloped RNA virus belonging to the family Influenza A viruses are zoonotic and as a group of viruses, they possess a wide host range including humans, at least 105 bird species, pigs, horses, dogs, cats, ferrets, mink and marine mammals. In the United States alone, more than 200,000 hospitalizations and 36,000 deaths annually are due to complications from seasonal influenza in humans. Globally, it is estimated that influenza causes 300,000 to 500,000 human deaths annually http://www.who.int/csr/don/2009_08_21/en/index.html). Therefore, enhanced surveillance of avian and swine influenza A viruses is necessary to provide an understanding of the ecology and evolution.From April 2009 a pandemic caused by a novel H1N1 virus has been ongoing. As of August 2009, there have been more than 182,000 laboratory confirmed cases of pandemic influenza H1N1, 1799 deaths, in 177 countries and territories have been reported to WHO  is time and resource demanding. For example, in the recent large-scale influenza sequencing project, 95 overlapping one-step RT-PCR were performed per sample to obtain the complete viral genome sequence Twelve samples including 10 virus isolates  and two cloacal swabs, were processed for pyrosequencing. Complete genomes (>99% Open Reading Frame) were obtained for all eight segments of each virus isolate and a cloacal swab . For theComplete genome sequences were obtained from the cloacal swab of A/green-winged teal/Minnesota/Sg-00131/2007(H3N2) and virus recovered using egg system. A comparison of sequences from each segment revealed 80–91% nucleotide identities  suggesting extensive variability or existence of quasispecies in the cloacal swab. In the second cloacal swab-virus isolate pair, complete sequences were obtained from the virus isolate, whereas only 15 influenza sequence reads were obtained from the cloacal swab. These 15 reads of ∼230 bp included sequences of PB2 (two reads), PB1 (four reads), PA (three reads), HA , NP (one read), and NS (two reads). Four sequences  had 100% identity with the virus recovered using embryonated chicken eggs, A/mallard/Minnesota/Sg-00133/2007(H4N6).Whole genome sequences using standard dye terminator chemistry were also available for four H3N2 viruses. Comparison of these sequences against the pyrosequencing data revealed eight single nucleotide mismatches . Six subA series of genomic subpopulations or quasispecies as identified by single nucleotide polymorphisms (SNP) at specific nucleotide positions was identified in five virus isolates (four egg grown avian influenza viruses and one cell cultured swine influenza virus) and a cloacal swab. All the above samples are H3N2 subtype and all quasispecies populations observed in this study originated from mutations in NP, PB1, PA, M, and NS genes . One exaA bald eagle isolate, A/bald eagle/Virginia/Sg-00154/2008(H1N1/H2N1) that was originally typed by sequencing segments of HA and NA as H2N1 showed evidence of mixed subtypes by whole genome analysis. Analysis of the HA sequences from the 454 data revealed that this isolate carried both H1  and H2  subtypesAs described above, from 15 influenza reads (∼230 bases each) that were realized for cloacal swab of A/mallard/Minnesota/Sg-00133/2007(H4N6), there was evidence of mixed infection with H1, H3, and H4 subtypes.Complete genome sequencing of influenza A viruses is essential to determine the genetic basis of pathogenicity, antiviral resistance, and understanding the evolution of viruses in a variety of hosts and environments. Previous studies on sequence-based detection of antiviral resistance and diagnostics routinely used amplification of short portion of NA or HA genes followed by pyrosequencing. Hoper et al. Application of GS De novo Assembler or GS Reference Mapper software for our 454 sequence analysis failed to identify full-length contigs. GS assembler yielded several short contigs and GS Reference Mapper produced a few false insertion/deletions . We, thePresence of mixed infection and quasispecies in influenza viruses has also been demonstrated by others using RT-PCR of a short segment of HA from cloacal samples This is in agreement with the study of Wang et al. A possible rationale for the relatively few influenza reads (15 reads) observed in one of the cloacal samples could be due to insufficient RNA in the original sample or RNA losses during processing for pyrosequencing. In the other cloacal swab of A/green-winged teal/Minnesota/Sg-00131/2007(H3N2), complete sequences were obtained and these sequences had 80–91% nucleic acid identities with the virus recovered using embryonated egg system. This result indicates that there was a mixed population of viruses in this cloacal swab but the H3N2 subtype possibly became the predominant subtype by out-competing other virus subpopulations in the embryonated egg system. More studies with larger numbers of matched-pair samples need to be performed to completely resolve this phenomenon.Complete genome sequences of A/bald eagle/Virginia/Sg-00154/2008(H1N1/H2N1) showed two virus lineages (H1N1 and H2N1). Using RT-PCR based HA and NA typing, this virus was identified as H2N1. In general, unambiguous indexing of mixed subtype infections would require sequential limiting dilution, PCR, cloning, and sequencing of several clones. To our knowledge, this is the first report of full genome sequencing of all eight segments from a mixed infection representing two lineages of the virus.In our analysis of 12 samples, quasispecies were identified from five samples (four egg grown waterfowl isolates and one cell cultured swine influenza virus). All these viruses were H3N2 and identified quasispecies originated from mutations in NP, PB1, PA, M, and NS genes but not in HA, NA or PB2 genes. The four waterfowl isolates used in our study were recovered at the same study site and on the same day. This result concurs with the study of Dugan et al., Inasmuch as the mutation rate for type A influenza viruses is estimated at one nucleotide change per 10,000 nucleotide during replication and most infections are caused by as many as 10 to 1000 virions which likely possess varying numbers of nucleotide differences in their genomes, one can expect that each influenza A virion is possibly a quasispecies. However, we identified relatively few quasispecies - probably because the currently available sequence analysis software do not allow robust quasispecies analysis and extensive manual curation is necessary. We believe that with the help of improved bioinformatic tools we would detect more quasispecies populations in our sample sets.The method described in the current study does not require virus propagation, sequence information and circumvents the need for cloning and library construction prior to sequencing. Thus the currently described method is simple and less time consuming compared to Sanger sequencing. Despite these obvious advantages the cost of equipment is high and requires extensive bioinformatic expertise for assembling and analysis of the contigs.In conclusion, using an unambiguous genome sequencing approach, we present evidence of quasispecies and mixed infections among influenza A viruses that could help shape our understanding of the ecology and evolution of these viruses. Future studies should be undertaken to - 1) strengthen the interpretation of culture and sequence data generated by current influenza A virus surveillance networks; 2) establish novel influenza sequence-based evolutionary analyses; and 3) provide an improved understanding of influenza subtype stability and transmission in a wide array of mammals and birds.Twelve samples, including eight avian influenza viruses grown in embryonated chicken eggs, two swine influenza viruses propagated in MDCK cells with trypsin, and two influenza A virus positive cloacal samples were used: 1) A/mallard/South Dakota/Sg-00125/2007(H3N2), 2) A/northern pintail/South Dakota/Sg-00126/2007(H3N2), 3) A/mallard/South Dakota/Sg-00127/2007(H3N2), 4) A/mallard/South Dakota/Sg-00128/2007(H3N2), 5) A/green-winged teal/Minnesota/Sg-00131/2007(H3N2), 6) A/mallard/Minnesota/Sg-00133/2007(H4N6), 7) A/bald eagle/Virginia/Sg-00154/2008(H1/H2N1) (mixed isolate), 8) A/swine/Minnesota/Sg-00239/2007(H1N2), 9) (A/turkey/Minnesota/1138/1980(H7N3), 10) A/swine/North Carolina/R08-001877-D08-013371/2008 (H3N2), 11) cloacal swab of A/green-winged teal/Minnesota/Sg-00131/2007 (H3N2), and 12) cloacal swab of A/mallard/Minnesota/Sg-00133/2007(H4N6). All avian isolates were grown in embryonated chicken eggs while swine viruses were grown in Madin Darby canine kidney (MDCK) cells with trypsin. All isolates were passaged once or twice only.5′-CCT TGT TTC TAC T-biotin-3′) at 70°C for 5 minutes followed by 15 minutes at 39°C. Equal volume (240-µL) of 2X binding and washing buffer containing 0.5 mg of washed Dynabeads MyOne Streptavidin C1(Invitrogen) was added to the above RNA samples and mixed thoroughly with a pipette. Fifty micro liters  of Dynabeads MyOne Streptavidin C1 beads were washed with 1X binding and washing (B&W) buffer as per the manufacturer's instructions and resuspended in 240-µL of 2X B&W buffer . The sample was incubated at room temperature for 30 min. with gentle shaking in the orbital shaker and then placed on a magnetic stand for 3 min. The supernatant was removed by aspiration with a pipette and the coated beads were washed four times with 1X B&W buffer. The captured RNA was eluted from the beads by incubating at 65° C for 5 min with 40-µL of 10 mM EDTA, pH 8.2, in 99% formamide. The tube was placed on the magnetic stand for 3 min. and the supernatant, containing enriched RNA, was aspirated with a pipette.Total RNA was extracted from allantoic fluid/cell culture/cloacal swab using QIAamp Viral RNA Mini kit (Qiagen) as per the manufacturer's instructions. To reduce the contaminating host nucleic acids commonly observed in viral RNA preparations, viral RNA molecules were captured and enriched through the hybridization of a biotin-labeled oligonucleotide directed to the conserved 5′-end of all eight segments of influenza A virus genome. Total RNA  