Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs), dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity.During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit,  The selective response of axons to elongation and guidance cues encountered along their paths enables the precise formation of neuronal circuits and the formation of topographic maps. During development, mechanisms that specify neuronal subclasses are coupled to those that specify their axonal response through the selective expression of transcription factors.Pax6 expression upon exiting the cell cycle. Consequently, very few postmitotic populations express Pax6. Notable examples are the retinal ganglion cells (RGCs), the dopaminergic neurons of the olfactory bulb and certain neurons in the basal telencephalon and midbrain Pax6 in neural precursors has been widely explored, revealing a central function in cell fate specification and cell cycle regulation Pax6 is a homeodomain transcription factor expressed in several territories of the developing nervous system, mostly in the proliferative regions containing neural precursors Pax6Pax6 expression in both developing and mature RGCs is graded, with higher levels in the ventro-temporal distal cells and lower in the proximal domains Pax6 populations project to distinct non-overlapping and complementary topographic regions of the superior culliculus and lateral geniculate nucleus (LGN), which suggests that Pax6 may contribute to control axon targeting. Indeed, RGCs of mice overexpressing Pax6 show disrupted axonal trajectories and abnormal bundle formation Pax6 is directly involved in the control of axon growth and what guidance cues, if any, depend upon its activity.RGCs are one of the best-characterized postmitotic populations that express Xenopus and chick retina. This activity is independent of Wnt signaling and modulated by extracellular matrix molecules Sfrp1 is expressed in several structures of the embryonic and adult eye and brain, including the pigmented retina, cornea, ciliary bodies, lens epithelium, the prospective thalamus and the proliferative regions of the telencephalon Secreted Frizzled-Related Protein 1 (SFRP1) is one of the factors known to stimulate the directional growth of RGCs axons in Pax6 in neuronal postmitotic populations is necessary and sufficient to confer neurons with a positive response to SFRP1. Over-expression of Pax6 rendered cultured cortical neurons competent to respond to endogenous and exogenous SFRP1. Conversely, knock-down of Pax6 in mouse retinal explants abolishes RGC axonal growth induced by SFRP1 without affecting RGC differentiation or their axonal response to a different guidance cue like sonic hedgehog (Shh) or Netrin. These results suggest that Pax6 and SFRP1 are pair regulators of axonal connectivity in the retina and reveal a new function of Pax6 in the postmitotic populations.Here we show that expression of Pax6 positive precursors, such as cerebral cortical neurons or neurons derived from cortical neural stem cells (NSCs), do not express Pax6Pax6 expression in postmitotic populations, we forced its expression in neurons derived from mouse NSCs. NSCs grown as neurospheres were nucleofected with CAG-empty vector or CAG-Pax6, together with a CAG-GFP that allowed the visualization of transfected cells. GFP staining demonstrated efficient transfection by FACs cytometry  of the dorsal telencephalon, which give rise to both cortical neurons and NSCs in vitro also express detectable levels of the SFRP1 protein . Furthermore, in non-stimulated explants, the length of GFP positive axons was similar in both control and Pax6 shRNA electroporated retinas . Furthermore, Pax6 knock-down had no effect on the response of dorsal retinal explants to exogenously added Shh . C57BL6 mice were obtained from Harlan Laboratories, Inc. . Morning of the day of the appearance of the vaginal plug was defined as embryonic day (E) 0.5.Mouse embryos were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline  and cryoprotected in 30% sucrose in PBS. 10–20 µm horizontal sections were produced and mounted on Superfrost plus microscope slides . Inmunostaining of tissue sections and explants, or cell cultures was performed as described Confocal microscopy was performed with a TCS-SP5  Laser Scanning System. Tissue sections of 50 and 20 µm were analyzed by taking 0.2 µm serial optical sections with the LASAF v2.2.1 software . Fluorescence microscopy was performed with a Leica DMRXA and images were captured with Leica CW4000 FISH Version V1.2 software.Pax6 were: TRCN0000075377:ICCGGCCTGAAGCAAGAATACAGGTACTCGAGTACCTGTATTCTTGCTTCAGGTTTTTG;ITRCN0000075376:ICCGGCATGGCAAACAACCTGCCTATCTCGAGATAGGCAGGTTGTTTGCCATGTTTTTG;ITRCN0000075375:ICCGGGACGGCATGTATGATAAACTACTCGAGTAGTTTATCATACATGCCGTCTTTTTG;ITRCN0000075374:ICCGGCCACTTCAACAGGACTCATTTCTCGAGAAATGAGTCCTGTTGAAGTGGTTTTTG.The hairpin sequences targeting Pax6 and green fluorescence protein (GFP) cDNAs were under the cytomegalovirus enhancer, chicken β-actin promoter, and rabbit β-globin poly (A) signal (CAG)  promoter. For knock-down experiments, a mixture of the selected shRNA lentiviral constructs (1 µg/µl) and pCAG-GFP (1 µg/µl) was used. For testing off-target effects, we co-electroporated a construct driving expression of a silent resistant form of Pax6  plasmids. Pax6 lentiviral shRNA constructs were obtained from Sigma-Aldrich Inc. . Purified recombinant SFRP1 protein was generated as previously described In utero and ex vivo electroporations were performed as described previously ex vivo electroporation the head of the E13.5 embryos were transfer to a dissecting dish where they were electroporated with the above conditions. Then, electroporated retinas were dissected and incubated in DMEM-F12  supplemented with N2  medium at 37°C, 5% CO2 for 24 hours.Pax6 in RGCs did not interfere with axonal initiation because the number of total axons per explants was not significantly different in control and Pax6 shRNA electroporated retinas (not shown). It was also not affected by treatment with SFRP1, Netrin1 or Shh. For SFRP1 experiments at least 50 axons per condition in each independent experiments (n = 6) or 60 neurons per condition (n = 4) in Shh and Netrin1 experiments. We chose to specifically study SFRP1 response in dorsal explants because this population expresses high levels of Pax6 and is homogeneously composed of contra-laterally projecting axons facilitating the analysis of SFRP1 response.Dorsal retinal microexplants were plated on glass coverslips coated with poly-D-Lys  and laminin  and grown for 24 hours in DMEM-F12 medium supplemented with N2 in the presence or absence of SFRP1 (2 ng/µl) or N-Shh (1 ng/µl). After 24 hours, explants were fixed in 4% PFA at RT for 30 minutes and immunostained with anti-β-tubulin III and anti-GFP antibodies. Axonal length was determined in confocal images employing LaserPix v.4 image software (Bio-Rad). In non-electroporated explants, axonal length was estimated by counting the number of β-tubulin III positive axons crossing 600 and 900 µm radius circumferences of a minimum of 100 axons in each of three independent experiments (n = 3). For electroporated explants, we measured the length of each individual GFP positive axon from the edge of the explant up to the distal tip of the growth cone. Knock-down of Pax6, cells were fixed after 6 or 9 days in culture. To compare neurons that exit at the same time from the cell cycle, postmitotic neurons were labeled with a 12 h pulse of Bromodeoxiuridine  (0.5 ng/ml) in the media and subsequently extensively washed. For blocking the endogenous SFRP1 activity a mix of anti-SFRP1 antibodies : rabbit anti-SFRP-1 and goat anti-SFRP-1  or a control IgG  were added to the culture media after nucleofection. For stimulation experiments, SFRP1 was added to the media 24 h after the nucleofection and the early axonal growth measured 3 days after the addition of SFRP1.Dorsal telencephalons of E13.5 WT embryos were dissected and cells were dissociated employing trypsin and a flamed-tip, glass Pasteur pipette. Dissociated cells were cultured in DMEM-F12 supplemented with N2 , heparin, recombinant human basic fibroblast growth factor (FGF-2) , and epidermal growth factor . Neurosphere cultures were passage every 3 days by mechanical dissociation. Dissociated cells were transfected using the nucleofector  with 5 µg of a 1∶1 mixture of CAG-GFP and CAG-Pax6 or CAG- empty vector. Nucleofection efficiency and GFP positive cells were analyzed by flow cytometer (Coulter Epix XL) and data were analyzed with the Win MIDI software. Nucleofected neurospheres were seeded on glass coverslips coated with poly-D-Lys and cultured in DMEM-F12 supplemented with N2, heparin and 1% fetal calf serum (FCS). Cells were fixed in 4% PFA at RT for 30 minutes, then immunostained with anti-β-tubulin III and anti-GFP antibodies. Axonal length was determined with the LaserPix v.4 image software  over images taken with a fluorescence microscopy Leica DMRXA. For analysis of the effects of Pax6 or the empty CAG- vector and CAG-GFP using lipofectamine 2000  and following manufacture procedure. Three days after transfection, cells were fixed with PFA 4%, and immunostained with anti-β-tubulin III and anti-GFP antibodies to measure their axonal length as describe next. GFP positive neurons did not incorporate BrdU demonstrating an effect on posmitotic neurons independent of cell cycle exit. As mentioned, SFRP1 was added to the media 24 h after transfection and the axons measured 2 days after the addition of the protein.Dissociated cells from the dorsal telencephalon of E13.5 embryos were seeded onto 24 well Poly-D-Lys coated plates in Neurobasal media supplemented with B27 complement  and BrdU (0.5 ng/ml) to test proliferation. 24 h after seeding, cells where co-transfected with CAG-In neurons differentiated from NSC or primary neurons, we identified the axon by morphological criteria and the specific staining of MAP1b, selectively localized to the distal part of axonal projections Quantitative results are expressed as the mean ± SD. Axonal length distributions of each population were compared using a Chi-square test. Differences in gene expression was analyzed were compared with Student's two-sample t test. P values are indicated in figure legends.Figure S1Efficiency of Pax6 overexpression and inhibition.a) Transfection of pCAG-Pax6 in NSCs results in efficient overexpression of Pax6 proteins in NSC derived neurons. Pax6 proteins are not expressed normally in GFP positive CAG-control targeted neurons, but are ectopically expressed (yellow) in cells transfected with CAG-Pax6 plasmid. Bar indicates 20 µm. b) Fluorescent-activating cell sorting (FACs) analysis demonstrates more than 20% transfection efficiency. c) Ectopic Pax6 expression in NSCs does not alter the total number of secondary and primary neurites per cell. The number of neurites, excluding the axon, was quantified 9 days after transfection. d) Transfection of shRNA lentiviral constructs efficiently suppresses the ectopic expression of Pax6 in CHO cells. CHO cells co-trasfected with CAG-Pax6; control shRNA and CAG-GFP show expression of Pax6 protein (red). Pax6 is down-modulated in cells transfected with CAG-Pax6 and shRNAs targeting Pax6. Number of cells >100 (n = 3). Bar indicates 30 µm. Graph represents the proportion of GFP positive cells expressing Pax6 protein. Data are expressed as the mean ± SD. (***) p<0.001.(TIF)Click here for additional data file.Figure S2Ectopic expression of Pax6 stimulates axonal growth in cortical neurons. NSCs were nucleofected with CAG-empty vector or CAG-Pax6 and co-electroporated with CAG-GFP. Graph shows the percentage of nucleofected neurons with respect to their axonal length after 6 days of differentiation. Results are equivalent to those obtained at 9 days. Over-expression of Pax6 increments the axonal length compare with control neurons. Data are expressed as the mean ± SD. (**) p<0.01.(TIF)Click here for additional data file.Figure S3SFRP1 stimulated axonal response in Pax6 over-expressing neurons but not in the control population. Graph represents the population distribution of neurons with axons longer than 100 µm or more. Data are expressed as the mean ± SD. (**) p<0.01; (***) p<0.001.(TIF)Click here for additional data file.Figure S4Knock-down of Pax6 did not affect the expression of Fz2 receptor.a) Micrographs show confocal images of GFP positives axons in retina explants that were electroporated with shRNA control or shRNA targeting Pax6, and with CAG-GFP. Both populations present low levels of Fz2 expression and a positive dotted pattern of Fz2 staining. GFP negative mature axons presented a stronger signal. Bar indicates 3 µm. b) Q-PCR detection of the relative expression of the mRNA of Fz1, Fz3, Fz5, Fz6, Unc5a, Unc5b, Unc5c, Unc5d, Neuropilin1 and PlexinD1 from primary cultured neurons. Expression levels are relative to GAPDH transcript and normalized to one control sample (see mple see . There a(TIF)Click here for additional data file.Text S1cDNA sequence for the silent mutant form of Pax6 resistant.(DOCX)Click here for additional data file.Text S2Methods for real-time quantitative RT-PCR (Q-PCR).(DOCX)Click here for additional data file.


