A total of 215 compounds inhibited E-S-FLU virus infection, while only 22 inhibited the control H5-S-FLU virus coated in H5 hemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action, e.g., calcium channel blockers, estrogen receptor antagonists, antihistamines, serotonin uptake inhibitors, etc., and this correlates with inhibitor screening results obtained with other pseudotypes or wild-type Ebola virus in the literature. The E-S-FLU virus is a new tool for Ebola virus cell entry studies and is easily applied to high-throughput screening assays for small-molecule inhibitors or antibodies.We have produced a new Ebola virus pseudotype, E-S-FLU, that can be handled in biosafety level 1/2 containment for laboratory analysis. The E-S-FLU virus is a single-cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza virus hemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU virus production. Infection of cells with the E-S-FLU virus was dependent on the Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. The E-S-FLU virus was neutralized specifically by an anti-Ebolavirus glycoprotein antibody and a variety of small drug molecules that are known to inhibit the entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPACIMPORTANCE Ebola virus is in the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates for Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus and screen for inhibitors. Here we characterized a new surrogate, named E-S-FLU virus, that is based on a disabled influenza virus core coated with the Ebola virus surface protein but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU virus uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU virus in biosafety level 1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small-molecule library of 1,280 candidates for inhibitors of viral entry. Filoviridae family  is the only protein present at the virus surface. EBOV-GP plays an important role in virus cell entry, and it is the key target for neutralization by antibodies . Currentelations . Therefoin vitro studies.Here we describe a new Ebola virus pseudotype (E-S-FLU) based on a nonreplicating influenza virus, the S-FLU virus . Influen13–The S-FLU virus is a pseudotyped influenza virus with its endogenous HA sequence removed, and it was previously pseudotyped with different subtypes of influenza virus HA , 22. To 50) dilutions of the E-S-FLU virus at 48 h were ∼1:8, compared to ∼1:500 for the H5-S-FLU virus (50 dilution and the number of cells per well (3 × 104), the 50% cell infective dose (CID50)/ml was calculated as ∼2 × 106 in the E-S-FLU virus batch and ∼1.6 × 108 in the H5 S-FLU virus batch. However, the harvested culture supernatant from E-SIAT cells contains adequate pseudovirions for 50 μl of a 1:4 dilution to give close to full infection of the 3 × 104 cells in a well of a 96-well plate. No further purification or concentration steps are needed for the screening of inhibitory drugs or antibodies. With the same protocol, we were also able to pseudotype the S-FLU virus with Bundibugyo, Sudan, and Mayinga-Zaire EBOV-GP to similar viral titers (data not shown).Cloned seed S-FLU virus that is coated in influenza virus HA was added to propagate in E-SIAT cells to produce the EBOV-GP-pseudotyped S-FLU virus. After 48 h, culture supernatants were harvested and titrated on indicator MDCK-SIAT1 cells for infection. Infected MDCK-SIAT1 cells express eGFP that is encoded in the virus. We named the new pseudotype E-S-FLU. The E-S-FLU virus reaches a lower titer than viruses grown in H5 HA-transduced producer cells and forms smaller, more diffuse plaques than the H5-S-FLU virus . The typLU virus . From thIt has been shown previously that the transmembrane and cytoplasmic domains of influenza virus HA are important for influenza viral particle assembly –30. To p–The Niemann Pick C1 (NPC1) protein has been identified as the key entry receptor for Ebola virus at the level of late endosome/lysosome membrane –33. We i–To further validate our new EBOV-GP-pseudotyped E-S-FLU virus, we tested it in the antibody microneutralization (MN) assay in comparison with control pseudotype H5-S-FLU virus and an EBOV-GP-coated lentivirus pseudotype (GP-lenti). The H5-S-FLU virus contains exactly the same S-FLU virus core as the E-S-FLU virus but differs at the surface glycoprotein. GP-lenti is a lentivirus pseudotyped with the full-length Zaire strain EBOV-GP (wt/GIN/2014/Kissidougou-C15) matched to the E-S-FLU virus.50 similar to what has been reported for wild-type Ebola virus , while in vitro. To verify the use of the E-S-FLU virus in drug screening assays, we selected several types of drug molecules that have been tested repeatedly in the literature and screened them against the E-S-FLU virus in our drug inhibition assay. 50s) were in the same range as those in the literature.In the literature, several drug screening studies have been done with wild-type Ebola virus –41 and E–50 and 95% confidence interval (CI). We also screened these drugs against the H5-S-FLU virus . Most of them did not inhibit the H5-S-FLU virus, except niclosamide at 2.64 μM. N-acetyl-d-glucosamine and sialic acid. Cells in good condition will remain adhered to the plate during analysis and can be detected with WGA. Dead or unhealthy cells will detach and be washed away. WGA staining allows quick indication of drug toxicity in high-throughput plate screening assays. This is verified by light microscopy (All of the screenings in croscopy  and D.N-ethyl-N-isopropyl)amiloride (EIPA)  , 46, MDLe (EIPA) , 47, E64e (EIPA) , 48–50, EIPA) 48–, 42. For1280 library (Sigma-Aldrich) in our drug inhibition assay to test their effects on E-S-FLU and H5-S-FLU virus infections. This library contains 1,280 pharmacologically active small molecules with different molecular targets. All of the drugs were screened in the first round at 100 and 5 μM. Selected drugs were then titrated once in a drug inhibition assay to calculate their IC50s.Having established that infection with the E-S-FLU virus was dependent on the NPC1 receptor and was inhibited by specific antibodies and established small molecules, we proceeded to a formal drug screening. We screened 1,280 drug molecules from the LOPACin vitro  as the drug concentration increased. Twelve drugs, however, caused a reduced overall fluorescence level of infected cells without reducing the number of fluorescent cells significantly. All 12 of these drugs also affected H5-S-FLU virus infection. This suggests that they acted at a postentry phase by limiting the expression of viral proteins. In 50s of all 228 molecules that inhibited the E-S-FLU virus are plotted in 1280 library and the additional 13 individual drugs we tested in addition to this library.The IC1280 screening only and 26 were positive in the screening done by Johansen et al. only. We used a two-tailed Fisher exact test for statistical analysis of the comparison, and the correlation was significant (P < 0.0001).We compared our screening results with those recently reported by Johansen et al. , who use50 of <10 μM in our assay (Table S1). For the drugs that were positive in the LOPAC1280 screening only, we selected ebastine (E9531), imipramine (I7379), and verapamil (V4629) and ordered them individually from Sigma to confirm their inhibitory effects. All three drugs had an inhibitory effect on the E-S-FLU virus with IC50s comparable to those in the LOPAC1280 library screening. All three drugs have previously been reported as inhibitors , bepridil (B5016), calcimycin (C7522), D609 (T8543), diphenyleneiodonium (D2926), doxylamine (D3775), linezolid (PZ0014), naproxen (M1275), nocodazole (M1404), perphenazine (P6402), rotenone (R8875), sanguinarine (S5890), serotonin (H9523), and thapsigargin (T9033).Of the 26 drugs that were positive in the screening done by Johansen et al. but negative in our LOPAC1280 library did not inhibit the E-S-FLU virus, but the individually ordered samples showed specific inhibition of the E-S-FLU virus . This indicates some variation due to false negatives in the library preparation from Sigma.Bepridil  and perphenazine (a cationic amphiphile) from the LOPACD609, doxylamine, disopyramide, linezolid, naproxen, rotenone, and sanguinarine were confirmed as noninhibitors of the E-S-FLU virus in our assay up to 100 μM. Auranofin, calcimycin, diphenyleneiodonium, nocodazole, and thapsigargin were toxic to MDCK-SIAT1 cells even at 0.2 μM, yet some cells were still infected. None of the drugs that were further tested inhibited the H5-S-FLU virus.The design of the E-S-FLU virus was based on a pseudotyped influenza virus, the S-FLU virus, that was previously developed by our group and is similar to other single-cycle influenza virus pseudotypes , 22, 51.To validate the E-S-FLU virus as a useful surrogate for Ebola virus, we showed that infection is fully dependent on the NPC1 receptor , is inhi37–in vitro by Ebola virus . Out of these, 442 molecules had concordant results in the two screenings. The correlation was statistically significant (P < 0.0001). It is noteworthy that we identified at least 17 calcium channel inhibitors as specific inhibitors of E-S-FLU virus infection. Independent evidence exists for a possible role for calcium channels in the lysosomal membrane in viral entry  and a hydrophobic end (high logP), and are therefore likely to concentrate in lysosomes –59. The –in vitro  and so mMany of these drugs have been found to have a risk of causing long-QT syndrome, such as amiodarone, toremifene, and clarithromycin , 64. Inh65–50s of these agents were similar for the E-S-FLU and H5-S-FLU viruses, which is consistent with their acting on functions controlled by the influenza virus core, which is common to both. We suggest that they affect the virus at a postentry level like faviparivir, a specific inhibitor of RNA-dependent RNA polymerase . In contrast, in some cases, the drugs did not reduce the number of infected cells but reduced the intensity of fluorescence expression in the infected cell population. Of the 228 drugs tested, 12 reduced the fluorescence intensity . All of lymerase –75 that nhibitor –79; and hibitor –, 80, 81,50s on H5-S-FLU virus (3.3 μM) and E-S-FLU virus (1.7 μM) entry. Arbidol may inhibit the fusion of viral and lysosome membranes in a manner related to its effect on influenza virus HA  and niclosamide may act by reducing the lysosomal proton concentration, which can influence the efficiency of cathepsin in endosome-digesting EBOV-GP . Niclosavirus HA –87. Althirus HA –; it may In summary, we have developed a new surrogate E-S-FLU virus for Ebola virus. It mimics the Ebola virus at the level of cell entry and can be a valuable tool for the screening of antibodies and therapeutic drugs, as well as studying the fundamental biology of the Ebola virus entry process.KJ660346.1) was human codon optimized and synthesized by GeneArt. Lentiviral vector pHR-SIN  that abrogates an out-of-frame ATG, which had interfered with downstream eGFP expression. All subsequent versions of the S-FLU virus contain this enhancement, which gives a sufficiently bright fluorescence signal from infected cells to read on the CLARIOstar fluorescence plate reader. Seed viruses were doubly cloned by limiting dilution before the seeding of E-SIAT or H5-SIAT producer cells.The E-S-FLU virus was generated on the basis of a nonreplicating pseudotyped influenza virus (S-FLU) that was previously described , 90. We 50) assay on MDCK-SIAT1 cells by fluorescence detection (as described below). Titration experiments revealed that a relatively high MOI of approximately 1 TCID50 of seed S-FLU virus per cell added to infect E-SIAT cells produced the highest titers of the E-S-FLU virus, whereas a lower MOI (∼0.01) was adequate for seeding of HA-SIAT cell lines  by a standard limiting-dilution method as described by Powell et al. . Medium was removed from the wells, and cells were fixed in 10% formalin (in PBS) for 30 min at 4°C. The virus titer was assessed by eGFP fluorescence analyzed with a plate reader (CLARIOstar). The dilution of virus giving 50% of the maximum plateau fluorescence signal (EC50) was calculated by linear interpolation. The EC50 dilution obtained by best fit of the titration curve to the Poisson distribution was very similar (data not shown). The typical EC50 dilutions for the E-S-FLU virus harvested after 48 h were ∼1:8, compared to ∼1:500 for the H5-S-FLU virus (50 dilution and the number of cells per well (3 × 104), the CID50/ml was calculated as ∼2 × 106 in the E-S-FLU virus batch and ∼1.6 × 108 in the H5 S-FLU virus batch. For inhibition assays, the H5-S-FLU and E-S-FLU viruses were used at a concentration that gave close-to-maximum plateau fluorescence in infected MDCK-SIAT1 cells . This value was used to normalize the fluorescence readout to a percentage of the maximum in the figures (Batches of the E-S-FLU and H5-S-FLU viruses were titrated for infectivity of MDCK-SIAT1 indicator cells after overnight infection. Harvested culture supernatants (100 μl) containing the E-S-FLU or H5 S-FLU virus were titrated in 2-fold dilutions in VGM across a flat-bottom 96-well plate . MDCK-SILU virus . From th figures .In this study, all of the evaluation and screening was done with the Zaire (C15) E-S-FLU and H5-S-FLU viruses in parallel.Fluorescence plate readout was performed with Costar 96-well cell culture plates (Costar no. 3799). We chose the COSTAR 96 microplate mode in the CLARIOstar software. For measurement of eGFP levels, fluorescence excitation was at 483 nm (bandwidth of 8 nm) and emission was at 515 nm (bandwidth of 8 nm) with a 499-nm dichroic filter. Each well had 50 flashes at a 4-mm diameter and fluorescence readout from the top optic, giving the orbital averaging value. For measurement of WGA (Alexa Fluor 647 conjugate) in the drug inhibition assay, fluorescence excitation was at 630 nm (bandwidth of 30 nm) and emission was at 678 nm (bandwidth of 20 nm). Each well had 53 flashes at 4 mm and fluorescence readout from the top optic, giving the orbital averaging value. The focal length and gain were adjusted before each plate was read. The generally optimal focal length was 5.0 mm, the gain of the eGFP channel was adjusted to 3,000, and the gain for WGA-647 was adjusted to 2,500.The NPC1 protein is the key receptor for Ebola virus cell entry at the level of the lysosomal membrane –33. Two –5 wild-type, ex2 NPC1-KO, or ex4 NPC1-KO HeLa cells were seeded into a 24-well tissue culture plate in 500 μl of D10 medium  the night before infection. Before infection, the medium was removed and cells were washed once with PBS. Cells were infected with maximal doses of 1 × 106 CID50 of the E-S-FLU virus  or 8 × 107 CID50 of the H5-S-FLU virus  and incubated overnight. After infection, cells were trypsinized and transferred to 5-ml polypropylene tubes. Percent infection was analyzed by counting fluorescent cells in a flow cytometer .A total of 1 × 10in vitro. The assay was done as described by Powell et al. (50/cell) and H5-S-FLU  viruses were diluted in VGM to give plateau expression of eGFP in 3 × 104 MDCK-SIAT1 cells after overnight infection in 96-well flat-bottom plates. A 50-μl volume of virus was preincubated with 50 μl of antibody at 2-fold dilutions for 2 h. A total of 3 × 104 MDCK-SIAT1 cells in 100 μl of VGM were then seeded into each well, and overnight infection was allowed . After infection, the medium was removed from the wells and the cells were fixed with 100 μl of 10% formalin (in PBS) for 30 min at 4°C; the formalin was then replaced with 100 μl of PBS. The plates were analyzed with a fluorescence plate reader (CLARIOstar) and by fluorescence microscopy. The IC50 was calculated by linear interpolation. KZ52 is a human MAb that specifically binds Zaire EBOV-GP and neutralizes wild-type EBOV in vitro (3CSY)  for 5 min, and then 50 μl of Bright-Glo (Promega) substrate was added for a 5-min enzymatic reaction. Solutions were transferred to opaque plates for luminescence measurement with the GloMax-Multi detection system (Promega).The EBOV-GP-pseudotyped lentivirus (GP-lenti) we used in this study is coated with the same EBOV-GP from Zaire C15 as the E-S-FLU virus. GP-lenti carries a firefly luciferase reporter gene. Fifty-microliter volumes of GP-lenti pseudotyped virus were preincubated with 50 μl of antibodies at 2-fold dilutions for 2 h, and 3 × 101280 library used in this study, were ordered from Sigma-Aldrich and stored in 10 mM dimethyl sulfoxide at −20°C, with the exception of AY9944, which was ordered from Merck , and trans-Ned19 . Drugs were diluted in VGM to give a starting concentration of 100 μM (unless stated otherwise) and then titrated in 50 μl of VGM at 2-fold dilutions across a 96-well plate. A total of 3 × 104 MDCK-SIAT1 cells were seeded in 50 μl of VGM and preincubated with drugs for 3 h . A 100-μl volume of the E-S-FLU  or H5-S-FLU  virus diluted in VGM to give plateau expression of eGFP in 3 × 104 MDCK-SIAT1 cells was then added for overnight infection. Cells were fixed with 100 μl of 10% formalin (in PBS) for 30 min at 4°C, the formalin was then replaced with 100 μl of PBS, and the plates were analyzed with a fluorescence plate reader (CLARIOstar) as for the MN assay. Infection and inhibition were quantified by measurement of the eGFP expression level. When more than three measurements were made for a drug, the 95% CI was calculated  was added to each well and incubated at room temperature for 15 min. Plates were then washed twice with PBS before being read (see the plate reader setting mentioned above).Because of the toxic effect on cells of many drug molecules at high concentrations, dead cells were washed away before plate reading. This may give a falsely low eGFP fluorescence reading in the plate reader but can be detected by visual inspection in a fluorescence microscope. To estimate the number of cells remaining in each well, plates were stained with WGA after cells were fixed and washed once with PBS. WGA is a lectin that binds to sialic acid and in vitro. In their Table S1, they listed all of the experimental data for the small molecules they screened in their study. Of the 2,635 molecules they tested, 538 were also found in the sigma LOPAC1280 library and were screened against the E-S-FLU virus in our study. We compared the inhibitory effects of these 538 compounds in both screenings  and performed a Fisher exact test to measure the significance of the association.Johansen et al.  screenedFor the drug molecules listed in


Lavidaviridae. Virophages are small viruses with approximately 35–74 nm large icosahedral capsids and 17–29 kbp large double-stranded DNA genomes with 16–34 genes, among which a very small set is shared with giant viruses. Virophages have been isolated or detected in various locations and in a broad range of habitats worldwide, including the deep ocean and inland. Humans, therefore, could be commonly exposed to virophages, although currently limited evidence exists of their presence in humans based on serology and metagenomics. The distribution of virophages, the consequences of their infection and the interactions with their giant viral hosts within eukaryotic cells deserve further research.Virophages replicate with giant viruses in the same eukaryotic cells. They are a major component of the specific mobilome of mimiviruses. Since their discovery in 2008, five other representatives have been isolated, 18 new genomes have been described, two of which being nearly completely sequenced, and they have been classified in a new viral family,  Formerly, viruses were considered to be small particles with the ability to pass through 0.2 μm pore filters and remain invisible under a light microscope, in contrast to microbes . The conAcanthamoeba polyphaga mimivirus (APMV) was the first giant virus of amoeba discovered . Si. Si17]. All isolated virophages are non-enveloped viruses with small icosahedral capsids approximately 35–74 nm in diameter. Three-dimensional structural studies have only been carried out on Sputnik virophages, using cryo-electron microscopy [The replicative cycle of virophages, including its effects on the host virus, has only been described for Sputnik, in association with mamavirus ,36. The The specific strategies concerning how the virophage can enter eukaryotic cells and replicate in its host giant virus factory are still not clear. Shortly before their entry into amoebae, virophages were observed attached to fibrils present on the surface of the giant virus, and it has been hypothesized that these fibrils were, therefore, essential to virophage entry ,36,37. IBetween one and two hours post infection (h.p.i.), endocytic vacuoles can be observed in the amoebal cytoplasm. Then, an eclipse phase occurs that lasts approximately 2–4 h. During the eclipse phase, genome replication, transcription, and translation initiate. Both Sputnik and mimivirus replication occur in clearly-defined dense cytoplasmic areas, apart from the nucleus of the amoeba, named viral factories, which are centered around a small spherical compartment. At this stage, it is not possible to observe and isolate a virophage particle.After the eclipse phase, the viral factory expands, and virophage progeny virions begin being produced at one pole of the giant viral factory, before the production of mimivirus virions . OccasioThe Zamilon virophage is selective and propagates only when associated with mimiviruses from lineages B or C . Its repCafeteria roenbergensis, a marine phagotropic grazer, in the virus factory of the only giant virus known to infect this eukaryote, Cafeteria roenbergensis virus (CroV). Transmission electron microscopy shows the mechanism of entry of mavirus, which differs from that of Sputnik due to the fact that mavirus is endocytosed independently of CroV, most likely via the clathrin-mediated endocytosis pathways [Cafeteria roenbergensis survival.The mavirus virophage replicates in pathways . As in SCurrently, 18 full or partial genomes of virophages are available in the GenBank database .Some of the virophages have been isolated with their giant virus host using co-culture on eukaryotic cells they co-infect.Since the discovery of the first Sputnik virophage in 2008, three other closely-related Sputnik-virophages have been isolated. Sputnik 2 was described in 2012 with lentillevirus, a mimivirus belonging to lineage A. It was isolated from contact lens storage case liquid of a 17-year-old myopic woman presenting with keratitis  Table 1Table 1. Acanthamoeba, and they lead to abnormal virions and decreased infectivity and lytic ability of these mimiviruses. All Sputnik isolates have circular dsDNA genomes. These genomes are made of 18,343 bp for Sputnik (NC_011132), 18,338 bp for Sputnik 2 (NC_023846), and 18,338 bp for Sputnik 3 (NC_023847); the Sputnik Rio Negro genome is not yet available. The Sputnik 1, 2, and 3 genomes differ from each other by fewer than 10 base pairs and they have a low G + C content, as in mimiviruses [Sputnik virophages replicate in the presence of mimiviruses of iviruses ,18,26,39iviruses .Mavirus was the second virophage discovered . Its virMimiviridae [Megavirus chiliensis  and one ORF has significant homology with a Moumouvirus monve ORF  [Thereafter, Zamilon (‘neighbor’ in Arabic) was co-isolated in 2013 from Tunisian soil with the Mont1 virus, which belongs to the lineage C of iviridae . Its virty, 72%) . MoreoveMegavirales sequences. A total of 13 complete and partial genomic sequences of virophages were assembled based on the metagenomic sequences generated from Organic Lake in Antarctica, Dutch coastal waters, Yellowstone Lake, Ace Lake, a bioreactor, Qinghai Lake, and Dishui Lake. However, in most cases, the cellular and viral hosts for these virophages are unknown.Several metagenomes have been investigated in previous studies and screened for the presence of virophages and The third virophage, Organic Lake virophage (OLV), was the first virophage detected in 2011 by metagenomics, from a sample collected in Organic Lake, a hypersaline meromictic lake in Antarctica . In thisPhaeocystis globosa virophage may have lost structural genes, apart from a distant version of the major capsid protein (MCP) encoding gene [P. globosa culture cells. Santini et al. suggested that this element replicates as a provirophage integrated in the genome of its host virus or as a linear plasmid [In 2013, analysis of metagenomes generated from Dutch coastal waters (southern North Atlantic and the North Sea) revealed the presence of a virophage-like genome. The assembly of Phaeocystis globosa virus strain PgV-16T (PgV-16T), a distantly related mimivirus, generated a 19,527-bp long scaffold in greater copy number than the PgV-16T virus corresponding to a virophage genome. Sixteen predicted ORFs were found in its genome but most of them had no significant matches in sequence databases. Three ORFs had homologs in mavirus, including an endonuclease, the predicted DNA primase and polymerase and one ORF had a homolog in the OLV virophage. The ing gene . No viri plasmid .In 2013, a nearly complete genomic sequence from a virophage, named Ace Lake mavirus (ALM), was retrieved from an Ace Lake sample in Antarctica . It was In 2013 and 2015, seven additional complete virophage genomic sequences (YSLV1-YSLV7) were obtained from Yellowstone Lake metagenomes. This suggested a substantial diversity of giant viruses and the eukaryotic hosts in this environment ,31. ThesRecently, Yutin et al. detected sequences of virophages corresponding to MCP from several metagenome datasets . Thus, 1In 2015, the assembly of the partial genome of a Zamilon-like virophage was described from a non-aerated bioreactor metagenome, in which Yutin et al. concurrently detected Zamilon-related capsids sequences . ComparaBigelowiella natans were described, and found to be highly transcribed [Megavirales members and repeated elements that matched with transpovirons. Based on these findings, it was suggested that the genome of these chlorarachniophytes could have recorded genetic prints of virophages to use them as a molecular weapon against giant viruses, conferring a sort of immunity.In 2015, virophage genomes integrated into the nuclear genome of the unicellular alga nscribed . In the A novel group of virophages was discovered in 2016 from Dishui Lake, an artificial freshwater lake in Shanghai, China. A complete genomic sequence from this virophage, named Dishui Lake virophage (DSLV1), was obtained . Its assIn 2016, another new virophage was described from a metagenome of a planktonic microbial community of Qinghai Lake, a Tibetan mountain lake. A complete genomic sequence from this virophage, named Qinghai Lake virophage (QLV), was obtained, which contains 23,379 bp, has a G + C content of 33.2%, and encodes 25 ORFs . QLV shaMany virophage capsid homologs were detected in different metagenomic datasets generated from microbial communities sampled in lakes, rivers, Antarctic lakes, and freshwater lagoons, . They are also detected from the metavirome of distinct Antarctic hyperarid desert soil communities, activated sludge metagenome, freshwater sediment metagenome, gut metagenome, marine metagenome, and wastewater metagenome, testifying to the widespread occurrence of virophages in the biosphere ,47. TakeLavidaviridae, for virophages, which includes Sputnik, Zamilon and mavirus isolates [Among the 20–34 ORFs present in virophage genomes, six genes are conserved in most or all virophages and are considered as virophage core genes . These sisolates .In a phylogeny reconstruction based on the MCP of all known virophages, all Sputnik and Zamilon virophages form a robustly supported monophyletic group . In addition, mavirus is clustered with ALM, DSLV1 is clustered with YSLV3, and YLSV2 is clustered with YSLV6, with high reliabilities  .Lavidaviridae (Lavida is for LArge VIrus-Dependent or Associated virus) for virophages, which has been officially recognized by the ICTV [The diversity of virophages has considerably expanded during the past eight years. A debate on the classification of virophages as a satellite virus occurred ,51,52. Tthe ICTV .Virophages are a component of the mobilome of mimiviruses, together with self-splicing introns and transpovirons . They caMimiviridae family [Since the discovery of the first giant virus, several other mimiviruses have been isolated from different environments ,55,56. Pe family ,58,59. Te family ,60, and e family . Like ane family . Howevere family . Levassee family . Its dise family . These se family . It has e family . FurtherDuring the past four years, metagenomic analyses have revealed the presence of sequences related to virophages in various environmental samples, from deep ocean to inland, and collected on different continents ,30,47. VThe association of virophages with humans is still poorly known. Human exposure to giant viruses and to virophages should be concomitant. Senegalvirus, Shan virus, and megavirus LBA111 represent the first cases of isolation of giant viruses from human fecal or lung samples ,58,65. IVirophages are recently discovered viruses with an apparent broad worldwide distribution. Their discovery has occurred consequent to that of mimiviruses. They have contributed to differentiating mimiviruses from other viruses, bringing them closer to microbes. Indeed, they have revealed that mimiviruses, similarly to prokaryotes, could be infected by other viruses that integrate into their genome, and that mimiviruses have developed a mechanism of defense against these virophages. The virophage diversity will probably expand considerably in the near future and stimulate research to determine to what extent they infect giant viruses other than mimiviruses. The study of virophage origins, of their involvement in sequence exchanges between organisms, and of their impact on ecosystems is of substantial interest. Finally, based on preliminary findings, their association with humans warrants further investigation.


Cryptococcus gattii-induced cryptococcosis is an emerging infectious disease of humans and animals with worldwide distribution and public health importance due to its significant morbidity and mortality rate. The present study aimed to report a case of pulmonary infection by C. gattii molecular type VGII in State of São Paulo, Brazil.Cryptococcus spp. Internal transcribed spacers and orotidine monophosphate pyrophosphorylase nucleotide differentiation demonstrated that the isolate corresponds to the C. gattii VGII molecular subtype.A 5-year-old goat showing intermittent dry cough, ruminal tympany, anorexia, fever, tachycardia and tachypnea was presented for necropsy at the Veterinary Hospital of the School of Veterinary Medicine and Animal Sciences, São Paulo University, São Paulo, Brazil. Postmortem examination revealed numerous 2.0–6.0 cm diameter yellow gelatinous pulmonary masses. Tissues were evaluated by a combination of pathological, mycological, and molecular diagnostic techniques. Microscopically, pneumonia granulomatous, multifocal to coalescing, moderate, with many intralesional carminophilic yeasts was observed. The immunohistochemistry and mycological culture confirmed C. gattii molecular type VGII in Southeastern Brazil. Our findings emphasize the need for an active surveillance program for human and animal new infections to improve the current public health policies due to expansion of the epidemiological niche of this important microorganism.To our knowledge, this is the first report of a pulmonary infection in a goat linked to  Cryptococcus neoformans and Cryptococcus gattii  and 99% homology to IDR1100011626 (GenBank acc # JN675352). Moreover, orotidine monophosphate pyrophosphorylase gene (URA5) signature sequence from this isolate exactly matched that of C. gattii corresponds with PCR-fingerprint molecular type VGII.The sequencing results showed 100% homology to a region of the internal transcript sequence 1 (ITS1) of the ribosomal DNA amplified with C. gattii is recognized as the main agent of endemic primary cryptococcosis in the North and Northeast (NE) Region of Brazil, where it is responsible for 89% of human cryptococcosis, and has the potential to cause life-threatening disease in immunocompetent hosts and [C. gattii infections, were described in children in Pará [C. gattii is an endemic primary mycosis affecting HIV-negative hosts, with most cases caused by molecular type VGII, including an unexpectedly high number of children, [osts and . The firosts and . In 1999 in Pará . In the hildren, , 23.C. gattii infections are caused by VGII [C. gattii has been the agent of one outbreak in captive psittacine birds in São Paulo, Brazil [Most of the clinical isolates of VGII in the states of Rio de Janeiro and São Paulo have been collected from patients born in Northeastern Brazil . In Sout by VGII . However, Brazil .C. gattii molecular type VGII was described as a primary emerging pathogen on Vancouver Island, Canada, and, subsequently, spread to people and animals in British Columbia, Washington and Oregon. Retrospective studies suggested that C. gattii may have circulated in Southern California for much longer. The mechanism of the switch from tropical to temperate climates is unknown. However, most eucalyptus associated outbreaks in Australia are of molecular type VGI, whereas 90% of isolates from the Pacific Northwest are type VGIIa, and southern California isolates are type VGIII, the genotype commonly identified in Mexico. This suggests that different genotypes have different biogeoclimatic distributions [Also, ibutions –28.C. gattii can acts as a primary pathogen and causes endemic cryptococcosis, including meningoencephalitis, in HIV-negative patients. However, fully competent immune response in these hosts has been questioned, since infection with C. gattii is increasingly linked to the presence of various types of autoantibodies, which interfere with the host defenses, like anti-granulocyte-macrophage colony-stimulating factor [g factor .Cryptococcus is the most common systemic mycosis in cats and often results in a severe disseminated disease in dogs [C. gattii can be attributed to three factors: environmental expansion, a lack of diagnostic tests, and the increased frequency of identification by molecular detection procedures [ in dogs . Accordi in dogs , the larocedures . Epidemiocedures . Byrnes ocedures , emphasiC. neoformans, infections with C. gattii might need a more aggressive antifungal therapy due to the higher probability of severe neurological complications and possible delayed response to used antifungal compounds in humans [C. gattii strains, which might affect the outcome of antifungal therapy, and which is an indication that in vivo differences may exist [Cryptococcus spp. was established on available literature in horses, cats and dogs [Treatment options for cryptococcal infections are dependent on the severity and localization of the infection. In humans, treatment guidelines from the Infectious Diseases Society of America (IDSA) recommend treatment of severe cases of cryptococcosis in immunocompetent and immunocompromised patients using induction therapy for 2 weeks with a combination of amphotericin B and 5-flucytosine, followed by 2 weeks of consolidation therapy using fluconazole . Comparen humans . It has ay exist . Dose anand dogs –39. Howeand dogs .C. gattii in South America. In fact, it behaves as a primary fungal pathogen and causes endemic cryptococcosis in immunocompetent hosts in the northern region of Brazil, where it is particularly well-adapted to environmental biotypes associated with wood decay. However, this molecular type may be adapting to new areas in the southeast due to human activities and global climate change, or it may be present at a low density in the southeast, causing occasional human infections [Despite the few epidemiological studies available in humans and animals, molecular subtype VGII does not seen to be a rare genotype of fections .C. gattii molecular type VGII in a goat in state of São Paulo, Brazil. Our findings emphasize the need for an active surveillance program for human and animal infections due to expansion of the epidemiological niche of this important zoonotic microorganism.Histopathology, immunohistochemistry, microbiology, and molecular test are important to determine infectious agents and their differential diagnosis in cases of systemic respiratory diseases in small ruminants. To our knowledge, this is the first report of a pulmonary infection linked to