was incubated in the presence of 200-µL of 6X SSPE buffer containing 0.1 units/µL of SUPERase-In (Ambion) and 0.5 µM of the 5′-Capture Oligo  was added to 20-µL of enriched viral RNA. The samples were mixed thoroughly by pipeting, incubated for 2 min at 82°C, and then immediately transferred to ice to stop the fragmentation reaction. The reaction volume was increased to 50-µL by adding RNase free water, purified with RNAClean (Agencourt) as per the manufacturer's instructions and eluted with 20-µL of RNase free water.5′-phosphate-NNNNNNN-3′) and Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) as per the manufacturer's instructions. Each reaction consisted of 7-µL of fragmented RNA and 2-µL of 500-µM primers. After reverse transcription, the RNA was removed by hydrolysis by adding 20-µl of Denaturation Solution  and incubating at 65°C for 20 minutes. The mixture was neutralized by adding 20-µl of 0.5 M HCI in 0.5 M Tris-HCl, pH 8.0. The resultant sscDNA was recovered with RNAClean (Agencourt) as per the manufacturer's instructions and eluted from the beads with 20-µL of RNase free water.The fragmented RNA sample was reverse transcribed in 20-µL final volume using random hexamer (5′-biotin- GCCTTGCCAGCCCGCTCAGNNNNNN-phosphate- 3′) and “sscDNA Oligo B-prime” (5′-phosphate- CTGAGCGGGCTGGCAAGG-dideoxyC-3′) which, after annealing, results in “sscDNA Adaptor B” with a 3′-random overhang of six nucleotides. Similarly, the 5′-end adaptor consists of “sscDNA Oligo A-prime” (5′-NNN NNN CTG ATG GCG CGA GGG AGG dideoxyC-3″) and “sscDNA Oligo A” (5′-GCCTCCCTCGCGCCATCAG-3′) which form “sscDNA Adaptor A” with a six nucleotide 5′-end overhang. The adapter ligation reaction was carried out using T4 DNA ligase (New England Biolabs) in a total volume of 30-µL, containing 3 µL of 10X ligase buffer, 1-µL of  adapter A, 1-µL of  adapter B, 5-µL (2000 cohesive end units) of T4 DNA ligase, 15-µL of sscDNA and 5-µL of water. The reaction mixture was incubated at RT for 2 hrs; the ligated sscDNA was recovered with Dynabeads MyOne Streptavidin C1 (20-µL beads per sample) and eluted by incubating at 65°C for 5 min with 40-µL of 10 mM EDTA, pH 8.2, in 99% formamide. The final sscDNA was purified with two rounds of RNAClean (Agencourt) and eluted in 20-µL of nuclease free water.For clonal amplification and sequencing on the Genome Sequencer FLX, the sscDNA required the addition of adaptors to each terminus. The adaptors have been designed to enforce directional ligation to the sscDNA, such that one will be uniquely ligated to the 5′-end (sscDNA Adaptor A) and the other to the 3′-end (sscDNA Adaptor B) of the sscDNA. Each adaptor is comprised of two complimentary oligonucleotides that are annealed together as described. The 3′-end adaptor consists of “sscDNA Oligo B” (5′-GCC TCC CTCGCG CCA-3′), 10-µL (10 µM) Primer B (5′-GCC TTG CCA GCC CGC-3′), 1-µL of 50X Advantage polymerase mix, 10-µL of sscDNA, and 12-µL of nuclease free water. The PCR conditions used were: 96° C for 4 min; 30 cycles of 94° C for 30 s and 64° C for 30 s; 68° C for 3 min; hold at 14° C. The PCR product was purified with two rounds of AMPure (Agencourt) as per the manufacturer's instructions. The double stranded DNA library was eluted with 20-µL of water and quantified with the Quant-iT Picogreen dsDNA Assay Kit (Invitrogen). Emulsion PCR amplification was carried out using either primer A or primer B or both for bidirectional sequencing. The sequencing reactions were carried out in small regions of the PicoTiterPlate (1–4 regions/sample) on the Genome Sequencer FLX (GS FLX) platform.The final adapted sscDNA was amplified using Advantage 2 PCR Kit (Clontech) in a total volume of 50-µL containing 5-µL of 10X Advantage 2 buffer, 2-µL of 50X dNTP mix (10 mM each), 10-µL (10 µM) Primer A .All the influenza reads were run in GS De novo Assembler with three sets of parameters: minimum overlap (MOL) of 40 nucleotides and 90% identity, MOL of 100 and 100% identity, and MOL of 200 and 100% identity. The larger contigs (>500 bases) obtained by the above method were BLAST analyzed using NCBI resources and the most closely related sequences, referred to as reference sequences, for each segment were downloaded. All the influenza reads were mapped with reference sequences in GS Reference Mapper. The contigs obtained from GS Assembler and the consensus sequences obtained from GS Mapper were reassembled in Sequencher 4.8. The new contigs were then examined for ambiguous bases  and particular base positions were manually examined for the presence of more than one kind of base (quasispecies) in GS Reference Mapper.All the eight segments of four AIV isolates - A/mallard/South Dakota/Sg-00125/2007(H3N2), A/northern pintail/South Dakota/Sg-00126/2007(H3N2), A/mallard/South Dakota/Sg-00127/2007(H3N2), A/mallard/South Dakota/Sg-00128/2007(H3N2) were sequenced by classical Sanger sequencing method using ABI PRISM 3730xl DNA Analyzer (ABI) and the results were compared with the consensus sequences of pyrosequencing obtained with GS Reference Mapper software.