In epilepsy, in spite of the best possible medications and treatment protocols, approximately one-third of the patients do not respond adequately to anti-epileptic drugs. Such interindividual variations in drug response are believed to result from genetic variations in candidate genes belonging to multiple pathways.ABCB1 c.3435C>T, ABCB1c.1236C>T, ABCB1c.2677G>T/A, SCN1A c.3184 A> G, SCN2A c.56G>A (p.R19K), GABRA1c.IVS11 + 15 A>G and GABRG2 c.588C>T. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods, and each genotype was confirmed via direct DNA sequencing. The relationship between various genetic polymorphisms and responsiveness was examined using binary logistic regression by SPSS statistical analysis software.In the present pharmacogenetic analysis, a total of 402 epilepsy patients were enrolled. Of them, 128 were diagnosed as multiple drug-resistant epilepsy and 274 patients were diagnosed as having drug-responsive epilepsy. We selected a total of 10 candidate gene polymorphisms belonging to three major classes, namely drug transporters, drug metabolizers and drug targets. These genetic polymorphism included CYP2C9 c.430C>T (*2 variant), CYP2C9 c.1075 A>C (*3 variant), P = 0.05). In drug transporter, ABCB1c.2677G>T/A polymorphism, allele A was associated with drug-resistant phenotype in epilepsy patients . Similarly, the variant allele frequency of SCN2A c.56 G>A single nucleotide polymorphism was significantly higher in drug-resistant patients . We also observed a significant difference at the genotype as well as allele frequencies of GABRA1c.IVS11 + 15 A > G polymorphism in drug-resistant patients for homozygous GG genotype  and G allele .CYP2C9 c.1075 A>C polymorphism showed a marginal significant difference between drug resistance and drug-responsive patients for the AC genotype (Odds ratio [OR] = 0.57, 95% confidence interval [CI] = 0.32–1.00; Our results showed that pharmacogenetic variants have important roles in epilepsy at different levels. It may be noted that multi-factorial diseases like epilepsy are also regulated by various other factors that may also be considered in the future. ABCB1) genes that are expressed widely, including capillary endothelial cells that constitute the blood–brain barrier responsible for ATP-mediated efflux of different compounds outside the brain, serving as a defense mechanism.[ABCB1 (MDR1) gene codes for prototype molecule P-glycoprotein (Pgp) that is recognized to be a key element in regulating access of a diverse array of therapeutic agents to the brain and other tissues.[ABCB1, and some of them have been shown to the influence dosage of AEDs. The role of voltage-gated and ligand-gated ion channels in epileptogenesis of both genetic and acquired epilepsies, and as targets in the development of new AEDs, is very important.[SCN1A p. Thr1067Ala or c.3184 A>G (rs2298771) and SCN2A p.Arg19Lys or c.56 G>A (rs17183814) gene polymorphisms have been found to have a functional significance in the different neurological disorders.[GABAA receptor subunits may also be involved in resistance to AEDs.[Epilepsy is a common, serious, but treatable neurological disorder, affecting at least 60 million people worldwide. Several echanism.10 The AB tissues.12 Severa tissues. A large mportant. Ionic cuisorders. In the cisorders. Recently56 G>A rs183814 geThe study was retrospective and consisted of patients with epilepsy from northern a Indian population. Patients were recruited from the outpatient department (OPD) of Neurology attending the clinics of SGPGIMS and CSMMU Medical University in Lucknow. The patients were subjected to detailed clinical examination, investigations and clinical and family history to ascertain the type of epilepsy and treatment protocol based on clinical proforma. Classification of epilepsy was based on criteria instituted by the International League against Epilepsy (ILAE). For follow-up patients, some of the information was taken from their hospital records. The records of medication, dose, adverse reaction and seizure frequency was maintained for each patient. Any change in medication, its dose and number of seizures was followed. Blood sample was collected from nonresponders and responder patients fulfilling stringent criteria of drug responsiveness and adverse drug reactions. Serum drug levels were determined in 20% of the cases to ascertain drug compliance, especially in those showing signs of adverse drug reactions (ADRs) or nonresponsiveness. This study was approved by the local ethics committees of both the participating institutions. The clinical profile of the drug-responsive and the drug-resistant epilepsy patients was based on hospital investigations. Initial diagnosis was based on two or more unprovoked seizures in the individuals. Both new and old patients were taken after informed consent.The main criterion for drug resistance was the occurrence of at least four seizures over a period of 1 year, with three appropriate AEDs at maximum tolerated doses.20 PatienA venous blood sample (5 ml) was collected from each subject and was kept frozen till DNA extraction. Genomic DNA was isolated from peripheral blood leukocytes by the salting-out protocol. ExtracteABCB1) and metabolizers (CYP2C9*2 and CYP2C9*3) of the principal AEDs for use in future pharmacogenetic studies. Details of candidate genes selected for the study are given in The candidate genes were selected on the basis of their functional role, current biological knowledge of epilepsy, a reported prevalence of at least 5% for the variant allele and published evidence of an association with epilepsy and other neurological disorders. We aimed to select, as candidate genes, major drug targets (voltage and ligand gated), multidrug transporters . The desired power of our study was set at 80%. Relative risks for power calculation were set at 2. Descriptive statistics of patients and controls were presented as the mean and standard deviations (SDs) for continuous measures, while frequencies and percentages were used for categorical measures. The relationship between various genotypes and responsiveness was examined using the binary logistic regression. Association was expressed as odds ratios (OR) or risk estimates with 95% confidence intervals (CI). The association was considered to be significant when the P-value was <0.05.The sample size was calculated using the QUANTO 1.1 program (P = 0.04). AED levels were also measured in 100 patients to confirm drug compliance. The mean carbamazepine, phenytoin and valproate levels were 8.26 ± 5.25, 11.27 ± 8.12 and 68.0 ± 36.22 µg/ml, respectively.A total of 402 epilepsy patients were enrolled in the study. Based on inclusion and exclusion criteria, 128 were diagnosed as multiple drug-resistant epilepsy and 274 patients had drug-responsive epilepsy . Among t2C9*2 (430 C>T) or 2C9*3(1075 A>C) [2C9*2 430 T variant  [CYP2C9 1075 A>C (*3 variant) polymorphism showed a marginally significant difference between patients having multiple drug resistance and drug-responsive patients for AC genotype . The CYP2C9*3 variant allele frequency was also a little higher in drug-responsive patients compared with drug-resistant patients , and seems to be contributing toward a lower risk for developing multiple drug resistance in epilepsy [In our study population, none of the individuals were homozygous for 075 A>C) . No sign = 0.36) . CYP2C9 epilepsy .ABCB1 3435C>T did not differ significantly in the drug-resistant versus the drug-responsive patients for CT  and TT  genotypes [P = 0.39, OR = 1.14, 95% CI = 0.84–1.54). In the ABCB1 c.2 677G>A polymorphism, allele “A” was associated with the drug-resistant phenotype in epilepsy patients . However, genotype frequencies of 2677G>T ABCB1 polymorphism did not differ significantly in the drug-resistant versus the drug-responsive epilepsy patients for GT genotype , for TT genotype  and for AT genotype  [ABCB1 polymorphism also did not differ significantly in the drug-resistant and drug-responsive groups for the CT  and TT  genotypes [The genotype frequencies of enotypes . No sign13–2.24) . Similarenotypes .SCN1A c.A3184G polymorphism did not differ significantly in drug-resistant versus drug-responsive epilepsy patients for AG  or GG  [SCN2A c.R19K also did not differ significantly in the drug-resistant versus the drug-responsive patients for RK  and KK  genotypes [P = 0.03, OR = 1.62, 95% CI = 1.03–2.56). This suggests that SCN2A c.R19K polymorphism may be involved in modulating the drug response in epilepsy patients.The genotype or allelic frequencies of 36–3.78) . Similarenotypes . HoweverGABRA1 A>G polymorphism in drug-resistant versus drug-responsive epilepsy patients for homozygous variant GG genotype  and G allele  [GABRG2, 588C>T did not show any significant differences in drug-resistant versus drug-responsive epilepsy patients either at the genotype or the allele levels [GABRA1 IVS11 + 15 A>G intronic polymorphism may be involved in the drug response in epilepsy patients.We observed a significant difference at the genotype as well as allele frequencies of 05–1.95) . Howevere levels . This suCYP2C9*2 (430 C>T) and CYP2C9*3 (1075 A>C) in epilepsy patients developing multiple drug resistance. These findings indicate the important role of CYP2C9 variants in conferring a multiple drug-resistant phenotype against AEDs. Our findings are in support of previous observations that identified CYP2C9 as a major drug metabolizer for commonly prescribed AEDs.[CYP2C9 is responsible for the hydroxylation of up to 90% of serum phenytoin. Among AEDs, phenytoin, carbamazepine and phenobarbital mainly undergo significant metabolism by cytochrome P450 isozymes. Earlier studies have also observed a strong association between CYP2C9 genotype and phenytoin maintenance dose requirement.[et al.[CYP2C9 allelic variants affect phenytoin dose requirement; for patients carrying at least one mutant CYP2C9 allele, the mean phenytoin dose required to achieve a therapeutic serum concentration was about 37% lower than the mean dose required by wild-type individuals. Another similar study from Taiwan revealed that the CYP2C9 and CYP2C19 polymorphisms have dramatic effects on the population pharmacokinetic parameters of phenytoin.[MDR1 (ABCB1) and MRP2 expression also influence carbamazepine and phenytoin disposition and may account for inter-individual pharmacokinetic variability.[ABCB1 gene have been linked to the difference in the drug responsiveness.Multiple genes are known to be responsible for drug responsiveness. For example, DMEs include bed AEDs. CYP2C9 iYP2C9*2 4 C>T and uirement. Weide etYP2C9*2 4 C>T and henytoin. On the biability. P-gp is ABCB1 3435C > T, 1236C > T and 2677G > T/A polymorphisms but failed to find a significant association with these polymorphic variations in refractory epilepsy in the north Indian population. A number of studies from different populations have also explored the role of these polymorphisms in ABCB1 genes on drug-refractory epilepsy, but have shown conflicting results. Many factors may be attributed for the nonuniformity and nonreplication of results from various studies, including low sample size and genetic heterogeneity within the groups. Also, there is no consensus on a single definition of intractability. Individual studies use different definitions, creating difficulties for comparisons of results across studies. We have used inclusion criteria for responders and nonresponders that were similar and consistent with the original study by Siddiqui et al.[SCN1A c.3184 A>G polymorphism was associated with generalized epilepsy,[et al.[SCN1A IVS5-91 G>A intronic polymorphism of the same gene has been reported to show a population-specific association with carbamazepine and multiple drug-resistance epilepsy.[GABRA1 IVS11 + 15 A>G polymorphism in modulating drug response in pharmacotherapy, while GABRG2 588C>T was not found to be associated with drug resistance in north Indian epilepsy subjects. Several mutations in this gene have also been reported to be involved in epilepsy causation that result in loss of function of GABAA receptors via a reduction in GABA expression and accelerated deactivation.[GABRA1 IVS11 + 15 A>G polymorphism with refractory phenotype in our study may occur due to changes in the structure and function of inhibitory GABAA receptors.[In this study, we analyzed three genetic polymorphisms of ui et al. Althoughepilepsy.–39 It isepilepsy. It is altivation. It is poeceptors. Excessiv


Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat ( Previous studies indicated that a novel influenza A virus (H17N10) was circulating in fruit bats from Guatemala . Herein, we investigated whether similar viruses are present in bat species from South America. Analysis of rectal swabs from bats sampled in the Amazon rainforest region of Peru identified another new influenza A virus from bats that is phylogenetically distinct from the one identified in Guatemala. The genes that encode the surface proteins of the new virus from the flat-faced fruit bat were designated as new subtype H18N11. Serologic testing of blood samples from several species of Peruvian bats indicated a high prevalence of antibodies to the surface proteins. Phylogenetic analyses demonstrate that bat populations from Central and South America maintain as much influenza virus genetic diversity in some gene segments as all other mammalian and avian species combined. The crystal structures of the hemagglutinin and neuraminidase proteins indicate that sialic acid is not a receptor for virus attachment nor a substrate for release, suggesting a novel mechanism of influenza A virus attachment and activation of membrane fusion for entry into host cells. In summary, our findings indicate that bats constitute a potentially important reservoir for influenza viruses. Influenza viruses originating from animals caused the three major influenza pandemics of the previous century. Swine were a gateway for avian influenza virus genes to enter human populations as reassortant viruses and also initiated the influenza (H1N1) pandemic of 2009 Artibeus planirostris) from Truenococha as positive for influenza virus. Of the other available specimens, i.e. liver, intestine and spleen tissues from bat PEBT033, the intestine tissue specimen was strong positive whereas others were negative. A BLAST search based on the 250 nt sequence of the PB1 RT-PCR amplicon showed that the influenza gene in the PEBT033 bat was most closely related (77% nt identity) to the PB1 genes of recently described bat influenza viruses, e.g. A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10). The RNA from bat PEBT033 rectal swab sample was analyzed by Sanger and deep sequencing as described previously We sampled 114 Peruvian bats captured during 2010 in Truenococha and Santa Marta, two communities located in the Loreto Department, Peru, a remote and sparsely populated area in Amazonia . The samPhylogenetic analysis indicated that all genes of A/bat/Peru/10 were most closely related to those of bat influenza viruses from Guatemala, although forming a distinct lineage . With thThe differing phylogenetic position of the bat HA genes relative to the other seven gene segments indicates that a reassortment event took place after gene divergence into the bat and non-bat lineages . In contOverall, the magnitude of the evolutionary distance between A/bat/Peru/10 and A/bat/Guatemala/164/09 in the HA and NA genes, previously classified as subtype H17N10, would support its designation as an H18N11 virus, comprising new HA and NA subtypes Key structural features of the Peruvian bat influenza virus genome, including RNA transcription and replication promoter elements, open reading frames and mRNA splice signals, and ribosomal frameshift element (PA gene), are nearly identical to the recently described Guatemalan bat influenza genomes and likely to serve equivalent functions The influenza HA and NA proteins are critical for interaction with the host and determining the subtype of influenza type A viruses. The mean pairwise amino acid sequence identity of the HA of A/bat/Peru/10 with Group 1 HA subtypes is 49.1% . DespiteThe amino-acid sequences of the internal genes of A/bat/Peru/10 retain most of the known functional sequence motifs of other influenza A viruses with few amino-acid substitutions compared to the consensus amino acids at these sites in non-bat viruses . To evalα r.m.s.d. of 0.25 Å and 0.34 Å between equivalent monomers and trimers). The overall H18 HA trimer is similar to other HA structures with a membrane-distal globular head comprising the RBS and the vestigial esterase domain, and a membrane-proximal stem region containing the putative monobasic HA1/HA2 cleavage site and fusion peptide   , S10 [22inkages) . The lacinkages) –E showinβ-sheets in a propeller-like arrangement . Five additional bat species also appear to be highly seropositive despite small sample sizes  and N11 NAL (rNAL) by indirect ELISA. Specific IgG antibody titers to bat rHA or rNAL  were detected among 55 of 110 bats from Peru . High tile sizes .The high seroprevalence of bat influenza in bats from the Loreto Department in Peru prompted analysis of 228 serum samples from eight locations in southern Guatemala in 2009–2010. Specific antibodies to bat H17 subtype rHA were detected by ELISA in 86 of the 228 (38%) sera from eight bat species . The temArtibeus planirostris) in the Amazon River basin in northern Peru. While our phylogenetic analysis indicated that the Peruvian bat virus shares common ancestry with recently identified Guatemalan H17N10 bat viruses, their genetic and phylogenetic divergence is such that we propose that their HA and NA genes be designated as new subtypes H18 and N11, respectively. A high frequency of antibodies is consistent with widespread circulation of these viruses in bat populations from the Americas.We have characterized a new influenza virus from a flat-faced fruit bat  in four of the eight gene segments, genetic diversity exceeds that observed in all other animal species combined; (ii) the divergence into multiple HA subtypes and utilization of alternative mechanisms for sialic acid-independent virion attachment to target cells and subsequent release; and (iii) the widespread geographic distribution in the Americas  combined with a high seroprevalence in several bat species are indicative of an established infection, while the observation that the bat viruses form a monophyletic group is suggestive of sustained transmission in this species. The postulated ancient relationship of influenza viruses with aquatic migratory birds is consistent with the optimized parasitic relationship of the virus with ducks, involving subclinical infections with multiple virus subtypes as a result of efficient fecal-oral transmission. Although necessarily preliminary in nature, the data presented here suggest that similar ecological and evolutionary strategies may have been exploited by the influenza A viruses of New World bats.All procedures reported herein were performed in accordance with institutionally approved animal care and use protocols . All aspects of the bat collections were undertaken with the approval of the a Peru Ministry of Agriculture (permit RD-0389-2010-DGFFS-DGEFFS) and following the American Veterinary Medical Association guidelines on euthanasia and the Guidelines of the American Society of Mammalogists for the use of wild mammals in research.CDC field sampling of selected mammals, such as bats, has been ongoing for several years as a means of background zoonotic surveillance for primary pathogen detection TNA extracted from the rectal swabs (n = 110) were screened for the presence of influenza virus RNA using pan-influenza (pan-Flu) reverse transcriptase PCR (RT-PCR) as described previously de novo with a minimum contig length of 75 bases. All contigs with a coverage depth ≥3X where submitted to BLASTn against the non-redundant (nr) NCBI database to identify influenza sequences. This process was repeated with tBLASTx to find segments that were not identified from nucleotide BLASTn. To increase the reliability of the sequence data from Illumina sequencing, the rectal swab of bat PEBT033 was also processed by Sanger sequencing on RT-PCR amplicons of genome segment. The viral genome was amplified directly from the TNA extracted from bat PEBT033 rectal swab suspensions using universal influenza A primers (FWuni12 and RVuni13) The pan-Flu RT-PCR positive rectal swab suspension was subjected to both high throughput next generation sequencing and RT-PCR amplicon-based Sanger sequencing as described previously http://platform.gisaid.org/epi3/frontend). Sequence alignment was performed on the amino-acid sequences of each gene segment using MAFFT v6.853b 8486 complete genome sequences of influenza A virus, comprising both avian and other mammalian hosts, were downloaded from the GISAID database (4+I).To assist our phylogenetic analyses of these sequence data, we further reduced the sample size to 50–70 representative sequences for each gene segment. Phylogenetic trees of these data were then estimated using the maximum likelihood (ML) method available in the PhyML package 2O2 and phosphate-citrate buffer, was added to each well (100 µL). The plates were incubated at 37°C for 10 minutes, the color change was stopped by addition of 100 uL of 3N HCl, and the plates read by a plate reader. The limit of detectable response (based on 490 nm at 0.1 s OD) for the ELISA was set as values above average background plus 2 standard deviations.An indirect ELISA using bat influenza neuraminidase and hemagglutinin was run to establish the seroprevalence of IgG antibodies to bat influenza within the Peru bat population. In brief, ELISA plates were coated with recombinant bat hemagglutinin , recombinant avian hemagglutinin (rHA5 and rHA1), or recombinant bat neuraminidase-like (rNAL) at a concentration of 1 µg/mL (100 µL per well) in PBS, pH 7.4 overnight at 4°C. The next morning, bat sera were heat inactivated at 56°C for 30 minutes. Plates were washed 3 times with PBS with Tween 20 (0.1%), pH 7.4 (PBST). Bat sera were diluted 1∶500 in 2.5% non-fat milk-PBST and added to each well in duplicate, two-fold dilutions. Plates were incubated for 1 hour at 37°C. The test sera were removed and the plates washed 3 times with PBST. Biotinylated protein G (MBL corp)  was added to each well and the plates were incubated at 37°C for 1 hour. The plates were washed 3 times with PBST. Streptavidin HRP (Millipore)  was added to each well and the plates were incubated at 37°C for 1 hour. The plates were washed 3 times with PBST and O-phenylenediamine (OPD), with H6-tag 6-tag. The cleaved trimeric H18 HA ectodomain was purified further by size exclusion chromatography on a Hiload 16/90 Superdex 200 column  in 20 mM Tris pH 8.0, 100 mM NaCl and 0.02% NaN3.The ectodomain  of the H18 protein from influenza virus A/flat-faced bat/Peru/033/2010  was expressed in a baculovirus system for structural and functional analyses. The cDNA corresponding