Costs with dementia have been the focus of research around the world and indirectcosts to the caregiver appear in the literature as responsible for the greatestimpact. In Latin American (LA) studies, indirect costs with dementia range from60% to 75% of family income.To present preliminary results of the study "Description of the methods andcost analysis with dementia" currently being conducted at the Behavioral andCognitive Neurology Unit of Hospital de Clínicas of University ofSão Paulo - HC-FMUSP.A cross-sectional study which, to date, includes interviews of 93 primarycaregivers. The research protocol includes a sociodemographic questionnaire,the Functional Assessment Staging (FAST) scale, the Burden Interview(Zarit), an economic classification scale, and the Resource Utilization inDementia (RUD) scale.Monthly indirect costs were US$ 1,122.40, US$ 1,508.90 and US$ 1,644.70stratified into mild, moderate and severe dementia, respectively. Theprojected annual indirect costs were US$ 13,468.80, US$ 18,106.80 and US$19,736.40, representing 69 to 169% of family income.This small sample showed that the impact of indirect costs with dementia inBrazil may be higher than that reported in other Latin American (LA)studies. These initial results may represent an important contribution forfurther research on costs with dementia in LA. Thus, at some point in thedisease course, the need arises for specific pharmacological and non-pharmacologicaltreatment given by a multidisciplinary team.10 In addition, informal caregivers (family member orclose friend) or formal caregivers (paid caregiver or nursing home placement) may berequired.12Dementia can be considered a multifactorial syndrome that rapidly or slowly imposeson individuals affected a serious loss of higher cortical functions.1 Informal care costs represent the greatest burden, which in 2013accounted for about US$ 216 billion, corresponding to 17.5 billion hours expended byinformal caregivers on patients.2Meeting these requirements renders the costs of the illness an important economic andsocial impact on monthly income of families, and exert a much larger social burdenthan some societies can afford, especially low income ones. With regard toexpenditure on dementia, recent data from international organizations has revealedoverall annual costs of US$ 604 billion, only 16% of which represents direct medicalcosts of health and social systems.13 Regardless of the differenteconomic and social models of countries of low, medium or high-income, the costsassociated with dementia vary. These disparities have been attributed to themethodology used, such as the bases for calculating hours dispensed by the caregiverand lack of standardization of criteria on conceptual understanding and definitionsof direct and indirect costs.11In Europe, a recent review estimated a total annual cost of € 692 billion forthe 19 subtypes of neurological disorders, including dementia. Of this total,€ 105.2 billion was allocated to direct costs of their treatment.14 Direct medicalcosts refer to the medical care system, such as costs of hospital care, drugs andvisit to clinics. Direct social care costs refer to formal services provided outsidethe medical care system and also out-of-pocket expenses of caregivers with items notcovered by the local health system. There is some consensus among researchersconcerning the difficulty making a clear distinction between social and medicalcare. Thus, direct costs are those related to social and public health coveragesthat are promoted by the Health System. Indirect costs include informal costs suchas hours dedicated by the caregiver in meeting the needs of patients for basicactivities of daily living (ADL), instrumental activities of daily living (IADL),supervision and total loss or reduction of formal working hours of caregiverproductivity. Finally, indirect costs can be considered as the sum of informal costs+ direct social costs to the caregiver.14 In this study, all indirect costs will be considered,excluding direct medical costs of the health system. As a basis for calculatingindirect costs, some researchers adopt the minimum monthly wage in the country andan 8-hour working day to obtain the financial value of the hours spent oncare.17According to Alzheimer Disease International - ADI, costs of dementia can besub-classified as direct medical costs, direct social care costs and indirectcosts.18 and therefore themain objectives of the present study were to ascertain:In LA, only a few studies have addressed costs with dementia[1] the monthly/annual average indirect cost with dementia;[2] the impact of this expenditure on the caregiver;[3] whether severity of dementia translates to higher indirect costs;and[4] Whether the financial burden correlates with higher overall burdenlevels for caregivers.This is a cross-sectional observational study. The project "Description of Methodsand Cost Analysis with Dementia" was submitted to the Research Ethics Committee ofHospital das Clinicas of the University of São Paulo - HC-FMUSP and approvedunder number 368.096/2013. In this paper, the preliminary results of the indirectcosts of patients and their caregivers evaluated until 12/31/2013 are reported.Participants. A convenience sample of outpatients followed at theneurology and cognitive behavior unit was assessed. All patients were previouslyassessed by a neurologist. At the end of the study, the sample will comprise 300caregivers.Inclusion criteria. Patients with a diagnosis of probable dementiaat mild, moderate or severe stages;19 - Informal Caregivers spending time with the patient at leastthree times a week.Exclusion criteria. Patients diagnosed with Mild CognitiveImpairment (MCI) and their caregivers.Professionals involved. A nurse specialized in dementia and aneurologist.Measuring instruments. A research protocol including:A semi-structured questionnaire, to collect sociodemographic and clinicalstatus of patients and caregivers containing: Gender, age, education,retirement, income, economic status, kinship, marital status, place ofresidence of the patient, medical diagnosis, disease progression,co-morbidities, caregiver disease, use of medication prescribed by aphysician. Direct monthly social care costs, out-of-pocket spending bycaregiver on: drugs, medical appointments/health insurance, transportation,food, diapers, clothing, paid caregiver and other expenses. Hours spent bythe caregiver on ADL, IADL, care and supervision.20Functional Assessment Staging - FAST Scale, to stratify severity ofdementia.21Economic Criterion Brazil scale, to identify the economic status of thecaregiver.22Zarit Burden Interview scale, to determine the levels of caregiverburden.23Resource Utilization Dementia-RUD scale, to evaluate resources used in directand indirect costs.Procedures. Caregivers were invited while waiting for medicalappointments - waiting room - by the nurse responsible for the interviews, or werereferred by the medical team after consultation. After presenting the study proposalto caregivers in detail, caregivers were given an Informed Consent Form andrequested to sign this, retaining a copy.1 USD on 12/31/2013.To determine indirect costs with items not covered by the health system and paidout-of-pocket by the caregiver, the hours spent on care were calculated, taking as abasis, the current minimum wage (MW), updated annually, with the value thus obtaineddivided by a period of 30 days. The figure obtained was then divided by 8,considering a working day of 8 hours. The result was then multiplied by the numberof hours of care dispensed by the caregiver to the patient thereby giving the valueof the hours/day. Subsequently, the product was multiplied again to obtain the totalmonthly and projected annual costs. The calculation used in this study was based ona MW of R$ 678.00 (Brazilian currency), valid until 12/31/2013. All resultant valuesobtained in Brazilian currency (R$) were subsequently converted into U.S. currency($) at the exchange rate of R$ 2.36:Dementia severity was obtained by the FAST classification and all patients werestratified into one of three stages: score 1-4="mild"; 5a-5e="moderate" and7a-7f="severe". The scores on the burden interview lay in the ranges 0-20="little orno burden"; 21-40="mild to moderate"; 41-60="moderate to severe" and 61-88="severe".In this study, the scores corresponding to 0-20="little or no burden", were groupedunder the "mild to moderate" rating. The economic classification was obtainedaccording to the sum of the scores of declared items in each household. Thus, ratingscores were: 42-46="A1"; 35-41="A2"; 29-34="B1"; 23-28="B2"; 18-22 "C1"; 14-17="C2";8-13="D"; 0-17="E" . The ratings "D" and "E" were grouped together in this study.For the final classification, this sum was added to the score corresponding to thedeclared level of patient education, where scores ranged from 0="illiterate/basic 1incomplete"; 1="basic 1 complete/basic 2 incomplete"; 2="basic 2 complete/incompletehigh school"; 4="complete high school/incomplete college"; 8="complete college".Caregiver education was obtained based on declared years of schooling.Statistics. To compare categorical variables between groups, theChi-square (χ2) test was performed while continuous variables wereanalyzed by one-way ANOVA, followed by Bonferroni analysis. To investigatecorrelation between financial burden, dementia severity, and caregiver burden,Spearman's R correlation analyses was performed. The level of significance was setat 0.05. Data were analyzed using the SPSS for Windows 20.0 software package.To date, a total of 93 caregivers have been interviewed. Demographic data on thethree groups are shown in 17The preliminary results of this study, as expected, showed a significant impact ofindirect costs with dementia in Brazil. Findings from recent studies have associatedoverheads of indirect costs with the advancement of the disease and the presentstudy found the same result.11Some sociodemographic variables of patients and caregivers were similar to thosefound in other studies, maybe owing to cultural identities, for example: gender,age, marital status, education, relationship, retirement, economic status andmonthly income of patients.24 withanxiety (96%) and depression (100%) + family dysfunction (26.5%) in 47% of primarycaregivers of patients with dementia.25 We found a 38% rate of reports of various diseases incaregivers that started after taking responsibility for the care of their illrelative, with a higher percentage of reports (16%) observed at the moderate stage.The loss or reduction of hours of productivity was found not to increase withseverity of the disease and it was noted that greater reduction or loss of formalworking hours occurred at mild and moderate stages. This finding may be due to thesmall sample of patients in the severe phase, but also suggests that the search formedical diagnosis and treatment in the early stages may explain the higherconsumption of caregivers hours as well as the reduction or even loss ofproductivity.26 In themoderate stage, the need for a caregiver generates higher consumption of hours ofinformal care and expenses related with direct social care costs and our findingscorroborate other studies showing increased burden as the diseaseprogresses.27 The reduction orloss of hours of productivity observed at the severe stage can be viewed as theconsequence of the high number of hours spent per month associated with the severityof dementia.15Regarding the clinical status of patients and caregivers, some significantdisparities were found, including higher prevalence of AD, duration of