Mice carrying only one copy of the foxa2 gene show abnormalities in motor behavior in old age and an associated progressive loss of dopamine neurons. Manipulating forkhead function may regulate both the birth of dopamine neurons and their spontaneous death, two major goals of regenerative medicine.Parkinson disease affects more than 1% of the population over 60 y old. The dominant models for Parkinson disease are based on the use of chemical toxins to kill dopamine neurons, but do not address the risk factors that normally increase with age. Forkhead transcription factors are critical regulators of survival and longevity. The forkhead transcription factor, foxa2, is specifically expressed in adult dopamine neurons and their precursors in the medial floor plate. Gain- and loss-of-function experiments show this gene,  foxa2 gene acquire motor deficits and a late-onset degeneration of dopamine neurons. This spontaneous cell death preferentially affects neurons associated with Parkinson disease. This work provides new strategies to generate neurons in the laboratory and to block their death in old age.The restoration of dopamine neurons is a major focus of stem cell biology and regenerative medicine. The gradual loss of these neurons is a hallmark of Parkinson disease. Dopamine neurons in the midbrain convey important sensory and motor functions to the forebrain. We show that the transcription factor FOXA2 plays a central role in the birth and death of dopamine neurons in the midbrain. By defining their precursors in the ventral midbrain, we show that dopamine neurons are derived from organizer cells in the floor plate . We also show that FOXA2 specifies the floor plate and induces the birth of dopamine neurons. Mice with only a single copy of the  The connection between development and neurodegeneration is emphasized via a new mouse knockout of a transcription factor that is critical for dopamine neuron specification, which produces a late-onset, asymmetric degenerative condition in a manner very similar to human Parkinson disease. Midbrain dopamine neurons play important roles in motor control, reward, addiction, attention, and cognition ,2. A profoxa genes and have a central role in cell survival, cancer, and the longevity of organisms ), and anti-FOXA2  antibodies. Double-staining is indicated by yellow.+ cells (oculomotor neuron precursors) are observed in an immediately adjacent, nonoverlapping domain.(A) LMX1b expression (dopamine precursors) occurs in a single domain at the ventral midline. PHOX2a(B) NKX2.2-expressing cells  occupy the domain immediately dorsal to the motor neuron precursor domain. Some expression of NKX2.2 can be observed in PHOX2a-expressing domain.+ cells are found in the medial FOXA2+ domain.(C–E) Dopamine precursors express the floor plate marker, FOXA2, and are found in the medial part of this domain. LMX1b+ cells are found in the lateral parts of the FOXA2+ domain. Numerous PHOX2a+ precursors coexpress FOXA2 at E9.5 (F). As these cells migrate to the pial surface, they down-regulate FOXA2 (G) and (H).(F–H) PHOX2a(1.1 MB PDF)Click here for additional data file.Figure S2+ floor plate in the midbrain, just as they do in the developing spinal cord.Transverse sections through mouse mesencephalon at E9.5 (B), E10.5 (A) and (C), and E11.5 (D), stained with anti-FOXA2 , anti-NKX2.2 (red in [A]), anti-NKX6.1 (red in [B–D]) antibodies. The NKX2.2-expressing cells in (A) flank the FOXA2(695 KB PDF)Click here for additional data file.Figure S3(A) GATA3 expression is observed in a subset of neurons differentiating in the NKX6.1 domain E10.5.+ neurons are located immediately adjacent to the NKX6.1+ motoneurons (B). Like GATA3, LIM1/2 is expressed in neurons immediately adjacent to the NKX6.1-expressing motor neurons (C). In (B) and (C), NKX6.1 expression is lost as progenitors differentiate to GATA3- and LIM1/2-positive neurons.(B and C) At E11.5, GATA3(D) At E12.5, motor neurons are identified by PHOX2a expression, and more lateral neurons by LIM1/2 expression. LIM1/2 expression at E12.5 expands dramatically, compared to E11.5 (C).(461 KB PDF)Click here for additional data file.Figure S4+/−foxa2 mice .(A) Widespread gliosis is seen in the ventral midbrain of +/−foxa2 mice (C).(B and C) Increased density of glia is easily seen in the red nucleus of wild-type (B) and +/−foxa2 dopaminergic system throughout the VTA, the SN pars compacta and reticulata, and red nucleus.(D) Quantitation of glia in wild-type and (2.3 MB PDF)Click here for additional data file.Figure S5+/−foxa2 (B), (D), (F), (H), and (J) mouse at the indicated level. There is an almost complete loss of nigral dopamine neurons on the left side, whereas the right side is relatively intact, quantitated in (K).Coronal sections through the midbrain of a wild-type (A), (C), (E), (G), and (I) and a (684 KB PDF)Click here for additional data file.Video S1foxa2 heterozygote in a C57BL/6 background that has developed the late-onset motor problems characterized, here. Note the asymmetric kinked posture and the labored gait in this mouse.This is a 20-mo-old (1.3 MB MPG)Click here for additional data file.