to the H18 HA ectodomain was inserted into a baculovirus transfer vector, pFastbacHT-A (Invitrogen) with an N-terminal gp67 signal peptide, a C-terminal thrombin cleavage site, a foldon trimerization sequence, and a His6-tag, a vasodilator-stimulated phosphoprotein (VASP) domain and thrombin cleavage site Sf9 cells (Invitrogen) in 3-liter shaking flasks (Corning Inc.) was recovered from the culture supernatant and purified by metal affinity chromatography, subjected to thrombin cleavage and gel filtration chromatography. The purified monomeric N11 NAL protein was buffer exchanged into 10 mM Tris-HCl, 50 mM NaCl, pH 8.0 with 1 mM CaCl2 and concentrated to 14 mg/ml for crystallization trials.The cDNA corresponding to the ectodomain of A/bat/Peru/10 NAL  from influenza virus A/flat-faced bat/Peru/033/2010  was cloned into the baculovirus transfer vector pAcGP67-B, in frame with an N-terminal cassette containing a His3 was crystallized in 0.1 M MES, pH 6.9, 5% (w/v) polyethylene glycol (PEG) 1000 and 27.5% (w/v) PEG 600 at 22°C. The A/bat/Peru/10 HA crystals were flashed-cooled at 100 K without additional cryo-protectant. Diffraction data were collected at beamline 23ID-D at the Advanced Photon Source (APS)  NaNce (APS) . Data fowww.jcsg.org). All figures were generated with Bobscript www.pymol.org).The A/bat/Peru/10 HA structure was determined by molecular replacement (MR) using the program Phaser Each trypsin cleavage reaction containing ∼2.5 µg of A/bat/Peru/10 HA0 or trypsin-digested A/bat/Peru/10 mature HA (HA1/HA2) and 1% dodecylmaltoside (to prevent possible aggregation with any post-fusion HA) was set up at room temperature (∼22°C). Sodium acetate was used as the pH 4.9 buffer and Tris was used as the pH 8.0 buffer. Reactions were thoroughly mixed, centrifuged at >12,000 g for 30 seconds and allowed to incubate at 37°C for one hour. After incubation, reactions were equilibrated to room temperature and the pH was neutralized by addition of 200 mM Tris, pH 8.5. Trypsin was added to all samples, except controls, at a final ratio of 1∶1 (w/w) for A/bat/Peru/10 HA. Samples were digested overnight (∼18 hours) at 22°C. Reactions were quenched by addition of non-reducing or reducing SDS buffer and were boiled for ∼2 min. Samples were analyzed by SDS-PAGE.Initial crystallization trials were set up using a Topaz™ Free Interface Diffusion (FID) Crystallizer system (Fluidigm Corporation). Crystals were observed in conditions containing various molecular weights of PEG polymer. Following optimization, diffraction quality crystals for A/bat/Peru/10 N11 NAL were obtained at 20°C using a sitting drop method. The crystallization condition was 0.2 mM calcium acetate, 10% (w/v) PEG 8000, 0.1 M HEPES at pH 7.5. Crystals were flash-cooled at 100 K using 20% (v/v) glycerol as a cryoprotectant. Data were collected at the Advanced Photon Source (APS) beamline 22-ID at 100 K and processed with the DENZO-SCALEPACK suite Vm) of 3.38 Å3/Da. The model was rebuilt by Coot The structure of A/bat/Peru/10 NAL was determined by molecular replacement with Phaser Methods used for H18 HA and N11 NAL proteins used for crystal form 2 were as described see .N-acetylneuraminic acid (4-MU-NANA) 2 0.02% NaN3 buffer with excitation and emission wavelengths of 365 nm and 450 nm, respectively. The reaction was conducted for 60 minutes at 37°C in a total volume of 80 µl for the N11 NAL protein with ectodomain plus stalk region that was expressed as a tetramer. The reactions were all performed in triplicate and were stopped by adding 80 µl of 1 M Na2CO3. To compare NA cleavage activities at 12 different NA concentrations, with a fixed substrate 4-MU-NANA concentration of 0.05 mM, the NA starting solution at 0.51 mg/ml was serially diluted 1∶2.NA enzymatic activities were measured by using fluorescent substrate 2′-(4-methylumbelliferyl)-α-D-www.rcsb.org .The sequence data reported in this paper were deposited in the GenBank database at NCBI under accession numbers CY125942-CY125949. The atomic coordinates and structure factors of the A/bat/Peru/10 H18 crystals 1 and 2, and A/bat/Peru/10 N11 in crystal forms 1 and 2 are being deposited in the Protein Data Bank, Figure S1Geographic locations of bat-sampling sites in Peru. Bats were captured at Truenococha and Santa Marta in the Loreto District Click here for additional data file.Figure S2RNA-dependent RNA polymerase activity of A/bat/Peru/10 ribonucleoprotein (RNP) complex proteins . A549 human lung cells were transfected with pPol1-A/bat-NS.NCR-Renilla (Guat/164.NS-NCR) or pPol1-A/WSN-NS.NCR-Renilla (WSN.NS-NCR) and pSV40-Luc reporter plasmids, together with plasmids expressing PB2, PB1, PA and NP from either A/WSN/33 (WSN-4P) or A/bat/Peru/10 (Peru/033-4P) or A/bat/Guat/09 (Guat/164-4P) viruses or without the PB1 expression plasmid (Guat/164-3P). Values shown represent the activities of each RNP and reporter relative to that of WSN virus (100%) with the homologous reporter. Error bars indicate 95% confidence intervals. Experiments were performed three times independently.(PDF)Click here for additional data file.Figure S3The structure of A/bat/Peru/10 HA in crystal 2. (A) One monomer is shown with the HA1 chain colored in green and the HA2 chain in cyan. The glycosylation positions are highlighted in magenta with the glycan in yellow. A/bat/Peru/10 HA has four potential N-linked glycosylation sites in HA1  and a further two in HA2 (Asn145 and Asn154). While position 21 is close to the HA1/HA2 cleavage site, position 242 is closer to the putative receptor binding pocket. Positions 264 and 289 are close together in the middle of the molecule around the vestigial esterase domain in HAs from other influenza A viruses. In the HA2, positions 145 and 154 are near the membrane-anchoring region. Asn154 is conserved in all HAs, while Asn145 is only found in HA sequences from three other group 1 subtypes . From these structures, interpretable electron density for one or two N-acetyl glucosamines was observed at all six of these putative glycosylation sites. However, due to crystal packing, density for the carbohydrate at Asn242 was well defined in all three monomers and could be visualized up to the first three mannoses of the glycan. (B) The putative receptor binding site with the three structural elements, the 130-loop, 220-loop and the 190-helix. The putative binding site residues are shown in sticks. (C) Superposition of receptor binding site region of A/bat/Peru/10 H18 (in green), 1918 H1 , 2009 H1 , swine H1 , human H2 , human H5 , swine H9 , human H3 , human H7  and mallard H14 .(PDF)Click here for additional data file.Figure S4Comparative surface representation of the receptor binding sites of bat and non-bat HAs. A/bat/Peru/10 H18 HA (in green), 1918 H1 HA , 2009 H1 HA , swine H1 HA , H2 HA , H5 HA , H9 , H3 HA , H7 HA  and H14 HA , with arrows indicating the receptor binding sites in other HAs.(PDF)Click here for additional data file.Figure S5α2-3 glycans, but A/bat/Peru/10 HA and NAL exhibit no specific binding to any glycans on the array, including natural sialosides with α2-3, α2-6, α2-8 and mixed linkages, and other glycans that might exist in mammals (see Tables. S9 and S10). (D to E) ELISA-based plate assay of A/bat/Peru/10 HA and control A/Vietnam/1203/2004 H5 to 3′-SLNLN (D) and 6′-SLNLN (E). The H18 HA does not bind to either 3′-SLNLN or 6′-SLNLN in the experimental conditions, while the control H5 HA binds more strongly to 3′-SLNLN as expected.Glycan binding analysis of A/bat/Peru/10 HA and NAL. (A to C) Glycan microarray analysis of A/bat/Peru/10 HA (A) and NAL (B), and control protein A/Vietnam/1203/2004 H5 HA (C) was performed on the CFG glycan microarray v5.1, which contains 610 mammalian glycans. Binding signals (black bars) are shown in relative fluorescence units (RFU). The H5 HA showed good binding avidity to (TIF)Click here for additional data file.Figure S6α r.m.s.d. of 0.7 Å. The structures of A/bat/Peru/10 NAL in two crystal forms are the same except for the 150-loop, which is flexible in crystal form 2 and not modeled. (B) Comparison with GU09-164 NAL in crystal form 1  with r.m.s.d. of 1.3 Å. The overall structures are very similar except for 150-loop which is closer to the putative active site in A/bat/Peru/10 N11 NAL, making the putative active site less open. (C) Comparison with GU09-164 NAL in crystal form 2  with Cα r.m.s.d. of 1.2 Å. The overall structures are very similar except for 150-loop which is further from the putative active site in A/bat/Peru/10 N11 NAL, making the putative active site more open. (D) Comparison with 1918 N1 NA  with Cα r.m.s.d. of 1.7 Å. As large conformational changes are observed in the 110-loop, 150-loop and 430-loop, as well as C-terminus, these regions were excluded from the RMSD calculation. Compared to 1918 N1 NA, the A/bat/Peru/10 NAL putative active site in crystal form 1 adopts a much more open conformation.Stereo view of superimposed A/bat/Peru/10 NAL in crystal form 1 (in grey) with other NA structures. (A) Comparison with A/bat/Peru/10 NAL in crystal form 2 (in green) with C(PDF)Click here for additional data file.Figure S7NA cleavage activity analysis of A/bat/Peru/10 NAL. Only extremely low sialic acid cleavage activity was observable with NAL concentrations as high as 100 µg/ml.(PDF)Click here for additional data file.Figure S82 dilutions of sera were performed, starting at 1∶500, and an absorbance reading was taken at 490 nm for 0.1 seconds. Samples represented are (A) Peru 017, (B) Peru 019, (C) Peru 020, and (D) Peru 031.Detection of antibody to HA subtypes in bat sera by ELISA. Plates were coated with 1 ug/mL of H18 rHA antigen (homologous), or H17, H5,H1 rHA antigen (heterologous) to ascertain levels of cross reactivity. Serial log(PDF)Click here for additional data file.Table S1Nucleotide sequence identity between A/flat-faced bat/Peru/033/2010 (H18N11) and A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10) genomes.(DOCX)Click here for additional data file.Table S2Mean amino acid identity between A/bat/Peru/10 H18 HA and influenza A subtypes H1–H17.(DOCX)Click here for additional data file.Table S3a.Sequence comparison after sequence and structural alignment of HA1 (top) and HA2 (bottom) of A/bat/Peru/10 H18 HA with bat H17 HAs and non-bat HAs (DOCX)Click here for additional data file.Table S4Mean amino acid identity between the A/bat/Peru/10 N11 NAL and representative NAs of influenza A and B viruses.(DOCX)Click here for additional data file.Table S5a.Sequence comparison of A/bat/Peru/10 N11 NAL with bat N10 NALs and other influenza NAs(DOCX)Click here for additional data file.Table S6Data collection and refinement statistics of A/bat/Peru/10 HA and A/bat/Peru/10 NAL.(DOCX)Click here for additional data file.Table S7α rmsd values (Å) of A/bat/Peru/10 HA  with other influenza A virus HAsa.Comparison of C(DOCX)Click here for additional data file.Table S8a.Conservation of key residues in influenza A HA receptor-binding site (DOCX)Click here for additional data file.Table S9List of glycans on the microarray.(XLS)Click here for additional data file.Table S10List of 25 glycans that give strongest binding signals in the microarray.(XLSX)Click here for additional data file.Table S11Conservation of key residues in the NA active site.(DOCX)Click here for additional data file.Table S12Seroprevalence of IgG in Guatemalan bats to H17 rHA by ELISA.(DOCX)Click here for additional data file.Text S1Supporting Text. Methods, figure legends and tables.(DOCX)Click here for additional data file.