the diseaseand the presence of comorbidities in patients, the most prevalent beinghypertension, pointing to the importance of primary care actions. Among caregivers,it was noted that their clinical status can change an average of 2-3 years aftertaking on the role of carer. There was a high prevalence of emotional stress oncaregivers, as observed in other studies. Few caregivers were classified as havinglow levels of stress as compared to other caregivers of patients without dementia. ALA study found a higher frequency of depression in caregivers than non-caregivers of44% and 27%, respectively,28 In this regard, awareness andeducation initiatives can be very useful in low and middle incomecountries.5 Nevertheless,it should be noted that the average number of working hours reduced or lost permonth due to care is high, and the values reached by the calculations can exceed themonthly income of many families, as was shown in this study. A high percentage ofcaregivers reported income of 1 to 3 MW, and declared income, in some cases, fellbelow of the amounts estimated for indirect costs. Our estimates showed that thesecosts exceeded, in some cases, the declared family income at rates of 69% to 106% inthe mild stage; 89% to 136% in the moderate stage and 106% to 169% in the severestage. Recent LA studies have found committed family incomes of 60%,18 61%,15 and 66%-75%.11 The direct social care costs of the caregivers in thepresent study were greater than the monthly income of some caregivers in some cases,totaling US$ 462.15 at mild, US$ 593.70 at moderate, and US$ 970.40 at severestages. The higher spending of caregivers seen in the severe stage of the disease isin agreement with the finding of other authors, being attributed to the greatercomplications observed at later stages.26Perhaps, indirect costs to the caregivers can be minimized by the establishment ofpharmacological and non-pharmacological therapies that may promote greaterflexibility and allow time for the caregivers to perform other tasks.10 However, comparison of theseresults were hampered by methodological differences in LA, lack of stratification ofseverity of dementia observed and by the fact that only indirect costs wereaddressed in the present study. Also, an international comparative cost evaluationwas not conducted. However, some studies in LA found lower hours of care than thatobserved in this study.18 In Europe,findings from a study that estimated hours of care stratified by disease severitywere similar to this study results, especially at the mild stage ofdementia.26 We foundaverage proportion of 29%, 63% and 75% of weekly hours; 45%, 63% and 75% of monthlyhours; and a total average days per months corresponding to 82.3%, 98% and 95%, inmild, moderate and severe phases, respectively.The average hours of care, weekly and monthly, was higher than hours seen in otherstudies and contributed to a greater caregiver burden than financial burden,suggesting that actions of support and education, as proposed in other studies,should be sought to reduce the burden of caregivers.27The high number of hours reported here could be related to exceeding of caregiverlimits and not to increased demands for end-of-life care, since a high percentage ofpatients were at mild and moderate phases.29 In Brazil, initiatives include supportive measuresand education of professionals and caregivers with some positive results in reducingthe burden of care.30 Further research and multicenter studies to compare theresults obtained here are necessary. This small sample showed that the impact ofindirect costs with dementia in Brazil may be higher than other previous LA studies.These initial results may make an important contribution to future research aboutcosts with dementia in LA.These findings reveal the extent to which these caregivers fail to earn an incomeowing to premature withdrawal from the labor market, and of the risk of themselvesbecoming dependent on the health system given that diseases such as diabetesmellitus, depression and hypertension are also diseases with high morbidity,responsible for other NCDs. The lack of social structure has led to greaterisolation of individuals that, with no alternatives, are forced to leave their workand life plans, to take care of their sick relative. In some cases, those with nomonthly income become dependent on the pension or allowance of the patient or relyon the help of others to ensure survival of the family.The main limitation of this study was its cross-sectional design. Most studiesassessing the direct and indirect costs of dementia in high income countries haveinvolved longitudinal studies. Recent literature on this issue alerts the attentionof researchers to the need for systematic actions that focus on early diagnosis andtreatment. This systematization of actions has been considered a possible approachfor reducing the direct and indirect costs associated with the disease.In our milieu, there remains much to investigate and achieve in the sphere ofnon-communicable diseases Despite the limitations noted , this is the firstBrazilian study to perform the monetary calculation of indirect costs associatedwith dementia, where the next step is to continue the research presented employingan appropriate methodological design.


Aedes spp., immunologically naive human populations, global population growth with increased urbanization, and escalation of global transportation of humans and commercial goods carrying vectors and ZIKV undoubtedly enhanced the emergence of ZIKV. However, flavivirus mutations accumulate with time, increasing the likelihood that genetic viral differences are determinants of change in viral phenotype. Based on comparative ZIKV complete genome phylogenetic analyses and temporal estimates, we identify amino acid substitutions that may be associated with increased viral epidemicity, CZVS, and GBS. Reverse genetics, vector competence, and seroepidemiological studies will test our hypothesis that these amino acid substitutions are determinants of epidemic and neurotropic ZIKV emergence.The unexpected emergence of Zika virus (ZIKV) in the Pacific Islands and Latin America and its association with congenital Zika virus syndrome (CZVS)  and Guillain-Barré syndrome (GBS) have stimulated wide-ranging research. High densities of susceptible  Aedes ZIKV-competent mosquito is present.Zika virus (ZIKV) was first described in the African forests, where it circulates between nonhuman primates and sylvatic mosquitoes . More th5–Two ZIKV lineages have been described: African and Asian. Strains that emerged in the Pacific Islands and Latin America belong to the Asian lineage. In late 2015, a ZIKV outbreak due to an Asian lineage strain was also associated with cases of microcephaly. It occurred on Cape Verde off the coast of Africa (How did ZIKV emerge from its sylvatic forest existence to cause major epidemics throughout the Pacific and the Americas? Similar to dengue virus (DENV), ZIKV had the potential to adapt from a sylvatic cycle involving sylvatic mosquitoes and nonhuman primates to an urban cycle involving urban/suburban mosquitoes and humans .One possibility is that the emergence of ZIKV is simply a consequence of the increasing global human population, increasing population of competent mosquito vectors, increasing urbanization, and increasing global transportation of commercial goods. Lack of ZIKV-specific population immunity was almost certainly a contributing factor to the emergence of ZIKV. Additionally, the possibility of immune enhancement arising from cross-reactions with related viruses such as dengue virus cannot be ignored . Howeverhttp://www.ncbi.nlm.nih.gov/genbank). At the time, only one isolate was available from French Polynesia (FP). To study the possibility that some FP isolates had the mutation M/T2634V first documented in the Latin American strains, an additional 13 isolates from various times and places in the FP outbreak were sequenced in house. The in-house next-generation sequencing was performed with the Ion PGM sequencer , and analyses were conducted with the CLC Genomics Workbench software. RNA was extracted from serum or cell supernatant medium using the EZ1 Mini virus 2.0 kit and the EZ1 advanced XL machine (both from Qiagen). Complete virus genomes were amplified in 3 or 12 fragments using specific sets of primers with the Superscript III one-step reverse transcription-PCR (RT-PCR) Platinum TaqHifi kit .To explore the temporal evolution of ZIKV, representative and robust complete ZIKV sequences available as of 29 May 2016 were retrieved from GenBank (n = 67 sequences), and the other alignment (see n = 84 sequences). In addition, separate alignments were constructed for the 5′- and 3′-UTR regions (see http://tree.bio.ed.ac.uk/software/figtree/). All computations were performed at the CIPRES web portal . This resulted in an explosive outbreak of ZIKV infection involving 73% of the immunologically naive population. However, as far as is known, this strain did not disperse further. Six years later, another epidemic Asian strain was identified in FP, documenting for the first time an association with microcephaly and GBS , 15, 16.Aedes aegypti  receptor loop (VEPG), which show a direct correlation between the receptor sequence and the ability of DENV-2 both to infect mammalian cells and to be transmitted by  aegypti . Two add aegypti .However, the Philippine and Micronesian isolates retained the African TM1 and TM2 amino acids (V763 and T777). The Cambodian isolate (node C) retained the African V763 TM1 amino acid but acquired the Asian 777M TM2 amino acid. The Thailand virus isolate identified by node D retained the Asian 777M and acquired the Asian amino acid substitution 763M and the second Thailand isolate identified by node E, retained the Asian 777M and 763M but in addition acquired Y2086H . Thus, tThe first appearance of a French Polynesian virus in the tree is identified by node F. Node F1 identifies a branch with 3 Samoan viruses and 6 FP viruses, and node F2 identifies another branch with 5 FP viruses. Node F3 distinguishes a branch with 2 more FP viruses, which are the most closely related FP viruses to the Latin American viruses, identified by node G. Thus, nodes F, F1, F2, and F3 define 14 FP and 3 Samoan viruses that evolved in FP/Samoa over a period of less than 3 years. Note that nodes F1 to F3 are distinguished only by synonymous and sequence-specific nonsynonymous mutations.Node F identifies the evolutionary point of emergence of epidemic neurotropic ZIKV, and this coincides precisely with the appearance of the amino acid substitution S139N in both the French Polynesian and Samoan isolates .Amino acid 139 is in the ZIKV M protein, a transmembrane (TM) protein. While the function of this amino acid has not been defined for ZIKV, in DENV2 the equivalent amino acid is involved in the acid pH-mediated viral surface maturation process . Thus, aNode G defines the point at which the substitution M/T2634V arises. Thus, the tree shows clearly that GBS and microcephaly, which were first reported in FP, arose following the S139N substitution in the ZIKV M protein. None of the FP isolates had the M/T2634V substitution found in the Latin American isolates. Although it will require further verification, the substitution most likely arose after ZIKV escaped the Pacific. Whether or not the M/T2634V substitution contributed to the recognized increased incidence of microcephaly in Latin America compared with the Pacific is a matter for conjecture until further relevant virus isolates become available or experimental evidence is obtained.Did the five unique amino acid substitutions defined above—i.e., D683E (box 2), T777M (box 3), V763M (box 4), S139N (box 6), and M/T2634V (box 7)—have an impact on mosquito vector competence of epidemic ZIKV and increased ZIKV neurotropicity? These questions will be investigated using reverse genetics methods combined with vector competence and seroepidemiological studies.The acquisition of four additional amino acid substitutions, including one in the NS4B protein A/G2283S) and three in the NS5 protein , distinguishes African/Malaysian from other non-African ZIKVs , box 2. 