V(D)J recombination is the assembly of gene segments at the antigen receptor loci togenerate antigen receptor diversity in T and B lymphocytes. This process is regulated,according to defined developmental programs, by the action of a single specificrecombinase complex formed by the recombination antigen gene (RAG-1/2) proteinsthat are expressed in immature lymphocytes. V(D)J recombination is strictly controlledby RAG-1/2 accessibility to specific recombination signal sequences in chromatin atseveral levels: cellular lineage, temporal regulation, gene segment order, and allelicexclusion. DNA cleavage by RAG-1/2 is regulated by the chromatin structure,transcriptional elongation, and three-dimensional architecture and position of theantigen receptor loci in the nucleus. Cis-elements specifically direct transcription andV(D)J recombination at these loci through interactions with transacting factors that formmolecular machines that mediate a sequence of structural events. These events openchromatin to activate transcriptional elongation and to permit the access of RAG-1/2 totheir recombination signal sequences to drive the juxtaposition of the V, D, and Jsegments and the recombination reaction itself. This chapter summarizes the advancesin this area and the important role of the structure and position of antigen receptor lociwithin the nucleus to control this process. Development of B and T lymphocytes occurs through a series of well-defined differentiation stages initiating from a common hematopoietic stem cell. Each of these differentiation stages involves the activation and repression of antigen receptor loci that influence cellular identity. Expression of these loci in immature lymphocytes requires the activation and silencing of genomic DNA rearrangements that are regulated through the accessibility of chromatin, transcriptional elongation, three-dimensional structure, and nuclear positioning during cell development [The immune system is considered one of the best models to study the molecular mechanisms of epigenetic control of cellular differentiation elopment , 2. In fV(D)J recombination is initiated through the action of the protein products of the recombination activating gene (RAG) 1 and 2; together, RAG-1 and RAG-2 form a specific endonuclease in immature lymphoid cells. The RAG-1/2 complex is responsible for the double-strand DNA cleavage between the segments that allows recombination through the recognition of specific recombination signal sequences (RSSs) that flank them ; these RIg and Tcr loci (which encode the variable regions responsible for antigen recognition) contains the V, D (only in some loci), and J gene segments  and lineage (B or T) specific regulation is defined by controlled RAG-1/2 accessibility to the specific locus chromatin in B and T lymphocytes precursors [Igh, Igk, and Igl) only rearrange in B-lymphocyte precursors, and the T-cell receptor loci  rearrange exclusively in T lymphocyte precursors. Additionally, there is temporal control of V(D)J recombination that ensures that this process occurs in a developmental stage-specific manner during lymphocyte development. During T-lymphocyte development, Tcrb, Tcrd, and Tcrg loci rearrange earlier than Tcra locus. Similarly, during B lymphocyte development, Igh rearranges earlier than the Igk and Igl loci. Furthermore, there is an additional developmental control imposed on sets of gene segments within each antigen receptor locus. For example, D-to-J rearrangements precede V-to-DJ rearrangements at the Tcrb and Igh loci. This locus-, lineage-, temporal-, and gene segment order-specific regulation of V(D)J recombination is mediated through the control of RSS accessibility to the RAG-1/2 proteins. Hence, the chromatin imposes a barrier to RAG-1/2 accessibility that is controlled through strict epigenetic control, which is dependent on the specific antigen receptor locus, gene segment, cellular lineage, and developmental stage. This is the basis for the accessibility model proposed 25 years ago by Yancopoulos and Alt [H gene segment recombination at the Igh locus coincided with VH germline transcription (the process of transcription of sterile transcripts at an un-rearranged locus originating from V-associated promoters) during B lymphocyte development [H gene segments reflects an increase in the accessibility of the VH gene segments to both the transcriptional and recombinational machineries (RNA polymerase II (RNAPII) and RAG-1/2 proteins, resp.). Since then, germline transcripts initiating at V, D, and J gene segments have been found to developmentally coincide with the activation of V(D)J recombination at each antigen receptor locus [H gene segments that correlates with VH to DJH recombination has also been reported [Ig or Tcr locus is equipped with at least one transcriptional enhancer in the vicinity of the constant region and numerous promoters associated with V, D, and J gene segments (in vitro studies have demonstrated that assembly of RSSs into nucleosomes inhibits V(D)J recombination [The 5′ region of the segments  that areecursors . This co and Alt . These ielopment . Based oor locus –9. In adreported . In agresegments . The essbination –20, suppbination , 21–23. bination . In thisbination . Hence, bination –30. in situ hybridization (3D-FISH) experiments using distal DNA probes, this contraction correlates with transcription and V(D)J recombination [Rag−/− mice. These contractions could be mediated by interactions between regulatory regions and/or by specific nuclear structures. Enhancer-promoter interactions are thought to direct the long-distance communications −/− and Tcrd locus enhancer (Eδ)−/− thymocytes revealed no significant differences between the cell types [Tcra locus contraction and RSSs synapse, but locus contraction might facilitate both enhancer-promoter interactions and RSSs synapse that are required for transcription and V(D)J recombination. These data suggest that a preexisting conformation of the locus that is mediated by an enhancer-independent mechanism may promote the enhancer-promoter interactions necessary to activate transcription and V(D)J recombination of the distant gene segments [Igh locus configuration, but it is not known how they do this [Igh and Igk loci in immature B lymphocytes [Igh locus in developing B lymphocytes [It is interesting that the antigen receptor loci undergo a process of gene contraction (juxtaposition of V and D-J regions), which is strictly regulated during development. As demonstrated by three-dimensional fluorescence bination , 31–33. ications . Comparasegments , 33. Hen do this –38. Addi do this –44. In fphocytes , 46. Conphocytes . The preIgk, Tcrg, and Tcrd loci [−/− mice displayed defective transcription and recombination of a subset of Vκ gene segments [γJγ recombination at the Tcrg locus in response to IL-7 [Tcr and Ig loci or inclusion of mutations at motifs for required transcription factors within these cis-elements inhibit both transcription and V(D)J recombination at each locus [The finding that germline transcription at a given antigen receptor locus occurs concomitantly with its recombination  suggestscrd loci , 49; Ocasegments ; Stat5 i to IL-7 , 52; andch locus . α-to-Jα recombination at the Tcra locus [α/δ gene segments in a 1 Mb region at the 5′ end of the locus and 61 Jα gene segments in a 65 kb region at the 3′ end of the locus [α-to-Jα recombination events depend on both Eα, located at the 3′ end of the locus [α gene segments [α56 gene segment (56R) in the endogenous mouse locus to block transcription originating at the upstream TEA promoters [α gene segments located immediately downstream of the terminator sequence. Thus, these experiments clearly demonstrated that transcriptional elongation by RNAPII is necessary for creating accessible chromatin for the RAG-1/2 proteins to initiate Tcra recombination. For many years, it was not clear whether germline transcription was merely a side effect of chromatin accessibility generated by the activation of enhancers and promoters or whether it was causal in the V(D)J recombination process itself. In an elegant and definitive study, Abarrategui and Krangel have proven that germline transcription is a key developmental regulator of accessibility for Vra locus . This lohe locus   dependent promoter tightly associated with the Dβ1 segment to confer accessibility to this segment [β1 promoter with Eβ to deliver SWI/SNF chromatin remodeling complexes that results in a decrease of nucleosome occupancy at the Dβ1 gene segment [in vivo because they are positioned far away from the promoters. Although instances of recombination in the apparent absence of transcription have also been reported, credible explanations for each of them can be found. For example, isolated nuclei of lymphocytes from cription ; howeversegments ; howeversegments , the end segment , 59, and segment , 30, 60.α cluster in TEA-T and 56R mice J recombination, as has been shown for class switching at the Igh locus, is not expected because the RAG-1/2 substrate is double strand DNA. More recent experiments with the TEA-T and 56R mouse models have definitively demonstrated that chromatin accessibility to the RAG-1 protein is mediated by transcriptional elongation itself . In this56R mice  inhibiteTcra locus requires that the elongation machinery travels or has traveled through the RSS's DNA to allow the RAG-1/2 proteins to access to the RSS's chromatin. However, the precise molecular mechanism by which transcriptional elongation directs the process of VαJα recombination is not known. Chromatin structure imposes significant obstacles on the passage of the RNAPII through the DNA. Because the elongation of transcription is associated with the transient disruption of nucleosome structure [Tcra locus causes covalent histone modifications related to opening of chromatin [α gene segment, Jα61 [Thus, Abarrategui and Krangel's studies , 5 have tructure  and becatructure , it is pnt, Jα61 . Gene transcription is initiated by the binding of transcription factors to promoters and enhancers . The binTcra locus [αJα recombination through RAG-1/2 recruitment mediated by transcriptional elongation [Abarrategui and Krangel observed that H3ac, H3K4me3, H3K4me2, and H3K36me3 were significantly reduced at the 3′ end of the terminator introduced immediately downstream of the TEA promoter at the ra locus  J recombination angement . Additioangement . Furtherbination , 82. Finbination –86. ThesIg and Tcr loci in the nucleus has an essential role in directing V(D)J recombination between distantly located gene segments. It is now accepted that, in addition to transcriptional competence, a particular locus or allele must move away from repressive chromatin to allow distant RSSs to form a recombinational synapse through RAG-1/2 binding that initiates V(D)J recombination [Ig and Tcr loci move away from repressive compartments such as the nuclear periphery or pericentric heterochromatin when they undergo recombination; the loci then reassociate with them following recombination [Igh, Igk, and Tcrb loci with repressive nuclear compartments seem to be responsible for the establishment of allelic exclusion [Over the last ten years, it has become clear that the position of bination . It is nbination , 87. Furxclusion , 86, 88.Igh and Tcrb loci in pro-B/pre-B and pro-T/pre-T lymphocytes, respectively, even when V-to-DJ recombination is inhibited and one or both alleles are associated with repressive compartments in both stages [Igh locus is tethered to the nuclear membrane through the distal VH region cluster, whereas the DHJH region is located away [Igh and Tcrb D and J gene segments in pro-B/T and pre-B/T lymphocytes might be consequence of RSS accessibility due to the spatial orientation of these loci within the nucleus [H and Vβ gene segment transcription and accessibility are reduced in the transition from pro-B/T to pre-B/T lymphocytes, respectively, and hence both parameters do seem to correlate with allelic exclusion of Igh and Tcrb loci at pre-B/T lymphocytes [Igh and Tcrb loci in pre-B/T lymphocytes, but not in pro-B/T lymphocytes, operates primarily on VH and Vβ gene segment accessibility, respectively. The different chromatin structure at the VH and Vβ gene segments in the two stages indicates clear differences about what might be happening in pro-B/T versus pre-B/T lymphocytes [Igk locus, which is also relocated during B lymphocyte development [Igk transgenes to centromeric chromatin and inhibits Vκ-to-Jκ recombination [Igk allele away from the pericentromeric heterochromatin [Tcrb locus, the helix-loop-helix protein, E47, is a good candidate to direct the interaction of this locus with pericentromeric heterochromatin since its dosage is rate-limiting with regard to V(D)J recombination and forced E47 expression interferes with pre-TCR-mediated feedback inhibition [Ig alleles occurs during recombination and is mediated by RAG-1/2 binding [ The mechanism by which locus association with repressive compartments inhibits V(D)J recombination without inhibiting transcription remains unknown. RAG-1/2 binding to D and J gene segments is robust at the h stages , 31, 81.ted away . Thus, p nucleus , 85. It phocytes –91. Thesphocytes –91. Idenbination ; anotherhromatin . In the hibition . Additiohibition . Consist binding , 96. Fur binding . Hence, s-factors that regulate V(D)J recombination in vivo. V(D)J recombination is essential for the development of adaptive immune responses in vertebrates. In developing lymphocytes, V(D)J recombination is subjected to very tight spatial and temporal regulation. The regulation of this process is very complex and involves nuclear dynamics and changes in higher-order chromatin architecture to create gene segment accessibility to RAG-1/2 proteins. Active chromatin is bound by RAG-2 through interactions with specific H3K4me3, whereas RAG-1 binds to accessible RSSs derived from transcriptional elongation in large loci. The correlation between transcription and V(D)J recombination in both the recombined and allelic excluded antigen receptor loci led to the studies that established that the differential positioning of such loci at transcriptionally repressive nuclear regions might be responsible for allelic exclusion. Allelic association with the repressive nuclear compartments can inhibit V(D)J recombination by a mechanism other than transcription or RAG-1/2 accessibility to chromatin. Future experiments should be focused on identifying the cis-elements and tran