D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the PolD714A mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced kpol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication  The gp43 DNA polymerase of the RB69 bacteriophage (RB69 Pol) bears primary responsibility for the faithful and efficient replication of the viral genome. RB69 Pol belongs to the B-class of DNA polymerases and is closely related to T4 phage DNA polymerase. It shares significant sequence similarity with eukaryotic replicases of the same family, such as the α, δ and ε polymerases, which carry out the bulk of chromosomal replication in higher organisms ,2. Sever8 replicated bases . ResD714Ain vitro and solved three X-ray crystallographic structures of the mutant polymerase. Taken together, our data demonstrate how subtle structural changes originating from the substituted D714 can “act at a distance”, and propagate through neighbor-to-neighbor interactions, eventually reaching the catalytic center of the enzyme to distort its geometry.To that end, we have biochemically characterized the RB69 Pol3′-5′-exonuclease activity of the enzyme. For the D714A RB69 DNA Pol variant, we repeated the construction of the D714A mutation for both expression plasmids using the Quick Site-Directed Mutagenesis protocol (Stratagene). We then reverted the mutated gp43 gene at the 714 site and used these plasmids in in vivo DNA synthesis experiments as independent controls to confirm the observed phenotype. Additionally, for crystallographic purposes the D714A substitution was combined with Y567A replacement. The presence or absence of mutations in protein expression vectors was verified by sequencing of the entire open reading frame. Enzymes that are reported in this study and designated Pol+Exo+, PolD714AExo+ or Pol+Exo-, PolD714AExo-, PolY567A/D714AExo- originate from the pCW19R and pCW.50 plasmids, respectively.Plasmids expressing alanine mutants in E614, N711, D714 and Y720 of RB69 DNA Pol were generous gifts from Jim Karam (Tulane University) and are derivatives of plasmids pCW19R and pCW.50R. Plasmid pCW19R encodes the wild-type RB69 gp43 gene under control of the T7 promoter in the pSP72 vector (Promega). Plasmid pCW.50R expresses a substituted RB69 DNA Pol with D222A and D327A replacements that eliminate the +Exo+, Pol+Exo-, PolD714AExo+, PolD714AExo- and PolY567A/D714AExo- gp43s were performed as previously described -ATP  by T4 DNA polynucleotide kinase (Takara) and annealed to the corresponding templates at a molar ratio of 1:1.3 in 10 mM Tris-HCl, pH7.5. Annealing reactions were heated at 85°C for 5-10 min. and allowed to cool slowly to 25°C. Samples were kept at 4°C or frozen prior to usage. The oligonucleotides used in crystallization experiments were synthesized using an in-house oligonucleotide synthesizer. The non-hydrolysable nucleotide analog dGpnpp -imido]triphosphate) was purchased from Jena Bioscience (Gmbh).All oligonucleotides used in this study, except for the nucleotides used in crystallization trails, were synthesized in the Laboratory of DNA Sequencing and Oligonucleotide Synthesis  and gel-purified. Primers were 5ʹ-end radiolabeled with  = A(1 – e-kobst), where A – amplitude, t – time (sec), to obtain kobs .Chemical quench experiments were performed at 10°C using a KinTek RQF-3 instrument (KinTek Corp.). Single turnover conditions were achieved by maintaining a 10-fold excess of RB69 DNA Pol over the 5ʹ-radiolabeled 13*/19-mer DNA duplex  of the concentrated polymerase (18.5 mg/ml) and the reservoir solution (1.6 M (NH4)2SO4, 0.01 M MgCl2 and 0.05 M sodium dihydrogen citrate, pH 5.6) were mixed and allowed to equilibrate against 0.5 ml of the well solution. The largest crystals (0.3 µm – 0.5 µm) grew within 3 – 5 days, were transferred to the cryoprotectant (1.65 M (NH4)2SO4, 0.01 M MgCl2, 0.05 M sodium dihydrogen citrate, pH 5.6, 3 mM DTT, 10 mM KCl, 20% (w/v) glycerol) and subsequently flash frozen in liquid nitrogen. The ternary complex of PolY567A/D714A was prepared by mixing the protein (20 mg/ml) with a freshly annealed 13/18-mer DNA duplex  in 1:1.2 molar ratio, respectively. DNA annealing buffer contained 10 mM CaCl2, 50 mM NaCl and 20 mM Tris-HCl, pH 7.6. The non-hydrolysable dGpnpp analog was subsequently added to the mixture to the final concentration of 2 mM. Crystals for the Ternary I structure were grown via hanging-drop vapor diffusion by mixing equal volumes of the ternary complex and the reservoir solution containing 10% (w/v) PEG 8,000, 0.2 M (NH4)2SO4 and 0.1 M sodium citrate, pH 5.6. The crystals appeared within two days and were cryoprotected in the reservoir buffer with increased 14% (w/v) PEG 8,000 and 25% (w/v) glycerol, and flash frozen in liquid nitrogen. For Ternary II, crystals were grown using the same method as Ternary I, but with reservoir solution containing 12% (w/v) PEG 8,000, 0.2 M (NH4)2SO4, and 0.1 M MES, pH 6.0. The crystals appeared within two weeks and were cryoprotected in the same manner as Ternary I.Crystals of exonuclease deficient RB69 PolD714A apoenzyme and both PolY567A/D714A ternary complex structures were solved by molecular replacement in Molrep [Y567A catalytic complex (PDB ID code: 3NGI) [X-ray data collection was performed at beamlines X12C and X25 of the National Synchrotron Light Source at Brookhaven National Laboratory . Datasets were processed using HKL2000  (apo strn Molrep  and PolYe: 3NGI)  as starte: 3NGI) , followee: 3NGI) . Validate: 3NGI) . Coordinin vivo T4 phage growth complementation assay that is mainly dependent on the catalytic activity of the mutant polymerase. We assessed the essentiality of E614, N711, D714 and Y720, for in vivo DNA synthesis by RB69 Pol using a combination of T4 phage growth complementation assay and radioactive thymidine uptake experiments, as described previously [in vivo. Briefly, the T4 phage 43amam mutant, whose endogenous DNA polymerase is inactivated by two amber mutations at the 202 and 386 codons of the T4 gp43 gene, is used to infect the E. coli BB strain. E. coli BB cells carry a plasmid expressing either wild type or mutant RB69 Pol, which in the absence of T4 Pol is the only enzyme capable of performing phage genome DNA replication [43amam phage genomic DNA was measured after the host DNA synthesis had ceased completely [D714A was severely inhibited in this assay, suggesting that D714 is essential for replication in vivo  that is almost indistinguishable from the Pol+Exo+ (8 x 10-4), and is similar to background values for uncopied M13mp2 DNA (5 – 7 x 10-4) [D714AExo- variant displays a mild but reproducible 4.8-fold antimutator effect compared to the wild type Exo- polymerase , suggesting that, in fact, the D714A substitution results in an enzyme with higher fidelity than the wild type polymerase.One possible reason for the reduced ability of Polon assay . Purifiein vitro . D714A g x 10-4) ,29. Morein vitro may result from increased exonuclease activity and/or decreased ability to form binary polymerase-DNA complexes [Dapp was calculated as described in Materials and Methods  remains within the experimental error range . The errors introduced by the pre- and post-chemistry steps (i.e. DNA association or release from the polymerase) are therefore eliminated from the calculated kl values .pol and Kd,dNTP measured for the wild type RB69 Pol in this study remain similar to the values observed by others for correct base pair formation [D714A exhibited only a 2-fold higher dissociation constant  for the complementary dATP compared to the wild type RB69 Pol  (kpol) of PolD714A was reduced by 8-fold, resulting in a considerable 12-fold decrease in catalytic efficiency for proper Watson-Crick base pairing  . However pairing . This suD714A apoenzyme , with an RMSD value of 0.4 Å for the backbone atoms  [It is possible that the long-distance perturbations observed in the Polenzyme   as a seaAs expected, the ternary complex structure reveals an assembled active site with dGpnpp occupying the incoming dNTP binding pocket. The conformation of the template and primer strand is essentially identical to that observed in other RB69 Pol ternary complexes . Strikin2+) metal ion is observed coordinating the β and γ phosphates , resulting in a different crystal-packing environment. However, superimposition of both ternary complexes with the wild type structure reveals an identical fold, implying that the observed conformations are not affected by crystal contacts .43amam phage growth  that contacts the bound DNA. Residue E614 belongs to a putative RNA binding motif (dark purple).(TIFF)Click here for additional data file.Figure S2D714A mutant in comparison to the wild type polymerase.Pre-steady state kinetics of the correct nucleotide incorporation by PolRepresentative gels and the progress curves for four different dATP concentrations  are shown. The goodness of the curve fit (R2) and the calculated observed rate constants (kobs) are included for each nucleotide concentration.(TIFF)Click here for additional data file.Figure S3Microenvironment near the A714 residue in wild type and ternary complexes I and II.Stereo images showing the details of the network surrounding residue 714 in the wild type (A), ternary complex I (B) and ternary complex II (C). The distances between D411 and E686 are indicated as an arrow (wild type) and dashed lines .(TIFF)Click here for additional data file.Figure S4Overlay of the wild type ternary structure with ternary complexes I and II.C-α traces of the wild type , ternary complex I (cyan) and ternary complex II (green). The overall fold of the protein is identical in our structures and the wild type structure with RMSD values of 0.6 Å for 763 C-alpha atoms and 0.5 Å for 815 C-alpha atoms for ternary complexes I and II, respectively.(TIFF)Click here for additional data file.