83S and tComparative analysis of the 5′ and 3′ termini that form secondary structures in the untranslated regions (UTRs) of the ZIKV genome revealed differences between African and all other ZIKV isolates studied  and S2. −4; 95% HPD, 5.5 × 10−4 to 7.1 × 10−4) were lower than those for the tree with African viruses excluded . Divergence of African and Asian ZIKV from a most recent common ancestor occurred around 1834 . Subsequently there was a significant increase in the mean evolutionary rate of the Pacific/Latin American variants of ZIKV, an observation consistent with marked increase in virus transmission and turnover accompanying the massive island and urban outbreaks. tMRCA estimates indicate that the introduction of ZIKV into the Americas (Estimated mean evolutionary rates (MERs) and times to most recent common ancestor (tMRCA) based on strict molecular clock estimates, using trees that include or exclude African viruses, are also presented  was located in the viral receptor site, two (T777M [box 3] and V763M [box 4]) were located in the viral envelope protein, and the final but possibly the most critical one (S139N [box 6]) was located in the M protein. These amino acids clearly play important roles in virus attachment entry, release, or maturation of the virus during the infectious process. Thus, three unique amino acid substitutions directly associated with virus infection and transmission efficiency occurred just prior to the emergence and widespread dispersal of the highly epidemic and neurotropic ZIKV. The appearance of the membrane protein substitution S139N coincided precisely with the appearance of GBS and CZVS in FP. While this does not prove cause and effect, it appears to be more than a coincidence. Another unique substitution, in the NS5 protein of ZIKV (M/T2634V [box 7]) was identified in all Latin American viruses and may also impact neurotropicity since the relative rate of CZVS increased significantly in Brazil compared with FP.Other substitutions in the prM and E glycoproteins could either be compensatory or could alter virus characteristics, such as hydrophilicity and charge. As reported for DENV mutants, the ZIKV mutants show efficiency for producing neurovirulence  and adsoAedes species vectors. The recent discovery that ZIKV is significantly more thermostable than DENV may also contribute to its success as an epidemic virus through its ability to reproduce to higher levels of viremia and to resist inactivation under unfavorable environmental conditions (The emergence of ZIKV and its widespread dispersion throughout the tropics and subtropics have undoubtedly been influenced by increasing human mobility, urbanization, and failure to reduce the spread and expanding population density of ZIKV nditions .in vitro and in vivo.The use of reverse genetics, vector competence studies, and seroprevalence analyses to gain an understanding of the epidemic potential of ZIKV will enable the validity of our observations and hypotheses to be tested both KX447509 to KX447521.The sequences of the additional French Polynesian isolates have been submitted to GenBank under accession no. Data Set S1 Nucleotide alignment in fasta format of coding regions of Asian, Pacific, and Latin American ZIKV isolates. Download Data Set S1, TXT file, 0.6 MBData Set S2 Nucleotide alignment in fasta format of all Zika isolates. Download Data Set S2, TXT file, 0.8 MBData Set S3 Nucleotide alignment in fasta format of 5′ UTR of Zika isolates. Download Data Set S3, TXT file, 0.1 MBData Set S4 Nucleotide alignment in fasta format of 3′ UTR of Zika isolates. Download Data Set S4, TXT file, 0.1 MBFigure S1 Alignment of 5′ UTRs of Zika virus isolates in PDF format. Download Figure S1, PDF file, 0.4 MBFigure S2 Alignment of 3′ UTRs of Zika virus isolates in PDF format. Download Figure S2, PDF file, 1.5 MB


These polymersomes form by self-assembly, under electroformation conditions, of amphiphilic, glycosylated block copolymers in aqueous solution. The glycosylated exterior of the resulting polymeric giant unilamellar vesicles (GUVs) allows their selective interaction with carbohydrate-binding receptor-functionalized particles, in a manner reminiscent of the cell-surface docking of virus particles. We believe that this is the first example of a simple protocell model displaying cell-like behavior through a native receptor-ligand interaction.Despite the low complexity of their components, several simple physical systems, including microspheres, coacervate droplets and phospholipid membrane structures (liposomes), have been suggested as protocell models. These, however, lack key cellular characteristics, such as the ability to replicate or to dock with extracellular species. Here, we report a simple method for the  Over the last decades, the structural and molecular basis of cellular function has been elucidated. Cells are complex, hierarchical entities, which perform a number of functions that include nutrient transport and secretion, evolution and differentiation, replication and division, as well as adhesion and arrest. How life arose from its prebiotic origins is still unknown, and possibly will not ever be elucidated24711et al.Recent developments in the field of cell biomimicry have made it possible to design advanced cellular structuresThe second rational step towards cell biomimicry is to introduce some “living” functional aspects  to the existing cellular structural models. One such aspect is cellular internalization, in which cells take up a variety of external species including macromolecules, nanoparticles (e.g. viruses) and bacteria. Internalization occurs by various mechanisms, including endocytosis, the key stage in which is the docking of an external species to the cell membrane, followed by an invagination of the fluid bilayer and complete wrapping of the species in question and ultimately its transportation to the intracellular milieu encapsulated within a vesicle16a ca. 103 M−1)a values (109–1012 M−1)23Carbohydrates are commonly encountered ligands for cell surface receptor proteins (lectins) and, indeed, many biological processes in mammalian cells, such as initiation of the inflammatory cascade, virus docking, fertilization and cancer cell metastasis, are mediated by carbohydrate-lectin interactions20At present, no structural cell mimics that can interact specifically with extracellular species in solution via receptor-ligand binding have been reported. Successful internalization of nanoparticles into liposomes19F-NMR spectroscopy, were achieved. Further evidence of successful attachment of the sugar moieties was provided by FTIR spectroscopy (see SI).We first utilized RAFT to polymerize an activated ester monomer, pentafluorophenyl acrylate (PFPA), followed by chain extension with n-butyl acrylate (n-BA) to produce a reactive block copolymer precursor for subsequent modification with amine-functionalized sugars . PFPA wab-BA) glycopolymers using the electro-formation method (295-b-BA50), led to the formation of stable glycosylated GUVs (glyco-GUVs) with high yields (77 ± 8 vesicles per square mm) and average diameter of 20.0 ± 2.0 μm  – functionalized particles, it was necessary to demonstrate the availability of the pendent glucose moieties present on the vesicles’ surface for lectin binding. A turbidity assay was performed whereby 240 μl of a GUV solution was added to 600 μl of a Concanavalin A (Con A) solution in HEPES buffer (2 mg/mL). A steady increase in Aurbidity . This isCon A–functionalized polystyrene (PS) beads were prepared as model extracellular receptor functionalized species to study their binding interactions with our glyco-GUVs ,B. Comme120 – functionalized PS beads (RCA120 has no affinity to β-linked glucose moieties). All experiments were replicated in triplicate with an incubation time of 18 h, to allow significant numbers of interactions between beads and GUVs to occur. Upon overnight incubation of the glyco-GUVs with the unfunctionalized PS beads, very few examples of a bead next to a GUV were observed; however, the majority of the beads were distributed randomly and remained at the bottom of the visualization chamber. The percentage of interaction between the glyco-GUVs and the unfunctionalized beads, defined as the percentage of glyco-GUVs with an adjacent bead, did not exceed 6.5% in each of the observed samples. Similarly, upon overnight incubation of the glyco-GUVs with the RCA120–functionalized PS beads, a small number of interactions between the two species were observed; however the majority of RCA120–functionalized PS beads were dispersed randomly in the sample. The percentage of interaction between the glyco-GUVs and the RCA120–functionalized PS beads varied from 6 to 9%, which is slightly higher than that determined for the unfunctionalized PS beads.We next studied the interaction between our glyco-GUVs and Con A–functionalized PS beads as model extracellular objects. Confocal microscopy was used to visualize the interactions. In order to eliminate any potential errors and misinterpretations of data produced by non – lectin mediated interactions, two types of control experiments were performed: glyco-GUVs incubated with unfunctionalized PS beads ; and glyco-GUVs incubated with RCA120 – functionalized PS (8.2 ± 1.4%) and eight times higher than those with the unfunctionalized PS beads (4.9 ± 1.0%)  gave consistent results. Based on the collected data, the average percent of interaction between the glyco-GUVs and the Con A – functionalized PS beads was determined to be 42.0 ± 7.8% which is approximately five times higher than those with the RCA ± 1.0%) .via a single bead – GUV connection. We presume that bead aggregation is caused by some free glycosylated polymer chains or nanostructures (eg micelles) that are too small to be observed by confocal microscopy. Time-lapse images show that the beads and GUVs move in concert, demonstrating that the strength and stability of the sugar-lectin binding interaction is sufficient to withstand translation from Brownian motion. Furthermore, the precise location of beads relative to GUVs was investigated by microscopy. Successive confocal microscopy images at different focal planes (Z-stack images) indicated that beads located adjacent to GUVs were indeed interacting strongly with the vesicle membrane  to the surface of mammalian cells, which leads to viral particle entry and infection. This study, which we believe is the first to demonstrate receptor-mediated particle binding to giant polymersome protocells, may provide important insights for future research on protocells and minimal cell systems.n-butyl acrylate, followed by transesterification of the PFP ester with 2′-aminoethyl-β-D-glucopyranoside and removal of the trithiocarbonate end group by treatment with AIBN. Polymers were characterized fully by NMR spectroscopy and SEC; in all cases, the obtained Mn agreed well with that predicted from the monomer to CTA ratio and dispersity values were in the range 1.1–1.2. Glyco-GUVs were prepared using an in-house fabricated electroformation apparatus consisting of two glycopolymer-coated indium tin oxide (ITO) glass slide electrodes, separated by a rubber O-ring spacer containing an aqueous sucrose solution, housed in PTFE and connected to an external AC power source. Lectins Con A or RCA120 were conjugated to commercially available FITC-labelled carboxylate-modified polystyrene latex beads by EDC/NHS coupling. Interactions between beads and glyco-GUVs were investigated by bright field and fluorescence confocal microscopy. The collected images were processed using ImageJ software. The PFPA was synthesized in a manner similar to that described in the literatureHow to cite this article: Kubilis, A. et al. Giant Polymersome Protocells Dock with Virus Particle Mimics via Multivalent Glycan-Lectin Interactions. Sci. Rep.6, 32414; doi: 10.1038/srep32414 (2016).