The LSMO nanolayer with In2O3 epitaxial buffering has a (110) preferred orientation. However, the nanolayer without buffering shows a highly (100)-oriented texture. Detailed microstructure analyses show that the LSMO nanolayer with In2O3 epitaxial buffering has a high degree of nanoscale disordered regions (such as subgrain boundaries and incoherent heterointerfaces) in the film. These structural inhomogeneities caused a low degree of ferromagnetic ordering in LSMO with In2O3 epitaxial buffering, which leads to a lower saturation magnetization value and Curie temperature, and higher coercivity and resistivity.MnO There devices ,4,5. To 2O3 epitaxial underlayering. These films did not suffer lattice stress. These results enable an analysis of the correlation between nanoscale crystal imperfections and manganite nanofilm physical properties.High-quality epitaxial manganite films with specific orientations are essential for the next-generation of microelectronic and magnetic devices. However, single-crystalline perovskite oxide substrates are expensive, and a large diameter substrate is currently technologically unavailable. These factors hinder the practical application of epitaxial LSMO films in the electronic industry ,6. Two fc-axis-oriented sapphire substrates with and without 40-nm-thick In2O3 (222) epitaxial buffering. The deposition of the In2O3 epitaxy layers and LSMO nanolayers was performed using a radiofrequency magnetron-sputtering system. During the deposition, the substrate temperature for the thin-film growth of the In2O3 epitaxy and LSMO nanolayer was kept at 600°C and 750°C, respectively. Moreover, the gas pressure of deposition was fixed at 10 mTorr with an Ar/O2 ratio of 3:1. The as-synthesized samples are further annealed in air ambient at 950°C for 30 min.LSMO nanolayers (the Sr content is approximately 39%) with thickness of approximately 60 nm were grown on the α radiation. The detailed microstructure of the as-synthesized samples was characterized by scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM). The composition analysis was performed using energy dispersive X-ray spectrometer (EDS) attached to the TEM. The surface morphology of the LSMO nanolayers was investigated by atomic force microscopy (AFM) with an area size of 2 μm × 2 μm. The surface current images of the LSMO nanolayers were also observed using conductive atomic force microscopy (CAFM) with PtIr tips. A superconducting quantum interference device magnetometer was used to measure the magnetic properties of the samples.The crystal structure of the samples was investigated by X-ray diffraction (XRD) with Cu K2O3 epitaxial buffering, respectively. In addition to Bragg reflection from the In2O3 (222) and Al2O3 (0001) crystallographic planes, clear Bragg reflections of (100), (110), and (200) were present for the pseudo-cubic LSMO in the XRD measurement range. The XRD results show a highly (110)-oriented crystallographic feature of the LSMO nanolayer grown on the In2O3 (222) epitaxy. By contrast, a highly (h00)-oriented crystallographic feature was observed for the LSMO nanolayer grown on the bare sapphire substrate. The LSMO nanolayers with and without In2O3 epitaxial buffering are in a pseudocubic structure with a similar lattice constant of 0.387 nm. This is similar to the bulk value [2O3 (0.292 nm) in comparison to the (100) LSMO plane. Moreover, a large lattice mismatch (approximately -13.2%) exists between In2O3 (222) and sapphire (0001) [2O3 (222) has a higher crystallographic compatibility degree during in situ crystal growth. Figure 2O3 epitaxial buffering, respectively. The grains are densely compacted, and no pores are found in the film surfaces. Furthermore, the grain size is more homogeneous for the LSMO nanolayers grown on the sapphire substrate. The LSMO grain sizes range from approximately 50 to 80 nm for the LSMO nanolayers on the sapphire substrate. The grains lying on the In2O3 epitaxially buffered sapphire substrate range from approximately 50 to 120 nm in size.Figure lk value , demonstAl2O3 000 crystall2O3 epitaxial buffering. The In2O3 epitaxy has approximately a 40-nm thickness and exhibits a columnar crystallite feature. The inset shows the In2O3 epitaxial high-resolution (HR) lattice fringes on the sapphire substrate. A clear interface was formed between the film and the substrate. The electron diffraction pattern taken from the interface of the In2O3 film and sapphire substrate also confirms that the In2O3 (222) epitaxial layer was grown on the c-axis-oriented sapphire substrate [2O3 epitaxy during thin-film growth. This caused the heterointerface between the LSMO nanolayer and In2O3 epitaxy to be rugged. Further investigation of the HR lattice fringes of one LSMO grain  that serves as a transition boundary was formed between the LSMO nanograins and In2O3 epitaxy . This is because the In2O3 epitaxy was grown under an oxygen-deficient atmosphere.Figure ubstrate . Moreoveaterials . This cr2O3 epitaxy. The insets show HR lattice fringes taken from different local regions at the interfaces between the LSMO nanograins and the sapphire substrate. Two types of heterointerface between the LSMO and substrate were presented. In the left inset, a thin (approximately 2 nm thick) transition layer formed at the heterointerface. By contrast, the right inset exhibits a high degree of interface coherency between the LSMO nanograin and substrate. The observation demonstrated that local single-crystal LSMO grains can be formed on the sapphire substrate with a sharp heterointerface during thin-film growth. The heterointerface between the LSMO nanolayer and the sapphire substrate is relatively flat and smooth in comparison to the one grown on the In2O3 epitaxy. This is believed to reduce the potential crystal defects at the heterointerface. Moreover, the FFT patterns and HR lattice fringes revealed that a thin disordered region was formed between the misoriented nanograins  roughness of 1.7 nm, the surface of the LSMO nanolayer grown on the bare sapphire substrate was smoother. The rms surface roughness of the film with In2O3 epitaxial buffering is 3.5 nm. As observed from the SEM images, the roughening of the LSMO nanolayer surface grown on the In2O3 epitaxy might be associated with its irregular grain sizes. Figure 2O3 epitaxy measured at a fixed applied bias during AFM scanning. The LSMO nanolayer current maps show that the dark regions only account for a remarkably small ratio over the area of interest, revealing that the LSMO nanolayer surfaces remain a conductive characteristic under 0.05V. In comparison, the LSMO nanolayer without In2O3 epitaxial buffering has a homogeneously spatial distribution of current spots over the measured area. The current mean statistic value distributed over the measured area is 30.3 and 38.8 pA for the LSMO nanolayers with and without In2O3 epitaxial buffering, respectively. The LSMO nanolayer with In2O3 epitaxial buffering is slightly more resistant than the film without buffering.Figure M-T) for the zero-field-cooled (ZFC) and field-cooled (FC) samples. The applied magnetic field was 1,000 Oe during the M-T measurements. The M-T curves demonstrated that the LSMO nanolayers have a sharp ferromagnetic to paramagnetic transition. Comparatively, a higher magnetization and Curie temperature was observed for the LSMO nanolayers without In2O3 epitaxial buffering. The Curie temperatures of the LSMO nanolayers with and without In2O3 epitaxial buffering were 290 and 323K, respectively. A higher ferromagnetic ordering degree causes the LSMO films to have a higher saturation magnetization value and Curie temperature [2O3 epitaxial buffering caused the double-exchange mechanism to have a greater depression degree [2O3 epitaxial buffering is slightly more resistant than the film without buffering. There is a large difference in the ZFC and FC curves’ low temperature range. ZFC curves display a broad summit peak. A larger difference in magnetization between the ZFC and FC curves in the low temperature region was observed for the LSMO nanolayer with In2O3 epitaxial buffering, which conveyed that randomly oriented magnetic domains are more difficult to align in the film. The subgrain boundaries among the LSMO nanograins, rough film surfaces, and interfaces caused an existence of disordered spins in the LSMO nanolayer. These disordered spins might play an important role in separating the magnetically ordered regions in the LSMO nanolayer [M-H) hysteresis curves at 50 K for LSMO nanolayers with and without In2O3 epitaxial buffering. The field was applied parallel to the substrates. The respective in-plane saturated magnetization value was approximately 500 and 625 emu/cm3 for the LSMO nanolayers with and without In2O3 epitaxial buffering, respectively. The LSMO nanolayers with and without In2O3 epitaxial buffering have coercive fields that are 90 and 72 Oe, respectively. The crystal imperfections, such as surface roughness, subgrain boundary, and heterointerface, play important roles in determining the coercivity [2O3 epitaxial buffering act as domain-wall pinning centers [M-H results.Figure perature . This ren degree . Moreoven degree . This isanolayer . This caercivity . Moreove centers . The rel2O3 (222) epitaxial buffering. The LSMO experienced improved (110) preferred crystal growth via In2O3 (222) epitaxial buffering. Comparatively, the surface grain size is more homogeneous for the LSMO nanolayer grown on the sapphire substrate. The rugged surface of the In2O3 epitaxial underlayer further incurred rougher surface morphology of the LSMO nanofilm. The columnar crystallite feature of the In2O3 epitaxial underlayer caused a relatively smaller lateral domain size of the manganite ultra-thin layer on it. Moreover, In2O3 epitaxial buffering resulted in rugged heterointerfaces between the LSMO nanolayer and In2O3 epitaxy. These factors contributed to a higher content of subgrain boundaries and incoherent interfaces on a nanometric scale in the LSMO nanofilm via In2O3 epitaxial buffering. These disordered regions caused disordered spins to exist in the LSMO nanolayer. Therefore, lower saturation magnetization value and Curie temperature, and higher coercivity and resistivity are found in the highly (110)-textured LSMO nanolayer.In summary, 60-nm-thick LSMO nanolayers were grown on sapphire substrates with and without InThe authors declare that they have no competing interests.YCL designed the experiments and drafted the manuscript. HZ carried out the thin-film preparation and material analyses. WKL analyzed the AFM and CAFM data. All authors read and approved the final manuscript.YCL is a professor of the Institute of Materials Engineering at National Taiwan Ocean University (Taiwan). HZ received his Masters degree in Materials Engineering at National Taiwan Ocean University (Taiwan) in 2013. WKL is a graduate student of the Institute of Materials Engineering at National Taiwan Ocean University (Taiwan).