Enterovirus 71 (EV71) outbreaks occur periodically in the Asia-Pacific region. In2006, Brunei reported its first major outbreak of EV71 infections, associatedwith fatalities from neurologic complications. Isolated EV71 strains formed adistinct lineage with low diversity within subgenogroup B5, suggesting recentintroduction and rapid spread within Brunei. Picornaviridae and thegenus Enterovirus, is a common cause of hand, foot, and mouth diseasein children. Infection with this virus is rarely complicated by severe neurologicdisease, such as meningitis, brain stem encephalitis, neurogenic pulmonary edema, andacute flaccid paralysis. EV71 was first isolated in 1969 (Enterovirus 71 (EV71), a member of the family Brunei is situated on the island of Borneo  and has apopulation of ≈370,000. From February through August 2006, Brunei experienced itsfirst reported major outbreak of EV71. More than 1,681 children reportedly wereaffected, with 3 deaths resulting from severe neurologic disease. We report thevirologic findings from this outbreak.During March through October 2006, samples from at least 100 patients from Bruneidiagnosed with hand, foot, and mouth disease or herpangina were received at theUniversity Malaya Medical Center, Kuala Lumpur, Malaysia. Samples were inoculatedinto Vero and A549 cell cultures for virus isolation. EV71 was isolated from 34patients (including 2 who died of severe neurologic complications), and anadditional 7 isolates were obtained from Malaysian patients seen at the UniversityMalaya Medical Center during the outbreak period in Brunei . AdenoviEnteroviral RNA was extracted from cell cultures using QIAamp Viral RNA Mini Kit, and reverse transcription–PCR was performed toamplify the viral capsid protein (VP1) gene at nt positions 31–861. Theprimers used were VP1F5′-CAGGCTAGCATGGGAGATAGGGTGGCAGATGTGATCGAGAGC-3′ and VP1R5′-GGTGGATCCCAAAGGGTAGTAATGGCAGTACGACTAGTGCCGGT-3′. The 831-nt partialVP1 gene fragments were sequenced, and phylogenetic relations of the sequences wereexamined using selected enterovirus reference strains obtained from GenBank . SequencThe phylogenetic tree, drawn on the basis of the alignment of the VP1 gene sequences,showed 3 independent genogroups  with the prototype BrCr strain as theonly member of genogroup A  among all strains in subgenogroup B5. AllBrunei B5 isolates were clustered in an independent lineage within subgenogroup B5(99.9% bootstrap support), separate from the established Sarawak and Yamagataisolates from 2003 (–2 substitutions per nucleotide, similar topoliovirus (The different genogroups of EV71 are widely distributed around the world (Other than its northern coastline, Brunei is surrounded entirely by the EastMalaysian state of Sarawak. In 2006, an outbreak of EV71 affected approximately14,400 children in Sarawak (In summary, the first reported major outbreak of EV71 in Brunei was caused by strainsfrom subgenogroup B5 that were distinct from other reported B5 isolates, suggestinga recent introduction from an as-yet-unidentified source. Hence, continued molecularsurveillance of EV71 in Asia is required to further our understanding of factorsinfluencing the evolution of the virus and its association with emergence ofoutbreaks in the region.