Drosophila genetics and the medical relevance of mosquito-transmitted viruses have made dipterans important model organisms in antiviral immunology. Studies of virus–host interactions at the molecular and population levels have illuminated determinants of resistance to virus infection. Here, we review the sources and nature of variation in antiviral immunity and virus susceptibility in model dipteran insects, specifically the fruit fly Drosophila melanogaster and vector mosquitoes of the genera Aedes and Culex. We first discuss antiviral immune mechanisms and describe the virus-specificity of these responses. In the following sections, we review genetic and microbiota-dependent variation in antiviral immunity. In the final sections, we explore less well-studied sources of variation, including abiotic factors, sexual dimorphism, infection history, and endogenous viral elements. We borrow from work on other pathogen types and non-dipteran species when it parallels or complements studies in dipterans. Understanding natural variation in virus–host interactions may lead to the identification of novel restriction factors and immune mechanisms and shed light on the molecular determinants of vector competence.The power and ease of  Drosophila and the medically important vector mosquitoes of the genera Culex, Aedes, and Anopheles. These fields have complemented each other, with interest in mosquito-vectored viruses guiding studies in Drosophila, and the well-annotated genome and genetic tools of Drosophila providing a model to guide laboratory experiments in the less tractable mosquito. The comparison of antiviral processes in these dipterans highlights a conserved yet dynamic immune system, with some antiviral mechanisms conserved from flies to mammals, whereas others have evolved since the expansion of dipterans.The vast majority of research on invertebrate antiviral immunity has used dipteran systems, including the fruit fly Drosophila, and through comparisons of vectored flaviviruses, rhabdoviruses, alphaviruses, and bunyaviruses—part of a larger group referred to as arthropod-borne viruses (arboviruses). These studies reveal a general requirement for antiviral RNAi in dipterans, virus-specific contributions of canonical antimicrobial pathways , a diverse transcriptional response, and emerging antiviral processes that have yet to be studied in a comparative context iewed in ).Ochlerotatus taeniorhynchus and Ae. albopictus, respectively ..OchlerotAe. aegypti strains from Vietnam and Thailand, possibly caused by insecticide use, were also found to be associated with vector competence for DENV ,137Drosofeasible . Comparampetence ,185. How


There are substantial socioeconomic inequalities in both life expectancy and healthcare use in England. In this study, we describe how these two sets of inequalities interact by estimating the social gradient in hospital costs across the life course.Hospital episode statistics, population and index of multiple deprivation data were combined at lower-layer super output area level to estimate inpatient hospital costs for 2011/2012 by age, sex and deprivation quintile. Survival curves were estimated for each of the deprivation groups and used to estimate expected annual costs and cumulative lifetime costs.A steep social gradient was observed in overall inpatient hospital admissions, with rates ranging from 31 298/100 000 population in the most affluent fifth of areas to 43 385 in the most deprived fifth. This gradient was steeper for emergency than for elective admissions. The total cost associated with this inequality in 2011/2012 was £4.8 billion. A social gradient was also observed in the modelled lifetime costs where the lower life expectancy was not sufficient to outweigh the higher average costs in the more deprived populations. Lifetime costs for women were 14% greater than for men, due to higher costs in the reproductive years and greater life expectancy.Socioeconomic inequalities result in increased morbidity and decreased life expectancy. Interventions to reduce inequality and improve health in more deprived neighbourhoods have the potential to save money for health systems not only within years but across peoples’ entire lifetimes, despite increased costs due to longer life expectancies. Healthcare systems in most high-income countries aspire to provide equitable care, adopting the principle of equal access to services for equal need,There is a widespread assumption that over the life course such systems disproportionately favour people lower down the socioeconomic scale, in terms of the imbalance between their contribution to the costs of health services and their use of those services.9Most studies, to date, on the costs and use of healthcare services by different socioeconomic groups have been cross-sectional. This is an important limitation, because morbidity and mortality may have opposing impacts on lifetime healthcare costs—greater morbidity will tend to increase lifetime costs, whereas dying younger will tend to reduce them. After early childhood, average current-year healthcare costs for individuals increase throughout life, rising dramatically from the age of 50.Consideration of these longitudinal relationships is necessary in order to determine the impact of socioeconomic factors on health system costs. Measuring the size of this impact is important not just to quantify the relative healthcare benefits received by different social groups, but to understand the costs borne by the health service as a consequence of social inequality. In this study, we aimed to measure the costs to the NHS of socioeconomic inequality, by estimating the lifetime inpatient hospital costs of the whole English population by socioeconomic status.et al.This study focuses on socioeconomic differences in inpatient hospital costs across the life course. Hospital admissions in England are recorded in the Hospital episode statistics (HES) data set used to reimburse hospitals for provided care. This data set contains details on every episode of care, and a new finished consultant episode (FCE) record is created for every new admission, and every time responsibility for the care of a patient passes from one consultant to another. The HES FCE records data about the patient  and their hospital stay . Using this information the FCE is allocated to a healthcare resource group (HRG), which collates hospital stays that use similar levels of resources—this is the English version of diagnosis related groups used in the USA. Hospitals are reimbursed by the NHS through the payments by results (PbR) system based on the HRG, adjusted for the specifics of the case—for example, a more complicated case with longer than usual length of stay attracts additional reimbursement. Reimbursement is also adjusted for local cost variations (termed ‘market forces factors’). Costs attached to each HRG for each year, and variations for more complex cases, are given in the NHS national reference costs.The basic geographical unit of analysis in this study is the lower-layer super output area (LSOA). The country is divided into 32 482 LSOAs each containing, on average, 1500 people (range 1000–3000). Population data for 2011/2012 are taken from Office for National Statistics (ONS) midyear population estimates split by LSOA, sex and age . This data estimates the total resident population, including homeless people and people living in institutions. Mortality data for 2011/2012 are taken from the ONS, split by LSOA, sex and age . Area deprivation for LSOAs is measured using the index of multiple deprivation (IMD) for 2010. The IMD includes seven domains: (1) income; (2) employment; (3) health deprivation and disability; (4) education skills and training; (5) barriers to housing and services; (6) crime; and (7) living environment. These domains are combined to produce an overall deprivation rank for each LSOA. We grouped LSOAs into deprivation quintiles based on this rank ranging from Q1 (the most deprived fifth of LSOAs) to Q5 (the least deprived fifth of LSOAs).We grouped HES inpatient data into age, sex and IMD quintile categories. Of the 18 808 903 episodes in our 2011/2012 HES data set, 1 659 295 episodes (8.8%) could not be grouped due to missing data on either age, sex or LSOA of residence, and were dropped from the analysis. We then calculated the total cost for each age, sex and IMD quintile group using the HRGs and the relevant reference costs. Market forces factors adjustments were not made as we are interested in the variation in resource use by deprivation group rather than local cost variations. We then inflated these costs by 8.8% to account for the missing data . Finally, we divided by the population in each age, sex and IMD quintile group using ONS population estimates to estimate average costs for each group:We used these average costs to calculate the total cost associated with inequality in 2011/2012 by comparing the costs as observed in the data with the costs calculated, by assuming that each individual experienced the average costs (split by age and sex) experienced in the least deprived fifth of areas:We used these survival curves to calculate expected cost at each age split by sex and IMD quintile group by adjusting the average cost for the probability of an individual from each group being alive to incur that cost. Finally, we summed across these age groups to get an expected lifetime cost for an individual in each sex and IMD quintile group :We repeated this analysis for emergency and elective hospitalisations, and also compared rates of outpatient hospital use among the different groups.https://github.com/miqdadasaria/cost-of-inequalityThe analysis was performed using Oracle 11g and R 3.2.3—the analysis code is available at In 2011/2012, there were 11 477 435 elective episodes and 7 914 736 emergency episodes to hospitals in England . Numbers of episodes decreased between the ages of 0 years and 10 years in both sexes, then, for men, increased up to the age of 70 years, before declining in the oldest age groups, and for women, spiked sharply between adolescence and the age of 40 years —reflecting admissions relating to reproduction—before gradually increasing up to the oldest age groups A. For agThe trends for average annual costs per head of population C closely). Compared with residents in the most affluent fifth of areas, residents of the most deprived fifth of areas had a 20% higher rate of elective episodes, a 71% higher rate of emergency episodes, and a 39% higher rate of episodes overall. Detailed age, sex and deprivation breakdowns of the different types of admissions are given in the online The social gradient in hospital episodes was evident for elective and emergency admissions, but the gaps were greater for emergency admissions . Compare10.1136/jech-2016-207447.supp1Supplementary appendixThe potential savings for the NHS if the costs associated with the age and sex-specific episode rates in the most affluent quintile in 2011/2012 were achieved in the other deprivation groups are given in Survival curves for men and women by deprivation quintile are shown in Combining data on survival and average costs, we calculated expected costs of hospital admission over the life course for each deprivation group, assuming survival and costs remained constant at 2011/2012 levels. Cumulative lifetime costs are shown in Analyses for emergency and elective admissions are presented in the online In this study, we aimed to quantify the hospital care costs to the NHS of socioeconomic inequality. As expected, we found that hospital admission