There is a long lasting debate about the latency of human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). Evidence indicates that these viruses are transcriptionally silent and replicate through mitotic division of infected cells . However, this model is inconsistent with the permanent and vigorous stimulation of the host immune response directed against these viruses.To address this apparent paradox, we studied the fate of cells in which viral expression was transiently induced. Using a dual fluorochrome labeling approach, we show that virus-positive and negative cell populations have different kinetics in BLV-infected sheep. Furthermore, cyclosporine treatment completely abrogates the difference in kinetics, consistent with a role of the immune response in controlling virus expressing cells.


Serum samples were collected before and after two, four, and six months of treatment. We found serum cobalamin to decline and reach significant lower levels after six months of treatment (p = 0.003). Despite the decline in serum cobalamin, we observed no reductions in the physiological active part of cobalamin bound to transcobalamin , or increase in the metabolic marker of cobalamin status, methylmalonic acid. Instead, the non-functional part of circulating cobalamin bound to haptocorrin declined (p = 0.0009). Our results have two implications: The data questions whether metformin treatment induces an impaired cobalamin status in PCOS patients, and further suggests that serum cobalamin is a futile marker for judging cobalamin status in metformin-treated patients.Treatment with the anti-diabetic drug metformin is followed by a decline in plasma cobalamin, but it is unsettled whether this denotes an impaired cobalamin status. This study has explored changes in the markers of cobalamin status in women with Polycystic Ovary Syndrome treated with metformin (1.5-2.5 g per day) ( Metformin is an oral anti-diabetic drug known to reduce serum cobalamin vitamin B12) concentrations in patients with type 2 diabetes mellitus 2 concent,4,5. Foret al. (2012) found metformin treatment to improve the intracellular cobalamin metabolism in type 2 diabetic patients despite a low serum cobalamin [Cobalamin is involved in a number of key metabolic processes including red blood cell production and nervous system function, and cobalamin deficiency can lead to anemia and neurological manifestations ,11. In tobalamin . In concobalamin .Here we expand previous studies by analyzing the serum concentrations of total cobalamin, holoTC, totaltranscobalamin , Cbl-HC, totalhaptocorrin , and the metabolic marker MMA in women with PCOS before and after up to six months of treatment with metformin or placebo.n = 23) treated with metformin and placebo. The inclusion criteria were women (aged 18–45 years) diagnosed with PCOS with a BMI > 25 kg/m2, and the exclusion criteria were diabetes mellitus, elevated blood pressure, abuse of alcohol and drugs, and chronic disease. The study was approved by the Central Denmark Region Ethics Committee (no. 2166-00 and 1-10-72-540-12) and carried out from 2001 to 2002. Cohort II (n = 6) was diagnosed with PCOS at the Department of Obstetrics and Gynecology at Aarhus University Hospital, Denmark, from 2011 to 2012. Before inclusion, these women were scheduled to initiate treatment with metformin (1.5–2.5 g per day) by their gynecologist. The inclusion criteria age range was 18–45 years, and the exclusion criteria were treatment with either metformin or cobalamin within the past year, chronic diseases, and pregnancy. The study was approved by the Central Denmark Region Ethics Committee (no. 20100283). Our study was performed within the confines of the Helsinki Declaration, and all participants gave their informed consent before inclusion.This study included two cohorts of women diagnosed with PCOS according to the Rotterdam criteria and treated with metformin. Cohort I came from a cross-over study on the efficacy of metformin (1.7 g per day) in obese and non-obese women with PCOS . From thNon-fasting serum samples were collected at baseline (cohorts I and II), and after two (cohort I), four (cohorts I and II), and six (cohort I) months of metformin treatment. Samples were stored at −80 °C at the Department of Obstetrics and Gynecology, Herning Hospital, Denmark (cohort I) and at −25 °C the Department of Clinical Biochemistry, Aarhus University Hospital, Denmark (cohort II), until usage. In-house experiments suggest that cobalamin and its binding proteins are stable for at least 10 years when stored at −25 °C, and this is supported by others . CollectTotal cobalamin was assayed on an automatic Cobas 6000  system that uses alkaline hydrolysis (sodium hydroxide and dithiothreitol) for cobalamin extraction, and sodium cyanide for conversion into cyanocobalamin. In the assay, cobalamin in the sample competes with biotin-labeled cobalamin for binding to ruthenium-labeled intrinsic factor. The assay has a total imprecision of 6.6%. TotalTC was measured by an in-house sandwich ELISA with a total imprecision of 4%–6% . HoloTC t-test or the non-parametric Mann-Whitney test when appropriate. Since no differences in serum cobalamin was found between the two cohorts at any time point, the datasets were combined and treated as one during the following statistical analysis. To compare baseline value between the combined metformin group and the placebo group, the two-tailed unpaired t-test or the non-parametric Mann Whitney test was used. To compare changes between baseline and a given time point for each group, we used the two-tailed paired t-test or the Wilcoxon signed rank test. p-values ≤ 0.05 were accepted as statistically significant. Data analysis was performed using the statistical software available in GraphPad Prism version 5. Data are given as median (range) if not otherwise stated.The D’Agostino-Pearson omnibus test was used to determine if data followed the Gaussian distribution. To test for baseline differences between the two cohorts of metformin-treated women, we used the two-tailed unpaired n = 29) were recruited from two different study cohorts, cohort I (n = 23) and cohort II (n = 6). The decision to merge the two cohorts was based on agreements in weight (89 (60–125) kg) and body mass index (BMI) (32 (21–46) kg/m2) at the time of inclusion; and more importantly the fact that none of the women took cobalamin supplements during the study. The only difference between the two cohorts was related to their age, 34 (19–44) years (cohort I) and 23 (20–37) years (cohort II). Accordingly, the demographic data for the placebo-treated group was 89 (60–119) kg (weight), 32 (21–46) kg/m2 (BMI) and 34 (19–44) years (age).The group of metformin-treated women with PCOS (p = 0.003). This decrease in serum cobalamin was accompanied by a decline in Cbl-HC that was significant after four months of treatment  (p = 0.02) was observed after four months of metformin treatment. For the placebo group, no changes in cobalamin, its binding proteins, and the hematological parameters were observed; however a small unexplained increase in MMA (p = 0.02) was seen after six months of treatment.Markers of cobalamin status were measured at baseline and after two, four, and six months of metformin or placebo treatment . The res 0.0009) D. No ove 0.0009) , or in cOur study investigates the cobalamin status during six months of metformin treatment, but provides no information on longer periods of treatment. Despite this limitation, several interesting result are obtained from this study. We confirm previous studies showing that metformin treatment decreases the levels of circulating cobalamin ,3,4,5,9.We observed that the decline in serum cobalamin was accompanied by a decrease in the amount of cobalamin bound to HC, Cbl-HC. This cobalamin is inaccessible to the cells, thus provides no relevant source of cobalamin for the body. The decline in Cbl-HC supports earlier findings from a small study on ten diabetic patients . We obseIn conclusion, our data show that metformin causes a decline in serum cobalamin after six months of treatment, while no significant change occur in any of the other markers of cobalamin status. Together with previous data, our study suggests the changes in plasma cobalamin to be of no relevance for the cobalamin status of metformin-treated PCOS patients, at least for the first six months of treatment. Because of this, it is recommended not to use plasma cobalamin alone when judging an impaired cobalamin status in patients treated with metformin. Additionally, the results question whether low plasma cobalamin in metformin-treated diabetic patients causes a true or false cobalamin deficiency—an important topic that warrants future investigations.