Influenza A virus encodes for eleven proteins, of which HA, NA, NS1 and PB1-F2 have been implicated in viral pathogenicity and virulence. Thus, in addition to the HA and NA gene segments, monitoring diversity of NS1 and PB1-F2 is also important.55 out of 166 circulating influenza A strains (31 H1N1 and 24 H3N2) were randomly picked during 2007-2009 and NS and PB1-F2 genes were sequenced. Phylogenetic analysis was carried out with reference to the prototype strains, concurrent vaccine strains and other reference strains isolated world wide.Comparative analysis of both nucleotide and deduced amino acid sequences, revealed presence of NS gene with A/PR/8/34(H1N1)-like mutations  in both RNA-binding and effector domain of NS1 protein, and G63E, the HPAI-H5N1-like mutation in NEP/NS2 of five A/H1N1 strains of 2007 and 2009. NS1 of other A/H1N1 strains clustered with concurrent A/H1N1 vaccine strains. Of 31 A/H1N1 strains, five had PB1-F2 similar to the H3N2 strains; six had non-functional PB1-F2 protein (11 amino acids) similar to the 2009 pandemic H1N1 strains and rest 20 strains had 57 amino acids PB1-F2 protein, similar to concurrent A/H1N1 vaccine strain. Interestingly, three A/H1N1 strains with H3N2-like PB1-F2 protein carried primitive PR8-like NS gene. Full gene sequencing of PB1 gene confirmed presence of H3N2-like PB1 gene in these A/H1N1 strains.Overall the study highlights reassortment event involving gene segments other than HA and NA in the co-circulating A/H1N1 and A/H3N2 strains and their importance in complexity of influenza virus genetics. In contrast, NS and PB1-F2 genes of all A/H3N2 eastern India strains were highly conserved and homologous to the concurrent A/H3N2 vaccine strains suggesting that these gene segments of H3N2 viruses are evolutionarily more stable compared to H1N1 viruses. Influenza A virus (IAV) is a cytolytic virus that is responsible for significant morbidity and mortality worldwide per year. The genome of IAV consists of eight single-stranded, negative- sense viral RNA segments encoding the subunits of the transcriptase complex PB1, PB2, PA), nucleoprotein (NP), the matrix protein (M1), two non-structural proteins (NS1 and NS2/NEP), three integral membrane proteins (hemagglutinin (HA), neuraminidase (NA) and proton channel (M2)) and the eleventh gene product PB1-F2 which is encoded by an alternative ORF of segment 2  Figure . In addiThus, it can be hypothesized that NS and PB1-F2 gene segments of H3N2 viruses are evolutionarily more stable. This is in contrast to the analysis of HA and NA genes in the region, where comparative amino acid mutation rates were observed in both H1N1 and H3N2 strains . ReassorThe study was approved by the Institutional Ethics Committee, National Institute of Cholera & Enteric Diseases . The concerned members of this committee are: Hon'ble Justice Pinaki Ghosh, Kolkata High Court (Chairman), Dr. D. Mahalanabis (Member), Prof. A. C. Ghosh (Member), Prof. Biswapati Mukherjee (Member), Prof. Mrinmoy Ghosh (Member), Prof. Mrinal Kanti Chatterjee (Member), Mr. M. A. Wohab (Member), Mr. Amitrajit Ukil (Member), Mrs. Debolina Sarkar (Member), Prof. Subir Kumar Dutta (Member) and Dr. Phalguni Dutta (Member Secretary). Written informed consent was obtained from all participants involved in this study.Nasal and throat swabs were collected in Viral Transport Medium (VTM) from patients with influenza-like illness reporting in outpatient's ward of two referral hospitals; Dr. B.C. Roy Memorial Hospital for children (BCRMHC) and R.G. Kar Medical College and Hospital (RGKMCH) during 2007- 2009, as described previously . Of 166 Extraction of viral RNA from the clinical samples was carried out using commercially available QiaAmp Viral RNA Mini Kit  according to the manufacturer's instruction.To study the genetic diversity, full length NS, PB1, M1 and PB1-F2 encoding gene segments were amplified by RT-PCR using RevertAid™ First Strand cDNA Synthesis kit and DreamTaq™ DNA Polymerase  as per kit protocol with specific primers for the mentioned gene segments ,34. PCR et al. (1977), in ABI Prism automated 3100 DNA sequencer  using Big-Dye Terminator Chemistry. Sequences were compared with published cognate sequences of corresponding genes. DDBJ (DNA Data Bank of Japan) Clustal W system (version 1.83) was used for multiple sequence alignment of different nucleotide sequences of eastern India strains with other reference strains. Neighbour-Joining (N-J) trees were generated using pair-wise gap deletion, Maximum Composite Likelihood as distance measure and 1000 boot-strap replicates (generated with MEGA4) with boot strap values ≥70%.Genes were sequenced following dideoxynucleotides chain termination method of Sanger http://consurf.tau.ac.il/) .Accession numbers of all newly reported sequences obtained for full length non-structural (NS) gene, full length polymerase basic protein 1 (PB1) gene, PB1-F2 gene and matrix (M1) gene in this study are: [GenBank: The authors declare that they have no competing interests.MS and MCS conceived and designed experiments. JM and AG were involved in screening patients in hospitals and collecting clinical data. Experiments were executed by MS and SC. Analysis of results and drafting manuscript was done by MCS and MSC. SC, ACM and MCS contributed towards inputs in data interpretation, reviewing final manuscript and overall administrative responsibilities of the project. All authors read and approved the final manuscript.


SLC22A1/OCT1 mRNA seems to be downregulated, but systematic protein expression data are currently missing. Moreover, the underlying molecular mechanisms responsible for altered SLC22A1 expression in HCC are not fully understood. Therefore, we investigated the role of DNA methylation in the transcriptional regulation of the family members SLC22A1/OCT1, SLC22A2/OCT2 and SLC22A3/OCT3 in HCC.Organic cation transporters (OCTs) determine not only physiological processes but are also involved in the cellular uptake of anticancer agents. Based on microarray analyses in hepatocellular carcinoma (HCC), n = 71) using tissue microarray analyses, and the results were correlated with clinicopathological features. DNA methylation, quantified by MALDI-TOF mass spectrometry and gene expression of SLC22A1, SLC22A2 and SLC22A3 were investigated using fresh-frozen HCC (n = 22) and non-tumor adjacent liver tissues as well as histologically normal liver samples (n = 120) from a large-scale liverbank.Semiquantitative immunohistochemistry of SLC22A1 protein expression was performed in paired HCC and histological normal adjacent liver tissues (P < 0.0001). SLC22A1 expression was significantly inverse correlated with expression of the proliferation marker MIB1/Ki-67 . DNA methylation of SLC22A1 was significantly higher in HCC compared with non-tumor adjacent liver tissue and was lowest in histologically normal liver tissue. Methylation levels for SLC22A1 in combination with RASSF1A resulted in a specificity of > 90% and a sensitivity of 82% for discriminating HCC and tumor-free liver tissue.Based on tissue microarray analyses, we observed a significant downregulation of SLC22A1 protein expression in HCC compared to normal adjacent tissue (SLC22A1 is associated with downregulation of SLC22A1 in HCC and might be a new biomarker for HCC diagnosis and prognosis. Moreover, targeting SLC22A1 methylation by demethylating agents may offer a novel strategy for anticancer therapy of HCC.DNA methylation of  Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third most common cause of cancer-related death. Understanding of the molecular pathogenesis of HCC has improved, and some progress has been made in translating these findings into clinical practice . TherapeSLC22A1), OCT2 (SLC22A2) and OCT3 (SLC22A3) are extremely diverse in different tissues [SLC22A1 is expressed mainly in normal human liver, and SLC22A2 is predominately expressed in kidney [SLC22A1 mRNA expression is downregulated in HCC [SLC22A1, SLC22A2 and SLC22A3 have not been performed so far in HCC. Moreover, the underlying mechanisms responsible for altered expression of SLC22A1 in HCC compared with normal liver are poorly understood.Expression profiles among the three OCT family members OCT1  are given in Table S2 in Additional file n = 15) and adjacent non-tumor liver samples were obtained from the same institution [SLC22A1, SLC22A2 and SLC22A3 in normal and tumor tissues, commercial arrays including cDNA from 381 human tissues normalized to beta-actin were used . The study conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committee of the University Hospital Regensburg , by the ethics committee of University of Tuebingen  and the Charite . Written informed consent was provided by each patient.An overview about different sample sets used in the present study is given in Table S1 in Additional file titution . As addititution . For sysTotal RNA was extracted from HCCs and non-tumor tissue using the mirVana™ miRNA Isolation Kit . High quality liver RNA was extracted as previously described , and onlLocalization of OCTs in liver and HCC tissues was analyzed by immunofluorescence confocal laser scanning microscopy  for 10 minutes. Slides were incubated for 30 minutes at room temperature with the primary antibody  in an antibody diluent (Dako Cytomation). For negative control the primary antibody was omitted. The REAL-HRP system  was used for detection of the antibody binding before sections were counterstained with hematoxylin and mounted. For semiquantitative immunohistochemical assessment first, staining intensity was graded according to the following scoring system: 0 = negative, 1 = low, 2 = medium and 3 = high. As previously described [TMAs were processed as previously described ,18. SLC2escribed ,22, the escribed . EvaluatQuantitative DNA methylation analysis was performed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry , as described in the additional methods in Additional file P < 0.05. For a detailed description, see the additional methods in Additional file Statistical analyses were performed with R-2.13.0, PASW Statistics 17.0.2 and GraphPadPrism 4.03 . Statistical significance was defined as SLC22A1 mRNA is expressed most prominently in normal liver and expressed at significantly lower levels in HCC tissues , which was determined in HCC tissue samples by immunohistochemical staining as previously described  compared with adjacent tissue. Generally, SLC22A3 mRNA did not differ significantly between HCC and adjacent tissue .To confirm the finding of a significantly lower expression of SLC22A1 in HCC, mRNA levels of e Figure . Again, SLC22A1 in tumor tissue, we studied tissue-specific DNA methylation. DNA methylation of SLC22A1, SLC22A2, and SLC22A3 gene promoters was studied by gene-specific amplification of bisulfite-treated DNA followed by in vitro transcription and MALDI-TOF MS analysis [in silico for the presence of CpG islands. In contrast to SLC22A2 and SLC22A3, no CpG island was predicted in the promoter region of SLC22A1, which is supported by genome-wide CpG island mapping analysis [SLC22A1, 6 sites for SLC22A2 and 57 sites for SLC22A3  compared with normal livers without primary liver tumors (n = 100). The SLC22A2 gene region was again highly methylated  and decreased progressively from adjacent to histologically normal tissue of patients without primary liver tumors from the IKP-liverbank  compared with normal tissues derived from patients with HCC  for both genes.To investigate whether DNA methylation status of SLC22A1 mRNA expression is decreased in HCC [SLC22A1 mRNA is expressed most prominently in liver and at significantly lower levels in HCC tissues  are substrates for OCTs -5,30. Ind in HCC -11. Thisrognosis ,31,32. Srognosis . However-/- knockout mice [SLC22A1 mRNA expression and poorer prognosis [Therefore, we investigated first protein expression in a set of 71 HCC tissues and adjacent non-tumor liver tissues by semiquantiative immunohistochemistry. These