rates generally increased with age, and were higher in women during the reproductive years and higher in men at most other ages. For all ages, there was a clear socioeconomic gradient, particularly for emergency admissions, with the rate of admissions increasing with neighbourhood deprivation. The costs to the NHS associated with this inequality were partially offset by lower life expectancy in more deprived groups, but remained substantial: £4.8 billion per year at 2011/2012 levels.This is the first study based on comprehensive whole-population data in England to explore the relationship between lifetime hospital costs to the NHS and socioeconomic inequality. We used data at small-area level to minimise, as far as possible, the risk of ecological fallacy that may have masked inequality at larger and coarser geographical levels. Mortality data were used to extrapolate the results of the analysis across the patient lifetime to allow conclusions to be drawn on both cross-sectional and lifetime costs of inequality to the NHS.The study is subject to several limitations. First, we did not control for differing need for healthcare among the different groups, and so do not make any judgements on whether the different levels of healthcare use are ‘fair’ or appropriate, given differences in need. For example, it may be the case that for any given level of morbidity, poorer patients use less healthcare than richer patients, and hence, our estimate of the cost of inequality to the NHS, while representing current practice, underestimates ideal practice where patients receive equal treatment for equal need. Second, the focus of our analysis was inpatient care, but healthcare costs are also incurred through outpatient appointments and in primary care. In 2011/2012, inpatient costs and primary care costs each constituted 22% of the total NHS budget of £101.42 billion.et alet alAs far as we know, this is the first published analysis of the inpatient costs of socioeconomic inequality in England. The 2010 Strategic Review of Health Inequalities (the Marmot Review) estimated the cost of inequality to the NHS to be £5.5 billion per year,Socioeconomic inequalities in the determinants of health result in both increased morbidity and decreased life expectancy. We found that the substantially higher healthcare costs accrued by residents of deprived areas throughout their lives are only slightly offset by their lower life expectancy. Evidence suggests that even in a country with universal access to healthcare, more affluent groups benefit more,21Rising healthcare costs in older age are largely driven by the onset of chronic disease, and the earlier onset of these diseases in poorer populations shifts the healthcare costs to younger age groups. Better primary and secondary prevention, progressively weighted towards more deprived populations, is an obvious response, but one that has proved hard to achieve. Anticipatory interventions to tackle the onset of chronic conditions in deprived neighbourhoods can result in significant patient benefit,30–32Poorer people tend to use more healthcare at any given age, because they are sicker, but also tend to have shorter lives.It is not known how these two sets of inequalities interact to produce lifetime healthcare costs for different socioeconomic groups.There is a social gradient in both current and lifetime hospital costs. Despite dying at a younger age, people from more deprived neighbourhoods tend to require more healthcare, and cost the National Health Service (NHS) more over their lifetimes than people from more affluent neighbourhoods.Socioeconomic inequality cost the NHS in England £4.8 billion in 2011/2012 as a result of excess hospital admissions.There is a financial as well as a moral case for tackling socioeconomic inequality: reducing socioeconomic inequalities in health would reduce the excess morbidity of the poor, resulting in longer lives. Our modelling suggests that the reduction in healthcare costs resulting from reducing morbidity among the poor would outweigh the increase in healthcare costs resulting from their increased longevity.


Rupture of membranes (ROM) before the onset of uterine contractions, particularly in pregnancies less than 37 weeks gestational age, is a common diagnostic problem in obstetrical practice. Timely detection of ROM is vital to support gestational age-specific interventions to optimize perinatal outcomes and minimize the risk of serious complications such as preterm delivery, fetal distress and maternal/fetal infections. Rapid bedside immunoassay tests designed to detect amniotic fluid proteins in cervicovaginal fluids have emerged as valuable clinical tools to provide timely ROM diagnosis.In this prospective observational study, two commercially-available immunoassay tests  were evaluated concurrently in 111 pregnant women who presented with the chief complaint of ROM. Immunoassay results were compared to clinical parameters for determining ROM via comprehensive, retrospective clinical chart review. Diagnostic performance characteristics were calculated including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy.Overall, diagnostic performance characteristics were robust and similar between ROM Plus® and AmniSure®, respectively: sensitivity (96.4 and 89.3%), specificity (98.8 and 100%), PPV (96.4 and 100%), NPV (98.8% and 96.5) and accuracy (98.2 and 97.3%). For term patients (≥37 weeks gestation), the sensitivities were 93.8 and 81.3% and specificities were 97.1 and 100% for ROM Plus® and AmniSure®, respectively. For preterm patients (<37 weeks gestation), both immunoassay tests provided exact concordance with clinical confirmation of ROM resulting in 100% diagnostic accuracy.Both rapid immunoassay tests provided similarly excellent diagnostic accuracy for the rapid detection of ROM with only two discrepant results for ROM Plus® and three discrepant results for AmniSure® compared to clinical confirmation. The findings from this study recommend these tests for pregnant women presenting with suspected ROM to guide correct clinical management decisions to improve obstetrical and neonatal outcomes.NCT02208011 (1 August 2014).ClinicalTrials.gov,  Rupture of membranes (ROM) has significant clinical implications particularly if it occurs prior to 37 weeks gestation where it is accountable for 20–40% of preterm deliveries –3. In faWhen ROM is suspected, traditional diagnostic methods include the use of a sterile speculum examination to visualize leakage of amniotic fluid from the external cervical os or pooling of amniotic fluid in the posterior vaginal fornix, coupled with a microscopic evaluation of a collected specimen for evidence of ferning/crystallization, commonly referred to as the fern test, and pH testing of the fluid with nitrazine test paper , 9, 10. Several point of care immunoassay tests have been developed, validated and commercialized for the rapid and accurate diagnosis of ROM. These tests detect specific proteins, such as alpha fetoprotein (AFP), found in high concentrations in amniotic fluid but at extremely low background concentrations in cervico-vaginal secretions . The firThis study compared the diagnostic performance characteristics between two commercially-available, immunochromatographic methods for the detection of ROM: 1) a monoclonal immunoassay that detects the PAMG-1 , and 2) a monoclonal/polyclonal immunoassay that detects the presence of IGFBP-1 and AFP .One hundred eleven (111) pregnant women, ≥ 15 weeks gestation presenting with a complaint of ROM were enrolled in this study between August 2014 and June 2015. Pregnant women with known placenta previa and/or active vaginal bleeding were excluded. This research adhered to Good Clinical Practice (GCP) guidelines and followed the recommendations of the Helsinki Declaration. Each patient provided informed consent before any study-related procedures were performed. This trial was prospectively registered at ClinicalTrials.gov (NCT02208011).At the time of clinical presentation, both immunoassay tests were performed simultaneously on each patient to determine presence or absence of ROM. The methodological and procedural details of both tests have been detailed previously , 23, 24.AFI < 7 or no 2 × 2 pocket<48 h from initial exam to deliveryevidence of chorioamnionitis and/or endomyometritisThe following major criteria were used in the clinician’s review process as highly suggestive of ROM:Clinical confirmation, the final diagnosis of ROM, required the presence of at least two major criteria.Diagnostic performance characteristics with corresponding 95% confidence intervals (CI) were calculated for each test including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy  , 26. PerOf the 111 pregnant women in this study, 51 (46%) were term patients (≥37 weeks gestation) and 60 (54%) were preterm (<37 weeks gestation). For the entire patient population, comparing ROM Plus® results with clinical confirmation per retrospective chart review resulted in overall excellent diagnostic accuracy with a sensitivity of 96.4%  and a specificity of 98.8%   but exact specificity  Table . The corThe diagnostic performance results for the subgroup of term patients are provided in Tables For preterm patients, both immunoassay tests provided exact concordance with clinical confirmation of ROM 100%, 60 of 60) Tables  and 6.Ta0 of 60 TThere were 73 patients (66%) who underwent a sterile speculum exam in this study as a part of their initial evaluation. Compared to clinical confirmation, the sensitivity and specificity of the speculum exam were 72.2%  and 96.4%  with a PPV and NPV of 86.7%  and 91.4% , respectively. There were 7 discordant measurements resulting in an accuracy of 90.4%.Point of care diagnostic testing has revolutionized the medical management of patients with emergent conditions in the acute care setting , 28. RapThis is the first report of a concurrent evaluation of these two commercially-available immunoassay tests for the diagnosis of ROM. Comparable diagnostic accuracy was achieved by both assays with wide and overlapping confidence intervals. The findings are similar to previously published results showing strong diagnostic accuracy  for the more newly introduced ROM Plus® assay . AdditioBoth assays provided exact diagnostic accuracy in preterm patients with all measurements in complete agreement between tests and uniformly concordant with clinical confirmation. The risk of complications and worse outcomes increases significantly with lower gestational age making early ROM diagnosis, especially in very early preterm  patients, imperative .Achieving a high level of diagnostic accuracy is particularly important in cases of equivocal membrane rupture as nearly one-quarter of all pregnant women ultimately diagnosed with ROM do not present with overt clinical evidence of ruptured membranes on initial presentation . We founBoth rapid immunoassay tests evaluated in this study provided excellent diagnostic accuracy for the rapid detection of ROM with only two discrepant results for ROM Plus® and three discrepant results for AmniSure® compared to clinical confirmation. Test performance was particularly robust in preterm patients. The findings from this study support a recommendation for these tests in patients presenting with suspected ROM to guide clinical management decisions to improve obstetrical and neonatal outcomes. Future studies with larger patients sample sizes and cost comparison analyses to traditional methods of ROM detection may be helpful as institutions decide on implementation of point of care immunoassays.