The endemic status of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 in Egypt continues to devastate the local poultry industry and poses a permanent threat for human health. Several genetically and antigenically distinct H5N1 lineages co-circulate in Egypt: Strains of clade 2.2.1 proper replicate mainly in backyard birds causing the bulk of human infections, while a variant lineage within 2.2.1 (2.2.1v) appears to be perpetuated mainly in commercial poultry farms in Egypt. Viruses of the 2.2.1v lineage represent drift variants escaping from conventional vaccine-induced immunity and some of these strains also escaped detection by commercial real time reverse transcriptase PCR (RT-qPCR) protocols due to mismatches in the primers/probe binding sites.We developed therefore a versatile, sensitive and lineage-specific multiplex RT-qPCR for detection and typing of H5N1 viruses in Egypt. Analytical characterization was carried out using 50 Egyptian HPAIV H5N1 strains isolated since 2006 and 45 other avian influenza viruses (AIV). A detection limit of 400 cRNA copies per ml sample matrix was found. Higher diagnostic sensitivity of the multiplex assay in comparison to other generic H5 or M-gene based RT-qPCR assays were found by examination of 63 swab samples from experimentally infected chickens and 50 AIV-positive swab samples from different host species in the field in Egypt.The new multiplex RT-qPCR assay could be useful for rapid high-throughput monitoring for the presence of HPAIV H5N1 in commercial poultry in Egypt. It may also aid in prospective epidemiological studies to further delineate and better control spread of HPAIV H5N1 in Egypt. The incursion of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 of phlyogenetic clade 2.2, subclade 2.2.1 . In ordend June 2010 [Despite intense control measures including blanket vaccination, surveillance and depopulation of infected poultry holdings, HPAI H5N1 has gained endemic status in Egyptian poultry populations -8. This une 2010 ,8,11,12.A number of RT-qPCR assays for diagnosis and characterization with respect to subtype and pathogenicity of HPAIV H5N1 have been published. These assays target the matrix gene ,14, the A multiplex RT-qPCR assay was designed to detect HPAIV H5N1 viruses and simultaneously differentiate the main two major lineages circulating in Egypt; classic 2.2.1p viruses and the newly evolving clade 2.2.1v of antigenically drifted variant viruses. A single reaction assay using multiplexed primers and probes (three colours) was developed. Analytical characterization of the assay was carried out using 50 Egyptian HPAIV H5N1 strains and other avian pathogens. Diagnostic performance was examined with 63 swab samples from experimentally infected chickens and 50 avian influenza virus (AIV)-positive swab samples obtained from different host species in the field in Egypt.10 dilution series of RNA extracted from A/chicken/Egypt/0879-NLQP/2008 (clade 2.2.1v), A/chicken/Egypt/NLQP-0918Q/2009 (clade 2.2.1p) and A/Whooper swan/Germany/R65/2006 (European clade 2.2) (data not shown). The concentration of primers and probes was optimized to increase the efficiency and sensitivity of amplification to final values shown in table n values for the multiplex RT-qPCR were observed using the following thermoprofile and the SuperScript III RT/Platinum Taq Mix chemistry: 30 min at 50°C and 2 min at 94°C, then 42 cycles of 94°C, 56°C and 68°C for 30 seconds each. No significant differences in sensitivity were evident when running the three RT-qPCRs separately indicating that the multiplex approach produced no relevant detrimental effects on amplification efficacy (data not shown).Two strategies were combined to sensitively detect and distinguish viruses of clade 2.2.1p from 2.2.1v escape strains  whereas 15 isolates reacted like 2.2.1v strains (FAM-positive)  originating from SPF chickens which were experimentally infected with A/chicken/Egypt/0879-NLQP/2008 (clade 2.2.1v) or A/chicken/Egypt/NLQP-0918Q/2009 (clade 2.2.1p) were examined by the multiplex RT-qPCR and compared to Ct values obtained with an H5-specific assay described by Slomka et al. . Results originatOropharyngeal or cloacal swabs were also sampled in commercial farms (n = 11) and backyard poultry holdings (n = 39) in Egypt. A total of 50 samples pretested by M-specific RT-qPCR  to be poWe report here the development of a multiplex RT-qPCR assay for detection and differentiation of Egyptian H5N1 HPAI viruses of clade 2.2.1 proper (p) and the emerging 2.2.1 lineage of viruses which escape standard vaccine-induced immunity (designated here 2.2.1v). The assay was shown to have a detection limit of 2-5 cRNA copies per reaction. Based on a phylogenetic analysis of 50 isolates tested ,8, the aIn terms of diagnostic performance regarding the HPAIV H5N1 strains currently circulating in Egypt, the multiplex assay was at least equal to standard RT-qPCRs targeting the M gene of AIV and superior to a generic H5 RT-qPCR  when exaReducing the amount of circulating HPAIV H5N1 virus by concerted actions of rapid and specific testing, culling and vaccination of poultry is the key to mitigate the risk of human infections and fatalities in Egypt. Controlling the endemic HPAIV H5N1 situation in Egypt is particularly painstaking because of  the inteThe necessity to update commercial and generic H5-specific RT-qPCRs