experiments indicated significantly decreased expression of SLC22A1 protein in HCC Figure . When weout mice  and micrrognosis .SLC22A1 promoter region. This prompted us to systematically quantify DNA methylation levels of SLC22A1, SLC22A2, and SLC22A3 in the surrounding 5' UTRs of the target genes by MALDI-TOF MS. Consistent with both low expression of mRNA and protein, SLC22A1 methylation was significantly increased in HCC tissues compared with adjacent non-tumor . As a consequence, for instance, pretreatment with decitabine may increase the intracellular concentration of the SLC22A1 substrate cisplatin, thereby resulting in enhanced cytotoxicity. Of course, treatment with decitabine will not only lead to re-expression of OCTs and additional factors might be important for cisplatin efficacy, for example, the expression of the copper transporter CTR1, which alters sensitivity to cisplatin [ABCB1, encoding P-glycoprotein, is hypomethylated in several cancer cell lines, thereby leading to a multidrug-resistance phenotype compared to drug-sensitive cell lines [Since OCTs are important targets for various therapeutic agents, such as metformin or the platinum drugs cisplatin or picoplatin ,40,41, tisplatin . In addiisplatin ). For inll lines . MoreoveOur study has some limitations that need to be considered. First, our study design of HCC samples was retrospective and, for instance, confounding of SLC22A1 expression cannot be excluded with certainty. Second, the number of HCC samples was small, and the relevance of the correlation analyses of distinct clinicopathological factors  with DNA methylation or expression is thus limited and needs further study. Nevertheless, our findings indicate for the first time that downregulation of the uptake transporter SLC22A1 in HCC is associated with DNA methylation as an epigenetic phenomenon. This finding has potential consequences for the diagnosis of HCC and for future therapeutic concepts.SLC22A1 gene is associated with downregulation of SLC22A1 expression in HCC. Moreover, SLC22A1 methylation decreased from high levels in HCC to lower levels in histological tumor-free adjacent liver tissue and was lowest in tumor-free liver tissue not derived from patients with HCC. Therefore, early detection of aberrant DNA methylation of SLC22A1 might be beneficial for the diagnosis of HCC or for estimating risk for HCC in following patients at risk. Since platinum drugs are substrates of SLC22A1, the modulation of gene expression by pretreatment with demethylating agents may offer novel therapeutic options in HCC, which has to be proven in further studies.SLC22A1 protein expression is significantly decreased in HCC compared with tumor-free adjacent liver tissue. DNA hypermethylation of individual CpG sites within the HCC: hepatocellular carcinoma; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; OCT: organic cation transporter; PCR: polymerase chain reaction; SLC: solute carrier; TMA: tissue microarray; UTR: untranslated region.The authors declare that they have no competing interests.MS was responsible for study design. ES was responsible for study design and collected, analyzed and interpreted data. Data were collected, analyzed and interpreted by ATN, CH and SK. Statistical analysis was performed by SW. All other authors contributed samples for the study, and/or contributed to drafting the manuscript. All authors have read and approved the manuscript for publication.Additional methods, tables and figures. Methods: additional description of methods. Immunofluorescence microscopy of tissue samples was performed as previously described [RASSF1A was analyzed as described previously [P-values were adjusted according to Holm's multiple testing correction procedure [OCT1/SLC22A1, OCT2/SLC22A2 and OCT3/SLC22A3. Figure S1: differences in the SLC22A1 immunohistochemical score between the corresponding adjacent non-tumor tissue and HCC tissue, regarding the underlying etiology for HCC. Figure S2: Ki-67/MIB1 data for HCC tumor samples. Figure S3: association between SLC22A1 protein expression and tumor grade or tumor stage. Figure S4: DNA methylation profiles of SLC22A2 and RASSF1A in HCC and adjacent non-tumor tissue. Figure S5: association between SLC22A1 mRNA expression or DNA methylation and tumor stage or histological tumor grade. Figure S6: DNA methylation profiles of SLC22A1, SLC22A2, and SLC22A3 in non-tumor  liver tissues, as well as in non-tumor liver tissue derived from patients with HCC.escribed ,45. Primescribed . DNA meteviously . Statisteviously . Where irocedure . Table SClick here for file


Pandemic influenza A(H1N1) 2009 virus was first detected in Japan in May 2009 and continued to circulate in the 2010–2011 season. This study aims to characterize human influenza viruses circulating in Japan in the pandemic and post-pandemic periods and to determine the prevalence of antiviral-resistant viruses.Respiratory specimens were collected from patients with influenza-like illness on their first visit at outpatient clinics during the 2009–2010 and 2010–2011 influenza seasons. Cycling probe real-time PCR assays were performed to screen for antiviral-resistant strains. Sequencing and phylogenetic analysis of the HA and NA genes were done to characterize circulating strains.In the pandemic period (2009–2010), the pandemic influenza A(H1N1) 2009 virus was the only circulating strain isolated. None of the 601 A(H1N1)pdm09 virus isolates had the H275Y substitution in NA (oseltamivir resistance) while 599/601 isolates (99.7%) had the S31N substitution in M2 (amantadine resistance). In the post-pandemic period (2010–2011), cocirculation of different types and subtypes of influenza viruses was observed. Of the 1,278 samples analyzed, 414 (42.6%) were A(H1N1)pdm09, 525 (54.0%) were A(H3N2) and 33 (3.4%) were type-B viruses. Among A(H1N1)pdm09 isolates, 2 (0.5%) were oseltamivir-resistant and all were amantadine-resistant. Among A(H3N2) viruses, 520 (99.0%) were amantadine-resistant. Sequence and phylogenetic analyses of A(H1N1)pdm09 viruses from the post-pandemic period showed further evolution from the pandemic period viruses. For viruses that circulated in 2010–2011, strain predominance varied among prefectures. In Hokkaido, Niigata, Gunma and Nagasaki, A(H3N2) viruses (A/Perth/16/2009-like) were predominant whereas, in Kyoto, Hyogo and Osaka, A(H1N1)pdm09 viruses (A/New_York/10/2009-like) were predominant. Influenza B Victoria(HA)-Yamagata(NA) reassortant viruses (B/Brisbane/60/2008-like) were predominant while a small proportion was in Yamagata lineage. Genetic variants with mutations at antigenic sites were identified in A(H1N1)pdm09, A(H3N2) and type-B viruses in the 2010–2011 season but did not show a change in antigenicity when compared with respective vaccine strains. In late March to early April of 2009, a novel influenza virus of swine origin emerged in humans in Mexico and the USA and rapidly spread worldwide, prompting the WHO to declare an influenza pandemic In Japan, the first pandemic influenza case was reported in May 2009 In this study, we described the circulation patterns and genetic characteristics of viruses that circulated during the pandemic and post-pandemic periods. We focused on the comparison of pandemic influenza A(H1N1) viruses collected in Japan in the 2009–2010 and 2010–2011 seasons. In addition, we performed genetic analysis on A(H3N2) and influenza B viruses that cocirculated with the A(H1N1)pdm09 viruses in the 2010–2011 season.Seven hundred thirty three (733) patients with influenza-like illness who visited outpatient clinics in six prefectures in Japan  between July 2009 and February 2010, and 1,278 patients in seven prefectures  from December 2010 to March 2011 participated in our study. Among these patients, 4 from the 2009–2010 period and 14 from the 2010–2011 period were hospitalized after the initial assessment of the severity of infection at the outpatient clinic (data not shown). All of the hospitalized patients from the two periods fully recovered. There were no patients with lethal infection.Of the 733 respiratory specimens that were collected from patients with influenza-like illness in the 2009–2010 season, 601 A(H1N1)pdm09 viruses (85.4%) were isolated, as confirmed by the real-time PCR assays . A part Of the 1,278 respiratory specimens that were collected in the 2010–2011 season 972 (76.1%) viruses were isolated. There was cocirculation of different types and subtypes of influenza viruses in this season and were distributed as follows: 414 A(H1N1)pdm09 (42.6%), 525 A(H3N2) (54.0%) and 33 influenza B (3.4%) viruses . There wThere was variability in the influenza virus subtype predominance in each prefecture. A(H3N2) viruses were predominant in Hokkaido, Niigata and Gunma prefectures in the northern area of Japan, as well as in Nagasaki in the south. A(H1N1)pdm09 viruses were predominant in prefectures in the Kansai area of western Japan: Kyoto, Hyogo and Osaka .Among influenza B viruses, 31 out of 33 (93.9%) isolates belong to the Victoria lineage and 2 out of 33 (6.1%) isolates belong to the Yamagata lineage, according to cycling probe real time PCR results .All of the 601 A(H1N1)pdm09 viruses from the 2009–2010 period showed possession of H275 (wild-type) in the neuraminidase (NA) by screening with cycling probe real-time PCR and/or by genetic sequencing. These results suggested possible susceptibility to oseltamivir among the collected specimens.In the 2010–2011 season, 2 out of 414 (0.5%) isolates harbor the H275Y mutation in NA as shown by cycling probe assay and sequencing. These viruses were from primary respiratory specimens of patients with no prior treatment of oseltamivir.All of the 1,015 A(H1N1)pdm09 isolates in the two seasons were tested for the presence of M2-S31N substitution that confers resistance to amantadine using the cycling probe real time PCR method. All but two viruses possessed the M2-S31N change: A/Nagasaki/09N079/2009 and A/Kyoto/09K084/2009 had serine (AGT) at position 31 (as confirmed by sequencing of the transmembrane domain of the M2 gene) suggesting susceptibility to amantadine. All A(H1N1)pdm09 viruses from the 2010–2011 season harbor the S31N mutation in M2 .Among the A(H3N2) isolates, 520/525 (99.0%) had the M2-S31N substitution according to cycling probe real time PCR results , suggestSequence and phylogenetic analyses of the hemagglutinin (HA) and neuraminidase (NA) genes of 81 pandemic influenza A(H1N1) viruses from the 2009–2010 season and 55 from the 2010–2011 season were performed.In the 2009–2010 season, 80 out of 81 (98.8%) isolates had the S203T mutation in the HA that characterizes cluster 2 viruses A(H1N1)pdm09 viruses from the 2009–2010 season were located near the trunk of the HA phylogenetic tree. Comparison of the HA gene of the A(H1N1)pdm09 viruses with the vaccine strain  showed 2–14 amino acid mutations. Among these mutations, 5 are located in antigenic sites Ca  and Cb (L70F) An amino acid change from aspartic acid (D) to glutamic acid (E) was observed at residue 222 in the HA in 6 isolates from the 2009–2010 season. However, this amino acid substitution was not found in 2010–2011 isolates. Glycine (G) or asparagine (N) mutation at amino acid residue 222, which were previously reported to be associated with severe illness The NA phylogenetic tree was generally congruent with that of the HA . CompariTwo (2) oseltamivir resistant strains with the H275Y mutation in the NA collected in 2010–2011 were located in the same branch in the NA phylogeny. However, these two strains were found in different branches in the HA phylogeny .Two (2) amantadine-sensitive strains collected in 2009–2010 were distributed in different branches in both HA and NA phylogenies . In the The hemagglutinin (HA) and neuraminidase (NA) genes of 71 A(H3N2) viruses from the 2010–2011 season were analyzed. The HA and NA phylogenies showed that 48 isolates belonged to the A/Perth/16/2009 (Perth16) clade