Larus michahellis), a widely distributed, anthropophilic, and abundant seabird species. For 3 years, we sampled eggs from 19 breeding colonies in Spain, France, Algeria, and Tunisia. First, ELISAs were used to determine if the eggs contained antibodies against flaviviruses. Second, neutralization assays were used to identify the specific flaviviruses present. Finally, for colonies in which ELISA-positive eggs had been found, chick serum samples and potential vectors, culicid mosquitoes and soft ticks (Ornithodoros maritimus), were collected and analyzed using serology and PCR, respectively. The prevalence of flavivirus-specific antibodies in eggs was highly spatially heterogeneous. In northeastern Spain, on the Medes Islands and in the nearby village of L'Escala, 56% of eggs had antibodies against the flavivirus envelope protein, but were negative for neutralizing antibodies against three common flaviviruses: West Nile, Usutu, and tick-borne encephalitis viruses. Furthermore, little evidence of past flavivirus exposure was obtained for the other colonies. A subset of the Ornithodoros ticks from Medes screened for flaviviral RNA tested positive for a virus whose NS5 gene was 95% similar to that of Meaban virus, a flavivirus previously isolated from ticks of Larus argentatus in western France. All ELISA-positive samples subsequently tested positive for Meaban virus neutralizing antibodies. This study shows that gulls in the western Mediterranean Basin are exposed to a tick-borne Meaban-like virus, which underscores the need of exploring the spatial and temporal distribution of this flavivirus as well as its potential pathogenicity for animals and humans.In recent years, a number of zoonotic flaviviruses have emerged worldwide, and wild birds serve as their major reservoirs. Epidemiological surveys of bird populations at various geographical scales can clarify key aspects of the eco-epidemiology of these viruses. In this study, we aimed at exploring the presence of flaviviruses in the western Mediterranean by sampling breeding populations of the yellow-legged gull ( A number of zoonoses are (re)emerging worldwide, the majority of which present risks to animal and human health Flavivirus, including West Nile, dengue, yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses, cause significant disease burdens in humans and animals Arboviruses in the genus Culexe.g., Meaban virus) is circulating in and among seabird populations. While the pathogenicity of such viruses for birds and humans is still unknown, their potential capacity to spread is of concern, since they may be dispersed over large scales Some flaviviruses are of particular epidemiological concern. West Nile virus (WNV) is widely distributed across Africa, Europe, Australia, and Asia; more recently, it has spread to the Western Hemisphere. Over the last few years, WNV outbreaks have dramatically increased throughout the Mediterranean Larus michahellis michahellis) is a numerically abundant Laridae species Larus species, the ring-billed gull (Larus delawarensis), is susceptible to WNV infection The yellow-legged gull  and extensively sampling breeding sites along the coasts of the western Mediterranean Sea.Another benefit of using the yellow-legged gull in eco-epidemiological studies is that, in addition to being broadly distributed and numerically abundant, it has been able to colonize a number of habitats, from natural to urban areas and marine to terrestrial ecosystems Finally, the ability to detect the presence of disease agents in wildlife populations strongly depends on the performance of the various detection methods This study aims to detect the presence of flaviviruses in yellow-legged gulls as well as to provide some initial insights into the eco-epidemiology of flavivirus circulation in the western Mediterranean Basin. First, we screened eggs from yellow-legged gulls across the Mediterranean for flavivirus-specific antibodies. Then, to explore potential vectors in this system, we targeted an area in which flavivirus antibody presence was high; we analyzed nestling sera, soft ticks, and mosquitoes. This strategy enabled us to efficiently focus sampling and analytical efforts on the most relevant target colonies.From 2009 to 2011, in March and April, we sampled eggs from 19 breeding colonies located in France (n = 9), Spain (n = 5), Tunisia (n = 2), and Algeria (n = 3) , Table 1Flavivirus antibody prevalence was high in the Medes colony see . As a coOrnithodoros maritimus (soft ticks), which had previously been observed on Medes 2 were left running for 24 h in August 2011 in order to capture Culicidae mosquitoes. A total of 68 mosquitoes were sampled, morphologically identified and pooled by species into 10 groups containing up to 13 mosquitoes. The majority of them were identified as Culex theileri, a known vector of flaviviruses such as WNV in Europe sample/ODNC)×100. According to kit instructions, samples with PC values ≤40% were considered positive, those with PC values >50% were considered negative, and those with PC values between 40% and 50% were considered doubtful. In our statistical analyses, doubtful samples were grouped with negative samples.Egg extracts and gull chick serum samples were screened for antibodies directed against the WNV envelope protein, which contains epitopes shared with other viruses of the Japanese encephalitis serocomplex 90) was also performed on these eggs to detect antibodies against TBEV. A comparable test was performed on ELISA-positive eggs (n = 108) and serum samples (n = 18) to detect antibodies against Meaban virus.Virus neutralization tests were used to specifically identify the flaviviruses to which gulls had been exposed 5 Vero (TBEV) or SW13 cells (Meaban virus) per well 1 day before the neutralization assay. Diluted egg extracts  were incubated with TBEV (Hypr strain) or Meaban virus (Brest ART707 strain) in a suspension containing 400 plaque-forming units per mL of Dulbecco's Modified Eagle's Medium  for 1h30 in a CO2 incubator at 37°C. The culture medium was then removed from the wells, and 0.5 mL of the virus-serum mixture was added to the wells and left for 1h30 at 37°C. One mL gelosa was obtained by mixing equal volumes of carboxymethylcellulose  or Seaplaque agarose  with DMEM with 2% fetal calf serum. After 5 days of incubation at 37°C, the cells were rinsed twice, fixed with 4% paraformaldehyde , and stained with crystal violet  for the purposes of plaque counting. The extract was considered positive if it prevented the formation of viral plaques, i.e., if the number of viral plaques associated with the extract was less than 10% of the number counted in the control well (without serum or egg extract).Briefly, the neutralization tests for antibodies against TBEV and Meaban virus were conducted as follows. Six-well plates were seeded with 8×10Because maternal antibody concentration decreases with age in nestlings We used generalized linear models (GLMs), employing a binomial distribution and logit link, to assess whether flavivirus antibody prevalence in eggs sampled from the Medes colony differed among years. The most parsimonious model was selected using the Akaike information criterion (AIC). Analyses were performed in R 2.12.0 . For each of the three years during which eggs were sampled from the Medes colony, a spatial analysis was conducted to determine whether positive versus negative nests demonstrated spatial structure  that had been designed to detect the flavivirus ribonucleic acid (RNA) genome http://www.ncbi.nlm.nih.gov/) using the basic local alignment search tool (BLAST) to find the best match . Mean genetic distances were estimated using MEGA v. 5.2.2 NS5 sequences to previously identified flavivirus genospecies , a graphical front–end tool for RAxML-VI-HPC .Sequence chromatograms were manually verified and then assembled using Geneious v. 5.3.6 (Biomatters Ltd.). We compared the sequences we found to published sequences in GenBank  of eggs contained antibodies against the flavivirus envelope protein , a large proportion  or just data from the year with the greatest number of seropositive chicks . No positive spatial autocorrelation was present in the distribution of ELISA-positive eggs in the colony . Furthermore, antibodies against flaviviruses were present in the blood of 16 of the 501 Medes gull chicks . Two of the eight positive tick amplifications resulted in incomplete sequences that were of insufficient quality, and they were not used in subsequent analyses.In the Medes colony, flavivirus RNA was detected in 8 of the 135 tick pools tested . No flavivirus RNA was detected in the mosquitoes. All of the NS5 gene fragment found in Meaban virus (>95% similarity), a flavivirus first isolated in 1981 from O. maritimus ticks (Genbank accession number DQ235144 O. maritimus flavivirus sequences formed a single well-supported cluster (Bootstrap 70%) that was very closely related to sequences previously identified as Meaban-like  and Saumarez Reef (AF013403) viruses, both of which are tick-borne flaviviruses associated with seabirds. Other flaviviruses, such as WNV, USUV, or TBEV are far from the Meaban-like group in the phylogenetic tree  Phalacrocorax aristotelis) or while feeding on refuse dumps. Constraints could come from the vector; assuming that Ornithodoros ticks are the actual vectors of the Meaban-like virus, tick density is relatively large in the Medes colony compared to levels reported in other colonies , suggest these are the result of the circulation of a Meaban-like virus, hence a flavivirus closely related to one previously isolated from ticks of Although antibodies specific for flaviviruses had been reported in three yellow-legged gulls sampled in 2010–2011 in nearby areas et al.O. maritimus ticks found in seabird colonies in southern France and Corsica. However, because WNV has recently been detected in the Mediterranean Basin, notably in Spain The ELISA screening procedure we used detects antibodies against the flavivirus envelope protein, an antigen that is commonly used in WNV serodiagnosis in birds Culex theileri, the main mosquito species on Medes, did not play an essential role in transmitting viruses to gulls at the time of sampling. However, the number of mosquitoes tested was very low; therefore, we cannot rule out that this species or other mosquitoes present on Medes may be involved in the flavivirus epidemiological cycle. Conversely, RNA extracts from O. maritimus seabird ticks contained flaviviral RNA and, furthermore, the RNA sequence retrieved was more than 95% similar to a fragment of the NS5 gene of Meaban virus, a seabird tick-borne virus isolated from colonies of a very closely related gull species O. maritimus may infest up to 90% of the gull nests in the Medes colony Larus argentatus) colony in Brittany, France i.e., we did not check that the ELISA was more sensitive than the Meaban virus PRNT90). Neutralization tests for Meaban virus were likewise positive for the ELISA-positive eggs detected in other colonies in France and Spain  but were negative for Jijel in Algeria  e.g., Tyuleniy viruses) human pathogens Apart from the risk related to vector driven transmission, gulls may also serve as bridges for the spread of infectious agents. In particular, since yellow-legged gulls are known to colonize urban areas, it is important to consider the possibility that the virus may be dispersed to novel locations O. maritimus from additional locations and across different time periods. The host range of this Meaban-like virus also remains to be investigated, particularly since O. maritimus ticks occur on other avian species such as European shags In this study, we report the first detection of a Meaban-like virus in the western Mediterranean Basin. Despite widespread sampling, our results suggest that this flavivirus is present at mainly one location: the Medes Islands (Spain). Since antibodies were found in pre-fledging chicks and because of the fact that the prevalence of antibody-positive eggs increased over time in this colony over the three year study period, it would seem that the Meaban-like virus, whose zoonotic potential is unknown, is currently circulating among Medes gulls; isolation trials using ticks and gull serum are currently being conducted to test this hypothesis. In order to better predict the risk of spread of this Meaban-like virus will spread within the Mediterranean Basin, it would be interesting to conduct further investigations that target other bird species that nest on the Medes Islands, as well as to sample the seabird tick In conclusion, gathering further data on the diversity, distribution, and pathogenicity of viruses associated with seabirds is important, especially given the potential risks the viruses pose for human health. Our study has shown that a combination of population ecology and biomedical approaches can efficiently enhance our understanding of emerging infectious pathogens. Future studies should more thoroughly examine tick vector competence, infection rates, and population genetic structure Ethics committees present at each of the co-authors' institutions specifically approved this study. These include the following: Direction Régionale de l'Environnement, de l'Aménagement et du Logement (DREAL), Syndicat Mixte des Etangs Littoraux (SIEL), Conservatoire Etudes des Ecosystèmes de Provence-Alpes du sud (CEEP), Parc national de Port-Cros, Service Environnement de la Mairie de Gruissan (Aude), Departament d'Agricultura, Ramaderia, Pesca, Alimentació i Medi Natural de la Generalitat de Catalunya, Parc Natural de El Montgrí, les Illes Medes i el Baix Ter, Dirección General de Patrimonio Natural y Biodiversidad de Murcia, Conselleria de Medi Ambient i Mobilitat del Govern de les Illes Balears, Tomás Montalvo , Parc Natural del Delta de l'Ebre, Cabildo de Gran Canaria, Conselleria de Medi Ambient, Aigua, Urbanisme i Habitatge de la Generalitat Valenciana, the forest service in the Tunisian Ministry of Agriculture (permit number 518-28/02/2009), and the Algerian DGRSTD/MESRS and DSFP, King Saud University (Saudi Arabia). All efforts were made to minimize animal suffering and disturbance. In the Mediterranean Basin, the yellow-legged gull is a numerically abundant species and is subject to population control measures Table S1Reference sequences used in the phylogenetic analysis classified according to the flavivirus group sensu Heinz et al. 2001, Cook & Holmes 2001 see .(DOCX)Click here for additional data file.Table S2Detailed results of Meaban virus neutralization tests performed on ELISA-positive samples. The column ‘Titer >20’ corresponds to samples for which insufficient serum was available to make further dilutions.(DOC)Click here for additional data file.