for the Egyptian situation has been stressed recently . The curA panel of 50 HPAIV H5N1 strains isolated in SPF-chicken eggs in the National Laboratory for Quality Control on Poultry Production (NLQP) in Egypt was used for determination of the analytical specificity and sensitivity of the PCR assays  were evaluated. Reactions were carried out in a 25-μl volume on an MX3005P real time PCR machine (Stratagene).The concentration of primers and probes was optimized in separate PCRs  and re-adjusted when combined in the multiplex RT-qPCR to increase the efficiency of amplification. Likewise, different annealing temperatures ranging from 50 to 60°C and different chemistries (SuperScript III One-Step RT-PCR system with Platinum 1-107 copies) of cRNA.For preparation of standard controls, the cloned H5 gene segment from A/chicken/Egypt/0879-NLQP/2008 (clade 2.2.1v) and A/chicken/Egypt/NLQP-0918Q/2009 (clade 2.2.1p) was used for generating cRNA in vitro by run-off transcriptions performed as previously described [6.0 TCID50 of A/chicken/Egypt/0879-NLQP/2008 (clade 2.2.1v) or A/chicken/Egypt/NLQP-0918Q/2009 (clade 2.2.1p) . RNA was extracted from mixed oropharyngeal and cloacal swabs collected from individual birds 2 or 7 days post infection (dpi). RNA was used in quantitative RT-qPCRs described in this study and compared to M and H5 based RT-qPCR assays as previously described [All animal experiments were conducted following official German animal welfare regulations (LALLF M-V/TSD/7221.3-2.1-031/09). Six weeks old SPF chickens  were infected by the oculo-nasal route with a dose of 10escribed ,23.Tracheal and cloacal swabs were collected both from poultry at commercial farms (n = 11) and backyard holdings (n = 39) in the frame of the national surveillance scheme in poultry sectors in Egypt from 2008 to 2010. Oropharyngeal and cloacal swabs from five birds were pooled for RNA extraction and PCR analysis.Extraction of RNA from 140 μl of allantoic fluid  or swab fluid  was carried out using the QIAamp viral RNA mini kit  following the manufacturer's instructions. The RNA was eluted from the columns with 50 μl of DEPC-treated water and used immediately or after storage at -80°C. Likewise, DNA was extracted from bacterial species and DNA viruses (Additional file In addition to the descriptive evaluation of the test results, the technical sensitivity of the new multiplex RT-qPCR was investigated by comparing positive Ct-values with those of the standard H5-specific RT-qPCR test recommended by Slomka et al. . For thiAIV: avian influenza virus; DPI: days post infection; FLI: Friedrich-Loeffler-Institute; HPAI: highly pathogenic avian influenza; LPAIV: avian influenza virus of low pathogenicity; HA: hemagglutinin; NLQP: National Laboratory for Quality Control on Poultry Production; O.I.E.: World Organization for Animal Health; RT-qPCR: real time reverse transcriptase polymerase chain reaction.The authors declare that they have no competing interests.ESAW carried out part of the development studies, the analytical and the diagnostic evaluation; he performed the sequence alignments and helped to draft the manuscript. AME and ASA carried out parts of the analytical and diagnostic evaluation using Egyptian samples. MZ carried out the statistical tests. CG, MMA and HMH provided samples for analysis, participated in the design of the study and helped to draft the manuscript. TCH conceived and coordinated the study, and drafted the manuscript. All authors read and approved the final manuscript.Detection and differentiation of HPAIV H5N1 isolates collected from commercial poultry and backyard birds in Egypt in 2006- 2010 by multiplex H5 RT-qPCR and a generic H5-specific RT-qPCR [Click here for fileAnalytical specificity of the multiplex RT-qPCR for Egyptian HPAIV H5N1 using different avian influenza virus isolates and other avian pathogens.Click here for fileDetection and differentiation of HPAIV H5N1 in selected swab samples collected from SPF chickens experimentally infected with Egyptian clade 2.2.1p proper or 2.2.1v variant HPAI H5N1 virus strains by multiplex H5 RT-qPCR compared to a standard generic H5 RT-qPCR protocol.Click here for fileDetection and differentiation of HPAIV H5N1 in pooled swab samples collected from commercial poultry and backyard birds in Egypt in 2008- 2010 by multiplex H5 RT-qPCR compared to standard generic H5 and M gene RT-qPCR protocols.Click here for file


Enterovirus 71 (EV71) is an etiologic agent of hand-foot-and-mouth disease (HFMD), and recent HFMD epidemics worldwide have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case-fatality rates. EV71 contains a positive-sense single-stranded genome RNA of approximately 7400 bp in length which encodes a polyprotein with a single open reading frame (ORF), which is flanked by untranslated regions at both the 5' and 3' ends.in vitro synthesized RNA transcripts from the RT-PCR amplicons were then transfected into RD cells to produce the rescued virus. The rescued virus was further characterized by RT-PCR and indirect fluorescent-antibody (IFA) assay in comparison with the wild type virus. The rescued viruses were infectious on RD cells and neurovirulent when intracerebrally injected into suckling mice.A long distance RT-PCR assay was developed to amplify the full length genome cDNA of EV71 by using specific primes carrying a SP6 promoter. Then the