and 23 isolates belonged to the A/Victoria/208/2009 (Vic208) clade . When coMutations in the NA of A(H3N2) viruses ranged from 4–17 amino acid residues when compared with the vaccine strain . Of thesGenetic analysis of the HA and NA sequences of 29 influenza B viruses was performed. The HA phylogeny showed that 27 viruses belonged to the Victoria lineage; 26 viruses were closely related to the vaccine strain, B/Brisbane/60/2008 (Brisbane60) and 1 virus (B/Niigata/10F478/2011) was closely related to an earlier vaccine strain, B/Malaysia/2506/2004. The remaining 2 viruses belonged to the Yamagata-lineage and were B/Bangladesh/3333/2007-like (Bangladesh3333) . Within The NA phylogeny showed that all viruses belonged to the Yamagata-lineage but the clustering into two distinct groups was similar to that of the HA . Within http://idsc.nih.go.jp/yosoku/Flu/2010Flu/Flu10_2.html, [Japanese]).The pandemic influenza A(H1N1) virus first appeared in Japan in May 2009 and reached the pandemic stage in June 2009 http://idsc.nih.go.jp/iasr/influ-e.html) in Japan.Influenza A(H3N2) was the predominant influenza type-A virus that caused illness in the 2010–2011 season in our study. Strain predominance varied among prefectures but geographic clustering was evident. Prefectures in northern Japan had 4 to 7 times more A(H3N2) viruses detected than A(H1N1)pdm09 viruses. Prefectures in the Kansai area  had about 2 to 4 times more A(H1N1)pdm09 viruses detected than A(H3N2) viruses. Influenza virus peak activity also varied among the type-A viruses. The A(H1N1)pdm09 virus activity peaked in late January whereas A(H3N2) virus activity peaked in mid-February. We could not assess the peak of influenza B due to the termination of the study in early March. According to the National Influenza Surveillance in Japan, more influenza B viruses than influenza A viruses were detected after week 12 until week 20 (In the first year of pandemic (2009–2010), we did not detect any oseltamivir-resistant A(H1N1)pdm09 viruses. In the following season, we detected two (2) A(H1N1)pdm09 viruses  that possessed the H275Y substitution in the NA. These two isolates came from patients who had not received oseltamivir treatment, live in different areas and were infected at different times. There was no epidemiological link established between the two cases. These naturally-occurring oseltamivir-resistant A(H1N1)pdm09 viruses in untreated patients were previously reported in Hong Kong and Vietnam 50 (results not shown). DNA sequencing of the M gene of these two viruses showed that the sensitivity was due to a spontaneous mutation in the M2 segment and was not due to a reassortment with an amantadine-sensitive gene segment. From a CDC report The A(H1N1)pdm09 virus contains the M gene of the Eurasian swine lineage  and has the genetic marker (S31N in M2) for resistance to amantadine Based on the HA and NA sequence data of viruses collected within the pandemic period from July 2009 to February 2010, A(H1N1)pdm09 viruses are closely related to each other and to the vaccine strain, A/California/07/2009 and belong to cluster 2. The signature amino acid changes, HA-S203T and NA-N248D, of cluster 2 viruses that differentiate it from early pandemic cluster 1 viruses continued to persist throughout the duration of the pandemic and into the 2010–2011 season. One isolate, A/Nagasaki/09N083/2009, collected in November 2009 has a cluster 1 HA but a cluster 2 NA. This virus belongs to cluster 1.2 which mostly contained viruses from Japan isolated during the early phase of the pandemic Sequence analysis of A(H1N1)pdm09 viruses of the 2010–2011 season showed further evolution from viruses of the 2009–2010 season. These viruses were closely related to the previous season strains, A/Nagasaki/09N114/2009 and A/Fukushima/09FY090/2010. Viruses with the amino acid substitutions S185T and A197T had the greatest expansion and geographic spread. Structural analysis showed that S185T is found in the antigenic site Sb, which is located on the globular head of the HA . The amiThe amino acid substitutions HA-D222G/N in A(H1N1)pdm09 viruses were previously associated with severe infection A new amino acid mutation in the NA of A(H1N1)pdm09 viruses associated with reduced susceptibility to neuraminidase inhibitors were recently reported. A mild reduction in oseltamivir and zanamivir susceptibility was observed in viruses from Oceania and Southeast Asia with the S247N (serine to asparagine) mutation Our results showed the prevalence of amantadine-resistant A(H3N2) viruses is high (99%) in the 2010–2011 season. The trend of high prevalence of amantadine resistance among A(H3N2) viruses was observed in Japan after the 2005–2006 season in Japan, reaching 100% prevalence in the 2008–2009 season Genetic analysis showed there were two groups of A(H3N2) viruses detected in the 2010–2011 season, A/Perth/16/2009-like and A/Victoria/208/2009-like Our sequence analysis showed additional mutations in the HA of 2010–2011 viruses from previously circulating strains. Several amino acid substitutions in the putative antigenic sites in HA and NA were found in Perth16 and Victoria208 isolates when compared with the vaccine strain, A/Perth/16/2009. The mutations in the HA may have contributed to the reduced antigenicity against the vaccine strain of some Vic208 clade viruses There was cocirculation of Victoria-lineage and Yamagata lineage influenza B viruses in the 2010–2011 season. Most of the influenza viruses isolated belong to the Victoria lineage in the HA and were similar to the vaccine strain, B/Brisbane/60/2008. These reassortant Victoria lineage viruses that had a Yamagata lineage NA have been stably circulating worldwide since its emergence in 2002 In summary, all three predominantly circulating viruses in the 2010–2011 season demonstrated genetic variability from the previously circulating strains but were closely related to the 3 strains recommended by the WHO to be included in the vaccine for the 2011–2012 influenza season in the northern hemisphere: an A/California/7/2009-like virus, an A/Perth/16/2009-like virus and a B/Brisbane/60/2008-like virus It remains to be seen whether the pandemic A(H1N1) 2009 virus will follow the path of other pandemic viruses such as that of the Asian influenza A(H2N2) virus which survived shortly in the human population and disappeared 11 years after its emergence Eligible patients to this study were those who visited outpatient clinics presented with influenza-like illness symptoms  and had not been treated with amantadine, oseltamivir or zanamivir within the previous four weeks. The study period was between July 30, 2009 and March 22, 2011. The Niigata University Division of International Health  supplied viral transport media to clinicians participating in the Japanese Influenza Collaborative Study Group . Clinicians performed an influenza rapid diagnostic test, mainly by using the Quick-Ex Flu kit , on the first respiratory specimen, and collected a second respiratory specimen regardless of the rapid test results. The samples were stored in viral transport media for ≤72 hours at 4°C before shipment to our laboratory. Informed written consent was obtained from the patient or the patient’s guardian prior to specimen collection. The study was approved by the medical faculty ethics committee of the Niigata University Graduate School of Medical and Dental Sciences.About 100 µl of nasopharyngeal sample was inoculated onto Madin-Darby canine kidney (MDCK) cells. MDCK cell lines were kindly provided by Dr. Hidekazu Nishimura of Virus Center, Sendai National Medical Center, Miyagi Prefecture, Japan and were maintained as previously described Total RNA was extracted from 100 µl of clinical specimens and from 100 µL of viral culture using Extragen II kit  following manufacturer’s instructions. First-strand cDNA synthesis was performed using influenza A and B universal primers Cycling probe real-time PCR screening for mutations in M2 and NA genes that confer resistance to amantadine and oseltamivir, respectively, was performed on all 1,540 influenza A virus isolates (2009–2010 and 2010–2011) using fluorescent-labeled chimeric RNA-DNA probes and RNaseH . This assay utilizes a single nucleotide polymorphism (SNP) which can simultaneously detect amantadine-sensitive (S31) and amantadine-resistant (S31N) viruses as previously described Samples that were negative for the screening methods for A(H1N1)pdm09 and A(H3N2) were tested using a cycling probe real-time PCR method that can distinguish Victoria-lineage and Yamagata-lineage influenza B viruses according to the HA gene sequence.For the influenza B cycling probe real time PCR reaction, a commercially available cycling probe real time PCR kit, TaKaRa CycleavePCR®Core Kit  was used. The PCR reaction was prepared according to the manufacturer’s instructions and was supplemented with 5 pmol of each primer , 2.5 pmol of the carboxyfluorescein (FAM)-labeled probe (Victoria HA gene-specific), 2.5 pmol of the carboxy-X-rhodamine (ROX)-labeled probe (Yamagata HA gene-specific), and 1 µL of viral cDNA in a 25 µl reaction volume. PCR amplification and fluorescence detection were performed on Thermal Cycler Dice Real Time PCR System TP800 . Cycling conditions were as follows: denaturation at 95°C for 10 seconds followed by 40 cycles of denaturation at 95°C for 5 seconds, primer annealing at 57°C for 10 seconds, and extension and subsequent detection of fluorescence at 72°C for 15 seconds (primers and probes information are available on request).http://www.mbio.ncsu.edu/BioEdit/). Phylogenetic analysis was performed using MEGA 4.0 software The HA and NA genes of 136 A(H1N1)pdm09, 71 A(H3N2) and 29 influenza B viruses, as well as the M gene of 6 A(H1N1)pdm09 viruses from the 2009–2010 season, were amplified using gene-specific primers. PCR products were purified using MSBP Spin PCRapace kit  and directly sequenced. Sequencing reactions were carried out using BigDye Terminator v3.1 cycle sequencing kit  and sequencing products were run on an ABI Prism 3100 Genetic Analyzer. Sequences were assembled using Lasergene SeqMan Pro package version 7.2.1  and assembled sequences were edited using BioEdit software (www.gisaid.org) and from GenBank (www.ncbi.nlm.nih.gov/genbank).GenBank accession numbers of the HA and NA sequences of Japanese A(H1N1)pdm09 strains from the 2009–2010 season are CY066017–CY066182 and from the 2010–2011 season are JN790349–JN790440. The accession numbers for the HA and NA sequences of A(H3N2) strains from the 2010–2011 season are JN790441–JN790581, and for the influenza B strains are JN790295–JN790348. Vaccine strain sequences and supplemental sequences used for the phylogenetic trees were obtained from the GISAID EpiFlu™ Database (http://www.pdb.org) http://www.pymol.org).HA and NA amino acid sequences of influenza virus isolates in Japan were compared with the vaccine strains. Identified amino acid substitutions were mapped to reported HA antigenic sites Figure S1Phylogenetic analysis of the M gene of amantadine-sensitive A(H1N1)pdm09 viruses. Trees were constructed using the Neighbor-Joining method. Numbers at the nodes indicate confidence levels of bootstrap analysis with 1,000 replicates as percentage value. Human A(H1N1)pdm09 strains from the present study are in bold. Amantadine-sensitive strains (AmS) are indicated with filled triangles (▴). Other viruses included in the analysis were based on the study by Vijaykrishna D et al. (2010) (TIF)Click here for additional data file.Table S1Hemagglutination inhibition (HI) titer of selected A(H1N1)pdm09 viruses from the 2010–2011 season.(XLS)Click here for additional data file.Table S2Hemagglutination inhibition (HI) titer of selected A(H3N2) viruses from the 2010–2011 season.(XLS)Click here for additional data file.Table S3Hemagglutination inhibition (HI) titer of selected influenza B viruses from the 2010–2011 season.(XLS)Click here for additional data file.