Oligoryzomys fornesi rodents and is responsible for hantavirus pulmonary syndrome in this region.To confirm circulation of Anajatuba virus in Maranhão, Brazil, we conducted a serologic survey (immunoglobulin G ELISA) and phylogenetic studies (nucleocapsid gene sequences) of hantaviruses from wild rodents and persons with hantavirus pulmonary syndrome. This virus is transmitted by  Bunyaviridae, genus Hantavirus) cause a viral zoonosis transmitted by rodents belonging to the families Muridae and Cricetidae. Each hantavirus is predominantly associated with a specific rodent species in a specific geographic region. However, infection of other rodent species can occur as a spillover phenomenon  (Oligoryzomys fornesi (rice rat) rodents and Holochilus sciureus (marsh rat) rodents, respectively, and genetically characterized , are located in the western floodplain of the Maranhão River in Maranhão State, Brazil  and Santa Rita   are shown in The main findings of the serologic study are shown in <50 m from residences of 3 deceased HPS case-patients in accordance with accepted rodent capture and handling procedures and standard biosafety protocols for anesthetizing and killing rodents, and biometric analysis was conducted  and Akodon sp. rodents . The most common species captured in 2005 were N. lasiurus rodents  and O. fornesi rodents ; the remaining rodents were from other genera.Biologic samples (blood and viscera fragments) were obtained from 216 captured rodents: 96 (44%) captured in 2003 and 120 (56%) captured in 2005. The most common species captured in 2003 were O. fornesi rodents captured in 2005 and 6 (4%) of 167 N. lasiurus rodents (3 of 62 captured in 2003 and 3 of 105 captured in 2005)  .Virus RNA was extracted from IgM-positive human serum or blood samples and lung fragments from IgG-positive rodents by using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Nested reverse transcription–PCR and hemi-nested reverse transcription–PCR were used for amplification of partial nucleocapsid gene sequences from human and rodent samples, respectively, by using primers described .www.megasoftware.net) Estimated values were <45%, <25%, 22%, and 15% and were used for grouping viruses in clusters, clades, subclades, and species, respectively.Phylogenetic trees were constructed by using neighbor-joining, maximum-parsimony, maximum-likelihood, and Bayesian methods implemented in PAUP 4.0b.10  and had a genetic distance of 28.2% . The New World group was divided into clades I and II. Clade I was divided into 3 subclades (genetic divergence 23.5%), Ia, Ib, and Ic. Clade II was divided into 2 subclades (genetic divergence 23.7%), IIa and IIb. The strains used in this study were closely related to Anajatuba virus and were included in the IIa subclade (genetic divergence 2%) .Oligoryzomys, although sample Be AN 669104 was obtained from an N. lasiurus rodent, which suggests spillover transmission between rodent species.Nucleotide and amino acid homology between Anajatuba virus (O. fornesi rodents are its likely reservoir. Anajatuba virus infections of N. lasiurus were spillover infections. Human hantavirus infections are common among persons in the Baixada Maranhense region, but cases of HPS are rare. However, educational and health surveillance programs are needed to prevent hantavirus transmission.We showed that Anajatuba virus is responsible for human HPS cases and that Distribution of hantavirus groups and subgroups in the Western Hemisphere.


An error was introduced in the preparation of this article for publication. In the title, 'Thy1+ Nk Cells' should have an uppercase 'K'. The correct title should read: Thy1+ NK Cells from Vaccinia Virus-Primed Mice Confer Protection against Vaccinia Virus Challenge in the Absence of Adaptive Lymphocytes. The correct citation is: Gillard GO, Bivas-Benita M, Hovav A-H, Grandpre LE, Panas MW, et al. (2011) Thy1+ NK Cells from Vaccinia Virus-Primed Mice Confer Protection against Vaccinia Virus Challenge in the Absence of Adaptive Lymphocytes. PLoS Pathog 7(8): e1002141. doi:10.1371/journal.ppat.1002141


Background.Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching.Methods.Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events.Results.We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a.Conclusions.Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution. Streptococcus pneumoniae (the “pneumococcus”) is an asymptomatic colonizer of the human nasopharynx and a major cause of otitis media, sinusitis, pneumonia, and meningitis, resulting in approximately 14.5 million annual serious disease episodes globally among young children [cps) genes, located between dexB and aliA on the chromosome. The remaining 2 capsule types are synthesized through independent biochemical pathways.children –6. The cchildren , 8. Ninechildren , 9. The cps loci revealed variable lengths (approximately 10–30 kb) and a range of genes specific to capsule production [Sequence analyses of 88 reference oduction . The seroduction , 10–13.Serotype is a key determinant of invasive pneumococcal disease potential and prevalence; certain serotypes are more commonly associated with carriage and others more commonly with invasive pneumococcal disease , 15. A 7cps locus [cps locus, and studies of a limited number of such strains indicated that recombination fragment sizes varied from approximately 21.9 kb to approximately 56.5 kb [pbp2x and pbp1a genes .Surveillance following PCV7 introduction showed a decline of vaccine type (VT) and an increase of nonvaccine type (NVT) pneumococci in disease and nasopharyngeal carriage . This caps locus . (Serotyps locus  and was ps locus .) These ps locus , show seps locus , 24; any 56.5 kb –28. In sSupplementary Materials). In this study, we used serotyping, MLST, and whole-genome data to study a large, genetically diverse collection of historical and modern pneumococci, with the aim to better understand the mechanisms and role of capsular switching in pneumococcal evolution.Vaccine-induced selective pressure is contributing to the postvaccination changes of serotype epidemiology, but natural fluctuations in serotype prevalence also play a role. Many studies have documented prevaccine temporal changes in relative serotype prevalence: all but 3 included pneumococci isolated no earlier than 1969; only 2 studies provided genotype data  were previously serotyped by the Quellung reaction and genotyped by MLST [Supplementary Materials). CCs were named after the predicted founder ST. When no single founder ST could be determined, CCs were named NoneX, where X was the ST of lowest numeric value within the CC. Isolates of the same CC but with different serotypes were presumed to be capsular switch variants.A global collection of 426 pneumococci recovered during 1937–2007  on the Illumina platform as previously described [Supplementary Table 1).A total of 96 genetically diverse isolates from our collection were selected for whole-genome sequencing . MLST data were used to infer the CC most likely represented by the true donor isolate (the same as that of the donor representative). Recombination regions were identified as the minimum region over which the recombinant representative  differed from the ancestral representative  and was identical or highly similar (>99.7% sequence identity) to the donor representative . Where no suitably matched donor was identified, the maximum recombination region was estimated as the maximum length over which the recombinant differed from the ancestor .Eight CCs were represented by isolates subjected to whole-genome sequencing and were selected for further g MUSCLE  and impog MUSCLE  for visuAmong the entire collection of 426 pneumococci, 21 of 163 unique CCs were represented by isolates of 2–6 serotypes. At least 36 independent changes of serotype within CCs were represented in our collection, 34 of which predated the introduction of pneumococcal conjugate vaccines  cps locus sequences were unique , suggesting a possible recombination event between these CCs. The CC1514 representatives had a 432-bp deletion in the wciY gene, resulting in a predicted 250-amino-acid truncation of the protein. Isolate CGSP14 [wciY gene; if this represented a true deletion event rather than a sequencing/assembly error, the resultant protein would be further reduced to 38 amino acids.Seven of 10 CC15e CGSP14  also hadpbp2x, the cps locus, and pbp1a, differed from the CC667B isolate but was highly similar to a CC37829N isolate dated 1952  Within the cps locus, a second CC669N isolate differed from the oldest CC669N isolate by only 5 nucleotide substitutions .23F representative in our collection differed from the CC669N isolates from approximately 2 kb upstream of dexB to approximately 3 kb downstream of aliA, suggesting a potential recombination import of approximately 30.4 kb, but no suitable donor was identified.The single CC66wciX gene [35C isolates likely arose from a single capsular switch event , 35C n = 2), and 9V, 17F, and 18B (n = 1 each). Serotype 18C was assumed to be the ancestral serotype and was also represented by 1 of the 2 oldest isolates, both dated 1939. The change from serotype 18C to 18B was due to a single nucleotide substitution within the , and 9V,cps locus among 5 CC11318C representatives (Table wchA gene and by a putative import of approximately 1.1 kb (marked by 44 nucleotide substitutions) spanning the rmlA and rmlC genes .There was little to no variation at the cps sequences  representing serotypes 9V, 9A, and 19A were assigned to CC156/162. Four isolates were serotype 9V; the oldest was serotype 9A. The CC156/162–2006 repcps loci and flanking regions of the 2 CC156/16219A representatives (GenBank accession numbers AGOR01000001–AGOR01000024 and AGQA01000001–AGQA01000007) suggested a single capsular switch event. Approximately 50.4-kb regions of the CC156/16219A genomes were highly similar to those of a CC19919A isolate . Repeat motifs are difficult to sequence accurately, so further speculations about such changes cannot be made. Apart from the repeat region, the CC1917Fcps loci each differed by a maximum of 4 nucleotides (Table 3B).Seven of the CC191 representatives were serotype 7F, and the eighth, and oldest, was serotype 7A. Serotypes 7A and 7F differed by 3 nucleotides Table . There wpbp2x and approximately 4.8 kb downstream of pbp1a, CC2187F and CC1917F sequences were very similar, although differentiating nucleotides were identified , but whether these events occurred before or after the cps switch is unknown. Note that approximately 585 bp were missing between pbp2x and dexB in both CC2187F and CC1917F representatives. Table cps loci of CC2187F and CC21812F isolates.Eight CC218 isolates represented serotypes 