Thus, we established a rapid method to produce the infectious full length cDNA of EV71 directly from RNA preparations and specific mutations can be easily engineered into the rescued enterovirus genome by this method. Enterovirus 71 (EV71) is the major causative agent of hand, foot and mouth disease (HFMD), and may cause various neurological diseases, such as aseptic meningitis, acute flaccid paralysis and fatal encephalitis . EV71 haEnterovirus within the family Picornaviridae, and it contains a single-stranded positive genome RNA of about 7,400 bp in length which is infectious when introduced into cell culture. The single open reading frame (ORF) encodes a polyprotein and is flanked by untranslated regions (UTR) at the 5' and 3' ends; a variable length poly-A tract is located at the terminus of the 3'UTR. The polyprotein can be divided into three genomic regions . The P1 encodes the capsid comprised of four structural proteins VP1, VP2, VP3 and VP4. The P2 and P3 encode the nonstructural proteins including 2A, 2B, 2C, 3A, 3B, 3C and 3D [EV71 belongs to human enterovirus species A of the genus Reverse genetics permits the use of cDNA copies of viral RNA genomes to produce detailed studies of molecular features of virus infection and replication. The reverse genetic of picornaviruses has been developed for a long time . In 1981in vitro transcription. After optimization of RT-PCR conditions, chiefly through turning down annealing temperature from 55°C to 52°C or 50°C, the full-length cDNA of EV71 AH08/06 strain was successfully amplified  containing 10% fetal bovine serum  plus 2 mM L-glutamine, 100 IU of penicillin, and 100 μg of streptomycin per ml at 37°C in the presence of 5% COVirus was cultured in RD cells and harvested by freezing and thawing for 3 times. Viral RNA was extracted from viral stock using RNeasy mini kit (QIAGEN) according to the manufacturer's instructions. The reverse transcription reaction was performed 1 hour at 42°C with Super Script II Reverse Transcriptase (Invitrogen) using RNA from viral stock as the template.ACGCGTATTTAGGTGACACTATAGGTTAAAACAGCCTGTGGGTTGCACCCACTC-3' and 5'-AGCTCCTCTAGATTTTTTTTTTTTTTTTTTT TTTTTTGCTATTCTGG-3' (underlined words indicate restriction sites). Thirty five cycles of PCR amplification were carried out with the LA Taq™ Polymerase (TaKaRa) on an automated thermal cycler (Perkin Elmer). Temperatures were 94°C during denaturation (30 sec), 50°C during annealing (30 sec) and 70°C during polymerization (8 min). The final extension time was 10 min at 72°C. The DNA products were purified using QIAquick PCR Purification Kit (QIAGEN) and further quantified on a NanoDrop Spectrophotometers (Thermo Scientific).Full-length cDNAs of EV71 were acquired after long distance PCR using specific primers as follows: 5'-AGCTCCin vitro Transcription Kit (Promega) according to the manufactory's instructions. The in vitro-synthesized RNA transcripts were then transfected into RD cells using Lipofectamine 2000 reagent (Invitrogen). Briefly, RD cells seeded in the 24-well plates with more than 90% confluency. Lipofectamine 2000 was diluted into OptiMEM Medium (Invitrogen) and incubated for 5 min following by mixture with the RNA transcripts for another 30 min at room temperature. Then the RNA-Lipofectamine 2000 complexes (50 μl) was added directly to the plate well of containing cells and incubate at 37°C in a CO incubator. When cytopathic effect (CPE) was observed, the rescued viruses were harvested and stored at -70°C for use.RNA transcription was carried out using the SP6 The rescued virus was identified by RT-PCR with specific primers targeted at the VP3-VP1 genes . The reaction parameters were as follows: 95°C during denaturation (20 sec), 45°C during annealing (25 sec) and 72°C during polymerization (30 sec) within 30 cycles. The final extension time was 10 min at 72°C. The DNA products were separated using QIAquick PCR Purification Kit and further verified by DNA sequencing (Invitrogen).RD cells transfected with the RNA transcripts were grown on a specific slide. RD cells were washed in PBS for 3 times and incubated with mice EV71 monoclonal antibody (Chemicon) in 1:1000 dilutions for 30 min at 37°C. Then the cells were washed and incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Zhongshan) diluted 1:200 in Evans Blue for another 45 min. Finally, the cells were rinsed for 30 min in PBS and visualized under a fluorescent microscope (Olympus) after dried at room temperature.Suckling Kunming (KM) mice of 1 day old were obtained from the . These newborn suckling mice were inoculated intracerebrally with 50 μl rescued viruses or RNA transcripts. The neurological manifestations and survival rate were observed and recorded for 2 weeks. The Institutional Animal Care and Use Committee approved all animal protocols.EV71: enterovirus 71; HFMD: Hand, foot and mouth disease; ORF: open reading frame; UTR: untranslated regions; DMEM: Dulbecco's modified Eagle's medium; SPF: specific pathogen free; IFA: immunofluorescent assay; FITC: fluorescein isothiocyanate.The authors declare that they have no competing interests.JFH and RYC designed and performed the experiments and drafted the manuscript. MY and XT participated in cell culture and animal experiments. EDQ and CFQ supervised the work and edited the final version of this manuscript. All authors read and approved the final manuscript.