12F (n = 5) and 7F n = 3). Between approximately 9 kb upstream of . BetweendexB to approximately 5 kb downstream of pbp1a. A region of the serotype 2 representative  was highly similar to that of a CCNone1282 representative dated 1916  are consistent with fragments detected in previously published studies [ps loci between CCs, as inferred by our data. Isolates identified as donors from our collection could possibly have represented different CCs than the true donors; however, even if this were true, our conclusions about the number of independent capsular switches and the range of recombination fragment sizes would remain unchanged.We studied 18 capsular switching events in detail. Three of these were presumably the result of nucleotide substitution and/or deletion, a finding consistent with previous work . The rem studies , 42, 43.cps locus, which had also not been indicated by any previous studies [cps locus) and did not show any trends toward increasing/decreasing lengths through time.Our analyses did not indicate any recombination breakpoint hot spots around the  studies , 28, 30.12F→7F and CC667B→9N events were characterized by large (>50 kb) recombination imports and must have taken place no later than 1952 and 1960, respectively. Large-scale recombination between pneumococci has therefore clearly been occurring for decades, although the technology capable of detecting and detailing such events has only recently become available [pbp2x + cps + pbp1a loci, as reported for other events [9V→19A).The CC218vailable , 30, 42.r events , 28 and pbp2x ± cps ± pbp1a recombination events were detected soon after widespread vaccination began in the United States. No such recombinants had been reported before in nature, and the imported pbp2x and pbp1a sequences conferred penicillin nonsusceptibility. Thus, it was theorized that vaccine-induced and/or antibiotic-induced selective pressures may play a role in driving these genetic changes [12F→7F and CC667B→9N events were both associated with penicillin-susceptible pneumococci and occurred before the widespread use of penicillins and before the introduction of PCV7. Our new data suggest that recombination of the cps locus and flanking regions might be “normal” biological processes, the evolution of which has undoubtedly been influenced by naturally occurring immunity and other selective pressures. Presumably, vaccine-induced immune pressures and/or the pressure of antibiotic use subsequently influence the spread and maintenance of advantageous genes  by selecting recombinants that are best able to survive. However, the potential negative effects should not be underestimated: the CC6954→19A “vaccine escape” capsular switches in the United States were first recovered from patients with pediatric invasive pneumococcal disease only 3 years after PCV7 introduction and, 2 years later, were the third-most common serotype 19A CC causing invasive pneumococcal disease among all age groups [pbp2x and pbp1a genes. Consequently, the increase in prevalence of these strains likely contributed to the increase in pneumococcal penicillin nonsusceptibility in the United States after PCV7 introduction [Recently reported in vivo  changes , 44. Pene groups . These soduction , 45.19A-like representative was the most probable donor of the 19A cps locus and flanking pbps to the CC156/16219A isolates described in this study. CC19919A representatives were also the donors of the cps locus ± pbps to the vaccine escape progeny [19→19Acps locus switch [19A representatives appear to be “good” cps locus donors.Another interesting finding was that a CC199 progeny , 28 and s switch . Future cps locus that did not result in capsular switching, among isolates belonging to the same ancestral lineages (CC11318C and CC1917F) and different ancestral lineages (CC1917F vs CC2187F and CC1514 vs CC12414). Similar events have been noted within the CC8123F lineage [cps locus. A comparison of the cps loci of CC1514 and CC12414 representatives also indicated a deletion in the CC1514wciY gene, resulting in a predicted truncation of the putative glycerol phosphotransferase encoded by this gene. Notwithstanding the deletion, the CC1514 isolates were successfully serotyped by the Quellung reaction, indicating successful capsule production. Indeed, an in silico analysis of the predicted cps protein coding regions failed to identify a specific reaction catalyzed by the serotype 14 version of this protein [Our analyses also revealed recombination at the  lineage  and amon lineage –12. We s protein .cps locus recombination, the associations between genotypes and serotypes are puzzling, as is our evidence of a high level of cps sequence conservation within some CCs . Perhaps there is some synergism between serotype and the genetic background of a strain that conveys an advantage to certain combinations over others. This sort of synergistic effect has been invoked to explain changes in the pneumococcal population in South Korea, where there was a prevaccine reduction of multidrug-resistant CC271/32019F pneumococci and replacement by similarly multidrug-resistant CC271/32019A pneumococci over several years [cps locus contribute to capsular expression and facilitate a genotype/serotype association [Given the overall capacity for al years . It is acps locus over approximately 70 years. Capsular switching with/without simultaneous pbp transfer has occurred regularly and prior to both PCV7 introduction and widespread antibiotic use. It is highly likely that the proliferation of newly generated NVT capsular switch variants will continue to be favored by PHiD-CV and PCV13 vaccination programs, as was the case after PCV7 implementation. Penicillin-nonsusceptible variants will have an even greater advantage. Although the magnitude of these selective forces relative to those favoring established genotype/VT associations remains unclear, the implementation of vaccine programs across the globe will most likely favor the intercontinental spread of NVT and penicillin-nonsusceptible pneumococci.Our collection of pneumococci provided a unique opportunity to study evolution at the Supplementary materials are available at The Journal of Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.


Acanthamoeba polyphaga Mimivirus), group B (includes Moumouvirus) and group C (includes Megavirus chilensis). Virophages have been isolated with both group A Mimiviridae (the Mamavirus strain) and the related Cafeteria roenbergensis virus, and they have also been described by bioinformatic analysis of the Phycodnavirus. Here, we found that the first two strains of virophages isolated with group A Mimiviridae can multiply easily in groups B and C and play a role in gene transfer among these virus subgroups. To isolate new virophages and their Mimiviridae host in the environment, we used PCR to identify a sample with a virophage and a group C Mimiviridae that failed to grow on amoeba. Moreover, we showed that virophages reduce the pathogenic effect of Mimivirus (plaque formation), establishing its parasitic role on Mimivirus. We therefore developed a co-culture procedure using Acanthamoeba polyphaga and Mimivirus to recover the detected virophage and then sequenced the virophage's genome. We present this technique as a novel approach to isolating virophages. We demonstrated that the newly identified virophages replicate in the viral factories of all three groups of Mimiviridae, suggesting that the spectrum of virophages is not limited to their initial host.The giant virus Mimiviridae family includes 3 groups of viruses: group A (includes  Legionella sp. Acanthamoeba polyphaga Mimivirus (APM) Megavirus chilensis) Free-living amoebas are ubiquitous protozoa that feed on microorganisms in their environment through phagocytosis. However, some microorganisms are able to resist digestion by this predator after phagocytosis Cafeteria roenbergensis virus, a giant virus that infects a marine phagocytic protist We isolated a small virus that co-isolates with Mamavirus and infects the Mamavirus virus factory. We named this new virus Sputnik and classified it as the first virophage A. polyphaga co-culture with each APM in our collection using quantitative real-time PCR and, more notably, to investigate if virophages previously isolated with group A APM can infect group B or group C viruses. Noting the inability to co-culture a group C virus with its virophage that was detectable by PCR, we decided to inoculate the sample on amoeba and Mimivirus to recover the virophage.The first aim of our study was to investigate the replication capacity of Sputnik 1 and Sputnik 2 in an 6 particles/ml and the Acanthamoeba polyphaga amoebal suspension at 5×105 cells/ml (strain Linc AP-1). After the complete lysis of the amoebas, each co-culture was centrifuged at 2000 rpm for 10 min to pellet the remaining fragments. The supernatant was filtrated through a 0.8 µm pore filter to remove residual amoebas and cysts. The supernatants were frozen at −80°C before being used for viral co-culture inoculation.Each virus in our collection  was prod5 cells/ml, 1 ml of giant virus suspension and 1 ml of each Sputnik strain. After the complete lysis of the amoeba, the purification was performed as described above, but the filtration was executed on three successive filters of 0.8-, 0.45- and 0.22 µm pore sizes to obtain a pure Sputnik suspension. This suspension was prepared before each inoculation assay. These tests were performed in triplicate.Sputnik 1 5 cells/ml and 1 ml of each Sputnik suspension. After 1 hour of incubation at 32°C, the supernatant was gently removed to eliminate the Sputnik particles that were not internalized by the amoebas, and 10 ml of fresh PYG medium was added. This time point was defined as H0. The Lentille virus, naturally infected by Sputnik 2, was used as a positive control.Each giant virus was inoculated separately into 10 ml of PYG medium containing 1 ml of amoeba suspension at 5×10Sputnik encoding the major capsid protein . For each sample, the cycle threshold (Ct) was correlated to a reference scale to allow the evaluation of the Sputnik concentration. A total of 200 µl of each co-culture was analyzed by real-time PCR directly after inoculation to assess the quantities of Sputnik 1 and Sputnik 2.After incubation, 200 µl of each co-culture was sampled for DNA extraction and real-time PCR at H1, H0 and Day 3 (to observe the complete lysis of amoebas). The DNA extraction was performed with a Qiamp DNA extraction kit  according to the manufacturer's instructions. Real-time PCR was performed with the LightCycler 480 SYBR Green I Master (Roche) according to the manufacturer's instructions. Sputnik was detected and quantified by using a primer pair targeting the ORF20 of After incubation, 200 µl of each co-culture at H0 and H6 was added to a Cytospin single chamber (Thermo), cytocentrifuged at 800 rpm for 10 min and fixed with methanol. Indirect immunofluorescence microscopy was performed with mouse anti-Sputnik serum as described previously. To label the virus factories and the cell nucleus, we used 5 µl of DAPI . The viable trophozoites were counted using trypan blue (Oxoid) to assess the speed of the lysis of the amoebas by each virus. The preparation of selected samples for transmission electron microscopy was performed as described previously The complete Pol B gene sequences for all the viruses used in this study were available from unfinished APM genome sequencing in progress in our laboratory. The sequences of DNA polymerase B were aligned using MUSCLE and trimmed by TrimAL after visual editing. The phylogenetic tree was built with a maximum likelihood using Phyml software.Megavirus chilensisSputnik, which encodes the major capsid protein described above.Tentative detection of APM and virophages was performed on 90 soil samples collected in Marseille and the surrounding areas (south of France). To each 15 g sample, 150 mL of sterile water was added. Decantation occurred overnight at 4°C, and then the samples were filtered, first through Wattman's paper and then through 0.1 µm membrane. Membranes were then put in 1 ml of Page's Amoeba Saline buffer (PAS) and vortexed. The membranes were removed, and the suspension was kept at −80°C prior to use. DNA was extracted with the QIAGEN© QIAmp Mini Kit following the manufacturer's protocol. A combination of several primer pairs was required because the low polB gene conservation in the available fragment Acanthamoeba polyphaga and Mamavirus (used as a reporter virus) A. polyphaga amoebae at a concentration of 5×105 cells/mL and 100 µL of Mamavirus (previously cured of its virophage) at a concentration of 106 pfu/mL. The Mamavirus was confirmed free of virophages by PCR amplification (using the primers described above) before inoculation. Every day, 750 µL of the suspension was mixed with fresh A. polyphaga and Mamavirus at the same concentrations. Putative growth of virophages was monitored daily by quantitative PCR.To date, virophage cultures have always been obtained by co-isolation with their viral hosts. However, in our study, all attempts to isolate the giant viruses with their virophages in the 5 samples that tested positive using amoeba co-cultures were unsuccessful. Therefore, we designed a new protocol to isolate virophages by inoculating samples on a co-culture of 2 were used. These plates were inoculated with 30 mL of fresh A. polyphaga (5×106 cells/mL) that was previously rinsed in PAS. After one hour of sedimentation, the liquid and surplus of amoeba were removed. Before inoculation, rinsed Mimivirus was suspended in PAS and quantified at 109 infectious particles using an end-point dilution assay. The Sputnik3 aliquots used for inoculation were diluted in PAS and previously filtered through a 0.2 µm pore-sized filter. Sputnik3 was quantified by inoculation of 50 µL of dilutions ranging from the original tube to 10−15 in 500 µL of fresh A. polyphaga (5×105 cells/mL) rinsed in PAS, infected by Mimivirus (50 µL at 109 pfu/mL). Five daily subcultures were performed in Mimivirus-infected A. polyphaga, then 7.5 µL of each well were mixed with 7.5 µL of Mimivirus at 109 pfu/mL and dropped off on plates. Observations three days after the deposits showed smaller diameters of the lysis plaque formations of the originally Sputnik3 not diluted to the 10−4 dilution, and almost the same diameters for the other dilutions of Sputnik3 and the Mimivirus-PAS controls: according to these results, we estimated the concentration of Sputnik3 at 104 pfu/mL. Sputnik3 was quantified at 109 particules by mL using a virus-titer estimation protocol with negative staining observed with electron microscopy. Fifteen-microliter deposits were made on amoeba plates, with 25 deposits per plate. Thirty-four deposits were made with 7.5 µL of Mimivirus at 109 pfu/mL and 7.5 µL of PAS, 34 with 7.5 µL of Mimivirus at 109 pfu/mL and 7.5 µL of Sputnik3 suspension. Deposits of PAS-only and Sputnik3-only were used as negative controls. The plates were checked every day for lysis plaque formation. As the plaques were circular, diameters were measured to calculate mean areas. Statistical comparison was performed by Student's t-Test, using R software (package stats version 2.15.0).Non-nutritive plates containing PAS with 1.5% Agar and 5% of Merck© Hemacolor® solution 3 in square Petri dishes of approximately 520 cmThe sequences of pol B gene of all the giant viruses available in our collection  were comEach of the 17 Mimiviridae in our collection  was inoculated with Sputnik. Using real-time PCR quantification, we demonstrated a 10- to 30-fold increase in Sputnik concentration in most viruses . HoweverImmunofluorescence observations using Sputnik antibodies and DAPI confirmed the results obtained with real-time PCR. We could observe clearly the viral factories (VF) and the production of Sputnik virions at one pole of the VF  as previMegavirus chilensis was used to build a phylogenetic tree (We tested 90 soil samples collected in Marseille and surrounding areas (south of France) for Mimiviridae and virophage using PCR. We detected 9 Mimiviridae: 8 group A strains and 1 group C strain. We detected 5 virophage sequences, one of which was associated with the group C virus mentioned above. Detection of the other 4 virophages was not associated with the detection of a giant virus. The available partial sequence together with both the sequences of the giant viruses that we studied previously and the recently described tic tree  [6], [9]A. polyphaga newly infected by Mamavirus in PYG . Daily subculture was performed at a 1∶2 ratio (a volume of culture in the double of fresh amoeba infected by Mamavirus). Later, we were able to propagate this virophage on Courdo11, a group C strain, and on Moumouvirus, a group B strain. The complete genome sequencing of Sputnik3 (GenBank accession number: JN603370) allowed a comparison with the genomes of virophages Sputnik (GenBank: EU606015) and Sputnik2 (GenBank: JN603369)  . SputnikN603369) . The sec2 to 1.21 cm2 with a mean area of 0.88 cm2 (±0.15 cm2), whereas the plaques for the Sputnik3-infected Mimivirus deposits ranged from 0.22 cm2 to 0.72 cm2 with a mean of 0.56 cm2 (±0.12 cm2) (−15). Cross dilutions of Mimivirus and Sputnik3 revealed that plaque size was not dependent on Sputnik3 concentration, but the deposits with 105 pfu/mL of Mimivirus plaques were smaller and difficult to measure. The effect of Sputnik3 on Mimivirus was confirmed by real-time PCR on co-culture  and extended until the third day –b. No pl.12 cm2) . These d-culture .Our study shows that Sputnik 1 and Sputnik 2 are able to replicate in all of the APM virus factories. Immunofluorescence analyses confirmed the polar production of Sputnik viruses in the periphery of the giant virus factory as described previously Mimiviridae. Sputnik 3 is the first virophage recovered from group C, which contains Megavirus chilensis. The growth of Sputnik 3 in Moumouvirus, the third group of Mimiviruses, further demonstrates that, like Sputnik 1 and 2, Sputnik 3 is able to infect the 3 groups of Mimivirus. This result supports the hypothesis of the ability of virophages to drive gene transfer, potentially contributing to the mosaicism of giant viruses' genomes. This fuels the debate on the real nature of virophagesThe isolation of an additional virophage, Sputnik 3, using an original procedure confirms the capability of virophages to be cultured in viruses of the 3 groups of APM. We speculated that we were not able to isolate the virus detected in the sample from which we isolated the virophage because the amoeba we used was not susceptible to this virus Finally, the procedure reported here, which uses a cultivable helper giant virus, paves the way for the isolation and discovery of new virophages without the isolation of their giant virus host. Isolating virophages using a reporter giant virus is significant because diverse virophage signatures have been identified in nearly all types of aquatic systems by searching the GOS data base and thus are likely to play a key role in the ecosystem regulation of aquatic environments by regulating host-virus interactions Figure S1Number of amoebas in PYG medium.A: The number of viable amoebas present during the infection by giant viruses. B: The number of viable amoebas present during co-infection with giant viruses and Sputnik 1. C: The number of axenic amoebas compared with the number of amoeba infected by Mamavirus . Statistical analysis was performed according to the Wilcoxon Rank Sum test performed with R software using the package stats version 2.15.0 5 .(TIF)Click here for additional data file.Figure S2Position of giant viruses detected in the soil compared to currently known Mimiviridae. Phylogenetic trees based on partial polB gene sequence showing the position of giant viruses detected in the soil  compared to currently known members of the Mimiviridae family presented in our former study .(TIF)Click here for additional data file.Figure S3Alignment of the genomes of the 3 isolates of Sputnik.(TIF)Click here for additional data file.Figure S4Sequences of ORF17 (Gp17), ORF20 (Gp20) and ORF21 (Gp21) in Sputnik, Sputnik2 and Sputnik3. The black line indicates that the sequence is the same that the one just above. The amino acids in red are those that are different between the three Sputnik. In the Gp21 sequence, for which predicted secondary structures are available Click here for additional data file.Figure S5Lysis plaque assay with Mimivirus and Sputnik3. (A) Scan of a colored lysis plaques with A polyphaga monolayer inoculated with Mimivirus  and Mimivirus and Sputnik3  3 days after inoculation; (B) magnification of a right spot; (C) difference of lysis plaques means measured on colored plates 3 days following inoculation, between 34 deposits of Mimivirus and 34 deposits of Mimivirus and Sputnik (Spt3).(TIF)Click here for additional data file.Figure S6Quantification of Mimivirus and amoebal lysis. Quantification of Mimivirus by real-time PCR (curves), with Sputnik3 (red triangle) and without (blue square) from H0 to H24 post-infection, from co-culture in Acanthamoeba polyphaga in PAS (non-infected amoebas were used as negative control and provided no amplification). The bars represent the number of amoebas for each time: Mimivirus-infected amoebas in blue, Mimivirus/Sputnik3-infected amoebas in red and non-infected amoebas in green.(TIF)Click here for additional data file.


Since April 2010, a severe outbreak of duck viral infection, with egg drop, feed uptake decline and ovary-oviduct disease, has spread around the major duck-producing regions in China. A new virus, named BYD virus, was isolated in different areas, and a similar disease was reproduced in healthy egg-producing ducks, infecting with the isolated virus. The virus was re-isolated from the affected ducks and replicated well in primary duck embryo fibroblasts and Vero cells, causing the cytopathic effect. The virus was identified as an enveloped positive-stranded RNA virus with a size of approximately 55 nm in diameter. Genomic sequencing of the isolated virus revealed that it is closely related to Tembusu virus (a mosquito-borne Ntaya group flavivirus), with 87–91% nucleotide identity of the partial E (envelope) proteins to that of Tembusu virus and 72% of the entire genome coding sequence with Bagaza virus, the most closely related flavivirus with an entirely sequenced genome. Collectively our systematic studies fulfill Koch's postulates, and therefore, the causative agent of the duck egg drop syndrome occurring in China is a new flavivirus. Flavivirus is an emerging and re-emerging zoonotic pathogen and BYD virus that causes severe egg-drop, could be disastrous for the duck industry. More importantly its public health concerns should also be evaluated, and its epidemiology should be closely watched due to the zoonotic nature of flaviviruses. Rimerella anatipestifer, and Escherichia coli etc. Duck farming is a traditional agro-business in China and Southeast Asia, and Peking roast duck is well known worldwide. Several infectious pathogens affect the duck industry, such as duck hepatitis virus, duck enteritis virus, Flavivirus, Family Flaviviridae with over 70 serotype members Flaviviruses are single-stranded positive-sense RNA viruses classified in the Genus Starting in April 2010, a severe viral disease spread around the duck-producing regions in China, even in autumn when there is low or no mosquito activity in northern China. The affected ducks  manifest a clinical symptom with heavy egg drop. On some duck farms, the disease was devastating, completely eliminating successful duck reproduction. Therefore this viral disease has caused a serious economic loss. We performed a systematic investigation, from epidemiology, pathogen isolation, virus characterization, disease reproduction by infection with the isolated virus (fulfilling Koch's postulates), to virus genome sequencing and found that the duck egg drop disease was caused by a new flavivirus, BYD virus, that is closely related to Tembusu virus.In April 2010, an egg drop syndrome of unknown etiology was found in several duck farms in Southeast China. Based on our preliminary epidemiological data, the disease quickly spread to most of the duck-producing regions in China including many of the coastal provinces and neighboring regions, Anhui Province, Beijing Autonomous City, Hebei Province, Fujian Province, Guangdong Province, Guangxi Province, Jiangsu Province, Jiangxi Province, Shandong Province and Zhejiang Province . The disAt necropsy, severe ovarian hemorrhage, ovaritis and regression were consistently observed in the affected ducks . RuptureHistopathologically, the most obvious features of the disease were ovarian hemorrhage, follicle atresia and rupture. The ruptured follicles and interstitial tissues were filled with round or granular eosinophilic bodies . Similar5.8ELD50/ml. The virus produced a marked cytopathic effect (CPE) in the primary duck embryo fibroblasts (DEFs) 36 hrs after inoculation. The infected cells ruptured, and eosinophilic particles were found with hematoxylin and eosin (HE) staining (Baiyangdian (BYD) virus (BYDV) after the region name where the virus was first isolated. We isolated a dozen of the BYDV strains  from the affected duck brain and ovary tissues in total but focused on a single strain (BYDV-byd1) for the following studies, including genomic sequencing.Conventional infections with known bacteria or viruses were ruled out from our bacteria/virus isolations, especially. the possible influenza A virus and EDS-76 adenovirus. Thus, we next attempted to isolate any new pathogens. Brain and ovary samples from affected ducks were processed for virus isolation using embryonated duck eggs and embryos died 3–4 days post-inoculation. The allantoic fluids were then harvested for passaging. At the fifth passage, the allantoic fluid had an infectivity titer of 10staining . The virstaining . The iso5.8. to <10 ELD50/ml, indicating that it was an enveloped virus. To determine the nucleic acid type, the virus was assayed in DEF cultures containing 5-bromo-2′-deoxyuridine (BrdU). The infectious titers of the BYDV-byd1, duck reovirus, and pseudorabies virus (RNA and DNA virus controls) in the presence and absence of BrdU were 2×106.4 and 2×106.2, 2×105.4 and 2×105.6, <10 and 2×104 TCID50/ml, respectively  of sera from the recovered ducks (n = 20) were 3-fold higher than the control group (n = 10). This further confirmed the BYDV infection in the duck population.We used the ELISA method for BYDV-specific antibody detection in the convalescent sera. The optical density at 450 nm  infection After the BYDV genomic sequencing, partial E genes (401-bp product) amplified from the suspected brain tissues of the affected ducks were used for clinical diagnoses and virus screening by RT-PCR using specific primer pairs. Representative results of samples from five different provinces are shown in The sudden outbreak and quick spread of an egg drop syndrome in the major duck-producing regions in China has resulted in serious economic loss. This prompted us to conduct a thorough investigation of the cause of the disease. From preliminary epidemiology, pathogen isolation, and reproduction of the clinical disease by the isolated virus, BYDV, Koch's postulates were fulfilled, and a new flavivirus causing a serious duck disease was identified. This is the first severe duck disease caused by a zoonotic flavivirus ever reported. It had a very significant effect on the duck industry, especially in Southeast Asia where duck farming is most popular and mosquitoes are active. Although the transmission vector of the BYDV was not defined in our study, most  of its closely related viruses are transmitted by mosquitoes or ticks. This should be addressed in the future studies . However, it should be noted that the virus infection in egg-laying ducks in this outbreak continued into autumn when mosquito activity is low in northern China.Flaviviruses are notorious for their severe infective and zoonotic nature. Indeed, they can cause serious diseases for both humans and animals. Infections of poultry by flaviviruses are not uncommon, such as West Nile virus The public health significance of BYDV isolation as a duck pathogen can not be underevalud. Potential infection or asymptomatic infection of humans must be evaluated as soon as possible because duck consumption in Asia, especially in China, is extremely heavy. Close contact between humans and ducks or duck products is inevitable. Therefore, for both potential public health concerns and the duck industry, vaccine development against BYDV should be considered. Indeed, several succussful vaccines for flaviviruses have been developed and widely used , so a vaccine for BYDV may be feasible.With frequent isolations, flaviviruses continue to threaten human health and animal industries. Expansion of dengue virus, West Nile virus, and Kyasanur forest virus to the northern hemisphere, as well as the emergnce of new viruses . Ducks in the affected flocks showing morbidity or death within 6 hrs were taken to our laboratory for necropsy. Tissue samples were fixed in 10% neutral buffered formalin solution for histopathology. Simultaneously, brain, ovary and spleen tissues were frozen at −80°C for further analysis. The fixed tissues were routinely processed and embedded in paraffin. Sections (4 µm thick) were prepared from the paraffin blocks and stained with HE.We were notified by the local veterinary laboratories and duck farmers of an unknown egg-drop disease. Clinical examination and the production records were checked for several affected flocks in different areas. To exclude avian influenza virus infection, serum samples were collected from the affected flocks, and antibody titers against H5 subtype avian influenza A virus were tested with hemagglutination inhibition (HI) assays according to the standard method  to give a 20% suspension (w/v). After centrifugation at 3,000 g for 20 min, the supernatants were filtered through a 0.2-µm syringe-driven filter. The filtered suspension was then inoculated into five 10-day-old Pekin duck embryonated eggs (0.3 ml/embryo) via the allantoic sac. Embryos were checked daily, and the amnio-allantoic fluids of the infected embryos were collected for further passage. A suspected virus was isolated and it was named BYDV (The strain for further study in this report is named BYDV-byd1).2 and checked daily for CPE. When 75% of the cells in culture demonstrated CPE, the cultures were harvested to infect fresh cells.For cell culture passage, DEFs were prepared from 12-day-old duck embryos according to standard protocols, and Vero cells were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin and 100 mg/ml streptomycin. Cell monolayers were infected with 10-fold diluted allantoic fluid from the first passage of the duck embryos. The cultures were incubated at 37°C with 5% COAllantoic fluid and the cell culture supernatants from the first five passages were tested for hemagglutinating activity with a 1% suspension of chicken erythrocytes to exclude avian hemagglutinating viruses .To determine whether the isolated virus had a lipid envelope, the allantoic fluid virus (fifth passage) was treated with 5% (v/v) chloroform and the infectivity was assayed by duck embryo inoculation as descried previously 2SO4. The OD450 in each well was measured using an ELISA reader (Thermo Scientific Multiskan MK3). Twenty of serum samples from ducks recovered from outbreaks were tested, and ten of serum samples from normal ducks were used as control. The results are reported as the OD450 of test well/OD450 of the control well.To detect an antibody response to the BYD virus in infected ducks, an ELISA was developed based on the method described by Hlinak et al. For EM, virus-infected and control DEF cells were fixed with 2.5% glutaraldehyde at 24, 36 and 48 hrs post-infection. Ultrathin sections were processed and stained with uranyl acetate and lead citrate. The samples were then examined with a Hitachi TEM H-7500 as described previously 5.8 ELD50/ml), respectively. Additionally, fifty ducks were mock-infected with sterile PBS in the same manner. Ducks were monitored daily for egg production and killed on days 3, 4, 5 and 6 post-infection. Tissues were collected for pathological examination and virus isolation. DEF cultures were used to recover the virus from brain and ovary samples. The recovered virus was confirmed by RT-PCR and subsequent sequencing.To evaluate the pathogenicity of the BYDV, experimental infection was conducted in 96 440-day-old healthy laying ducks (no SPF ducks were available). Ducks were transferred to our animal facility and adapted to the new environment for 5 days to minimize the effect of shipping stress. Forty-six ducks were simultaneously infected by intramuscular and intranasal injection with 0.6 ml and 0.4 ml of the BYDV-byd1 virus . Animal infection experiments were conducted under the protolcol (CAU-AEC-2010-0603) approved by the China Agricultural University Animal Ethics Committee.Ultracentrifuge-purified BYDV-byd1 virus (fifth passage on DEF monolayer) was used for virus genome amplification. Because all of the aforementioned measures indicated that BYDV was an RNA virus, viral RNAs were extracted using a QIAamp Viral RNA Mini kit (Qiagen) following the manufacturer's protocol. cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Initially we attempted to randomly amplify viral gene fragments by the random priming method


Until recently the function of gPr80gag was unclear, but we have recently shown that gPr80gag facilitates viral budding and release through lipid rafts. Glyco-gag mutant virus is less efficiently released from fibroblasts, mutant virus has lower cholesterol content (and higher buoyant density), and less Gag precursor polyprotein (Pr65gag) is associated with detergent-resistant membranes in infected cells. The mechanism by which gPr80gag facilitates virus release is under investigation. The N-terminal 88 amino acids of gPr80gag are sufficient to enhance virus release, indicating that the Gag sequences on this protein are not required. Cellular La protein is involved in gPr80gag function, since over-expression of La phenocopies gPr80gag in facilitating virus release, and knockdown of La abrogates gPr80gag-enhancement of virus release. MuLV glyco-gag can also facilitate release of HIV-1 particles from transfected cells, which suggests that these two viruses may share mechanisms for directing virus release through lipid rafts. gPr80gagalso counteracts the murine APOBEC3 restriction factor (mA3) since gPr80gagmutant virus shows a defect for establishment of infection in wild-type mice, but not in mA3 knockout animals.Murine leukemia viruses (MuLVs) encode an alternate form of Gag protein in addition to the standard Gag precursor polyprotein, glycosylated Gag (glyco-gag or gPr80