Strategies to inhibit the activity of such proteins are often sought and proposed as possible additions to increase efficacy of the traditional antiretroviral therapy. This requires the acquisition of an in-depth knowledge of the molecular mechanisms underlying their function. The Nef auxiliary protein is expressed uniquely by primate lentiviruses and plays an important role in virus replication in vivo and in the onset of AIDS. Among its several activities Nef enhances the intrinsic infectivity of progeny virions through a mechanism which remains today enigmatic. Here we review the current knowledge surrounding such activity and we discuss its possible role in HIV biology.The replication and pathogenicity of lentiviruses is crucially modulated by “auxiliary proteins” which are expressed in addition to the canonical retroviral ORFs  Initial studies suggested that 3′ORF encoded for a GTPase , because its over-expression was found to attenuate viral transcription and HIV replication in cell culture  pathway dysfunction. Nef can down-regulate the TCR/CD3 complex , a propeKey to these activities of Nef is the ability to form ternary complexes with cargo molecules and adaptor or coatamer complexes via a ExxxLL acidic di-leucin motif  a EE di-In addition to modulating receptor expression levels, Nef also hijacks signaling pathways and alters the activation threshold of lymphocytes  by interWith the term infectivity we here indicate the efficiency with which the virus establishes an infection event within a cell, which culminates with the integration of the virus genome into the host cell genome. This is therefore a parameter which does not depend on steps of the virus life cycle which follow integration, such as virus gene expression or virus release. Infectivity is measured by relating the number or the frequency of the infectious events produced by cell-free virus with the physical number of virus particles. To compare infectivity across different samples, infectious events are therefore normalized to the physical virus content in the inoculum, determined by quantifying the amount of p24 CA protein or the RT-activity of the virus. Most studies investigating HIV-1 infectivity employ infection assays limited to a single round of viral replication by using trans-complemented molecular clones or by the addition of AZT or entry inhibitors at various time points following infection with replication competent viruses, in order to avoid the contributions from successive rounds of replication to the overall phenotype. Steps of the virus life cycle where the effect of Nef on infectivity can be manifest include receptor interaction, entry, uncoating, reverse transcription, nuclear import and integration.The Guatelli lab was the first to report that HIV-1 lacking the ability to express Nef has lower infectivity compared with the Nef-positive counterpart . This obnef alleles derived from a large panel of diverse HIV and SIV isolates has demonstrated that the activity on infectivity is phylogenetically highly conserved .The effect of Nef on virus infectivity requires its expression in producer cells rather than target cells . Nef mig57L58 in the course of virion maturation makes it reasonable to hypothesize a specific role for virus-borne Nef in early steps of viral replication , no effect of Nef is observed with Env that requires virus endocytosis and endosome acidification to promote fusion such as that of VSV and RSV-A . This haNef might thus have an effect on fusion that correlates neither with the route of entry, nor with Env dependence on pH. Such effect has been investigated with responsive and non-responsive Envs and led to contradictory results. While a Nef-dependent increase of the fusion process extrapolated from virus/cell or intravirion fusion assays has been reported by some groups , others Besides these discrepancies on fusion, it has been shown that Nef affects the accessibility of neutralizing antibodies directed against the MPER region of gp41 in a cell/virus fusion context . Althougin vitro, an effect of Nef in vivo cannot be ruled out . Both sides of this same coin can then either involve cofactors recruited by Nef or inhibitory factors counteracted by Nef. So far, the lentiviral auxiliary proteins Vif, VpU, and VpX have been involved in the neutralization of an inhibitory factor (or restriction factor) such as Apobec3, Bst2, and SAMHD1. In the case of Nef both possibilities remain open. The Nef requirement for reaching optimal infectivity is highly variable and depends on the cell types from which virions are produced . Cellular pathways in target cells might then specifically assist Nef+ viruses in the early steps of the virus life cycle . Many observations converge toward a fundamental role of vesicular trafficking for the activity of Nef for infectivity. In particular: (1) HIV-1 Nef requires the integrity of the AP2 interacting motif ExxxLL in virus producing cells . (2) SimAs highlighted earlier, doubts remain on the possibility that Nef affects cytoplasmic delivery of HIV. In the hypothesis where Nef enhances the efficiency of fusion between the virus and the cell membranes, it might do so by promoting the endocytosis of a membrane-bound fusion inhibitor in virus producing cells otherwise incorporated into viral membrane. More generally, factors with potential inhibitory effect, or responsible for the recruitment of co-factors in target cells, might also be rerouted away from or to the viral assembly platforms through the deregulation of protein trafficking by Nef in infected cells.Figure 2B). Of note, similar infectivities of Nef– and Nef+ viruses might either reflect the inability of Nef to drive virus assembly to such compartments  or indicate that optimal assembly does not require the presence of Nef . Because cytoplasmic tail-deleted Envs are still Nef responsive, specific determinants might lie in the extracellular domain, and interact with a chaperone along its secretion and delivery to the plasma membrane. Although this awaits formal evidence, it is compatible with the recent identification of a V2 region in HIV-1 Env as a major determinant of responsiveness to Nef/glycoGag.Another interesting challenge is the identification of the cellular compartment where Nef+ virions acquire their phenotype. Nef might drive the viral components to the appropriate assembly platforms where virions acquire specific features responsible for their higher infectivity and, as suggested earlier, the nature of the protein that decorates virions might play a role in this sorting mechanism, independently of their involvement in membrane fusion. Despite some flexibility in virus pseudotyping, much evidence suggests that the acquisition of Env by budding virions is somewhat regulated . Env migde facto the possibility to identify HDFs involved in the ability of Nef to increase virus infectivity . However, virions used in such HTS were either Nef- or VSV-G pseudotyped, excluding ectivity . A similBiochemistry-based approaches have been used in order to identify cellular factors involved in the many functions of Nef. Affinity tagging or immunoprecipitation followed by mass spectrometry  and convThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TaADF3) from wheat, which was detectable in all examined wheat tissues. TaADF3 is a three-copy gene located on chromosomes 5AL, 5BL, and 5DL. A particle bombardment assay in onion epidermal cells revealed the cytoplasmic and nuclear localization of TaADF3. The expression of TaADF3 was inducible by abscisic acid (ABA), as well as various abiotic stresses (drought and cold) and virulent Puccinia striiformis f. sp. tritici (Pst) but was down regulated in response to avirulent Pst. Virus-induced silencing of TaADF3 copies enhanced wheat resistance to avirulent Pst, with decreased reactive oxygen species (ROS) accumulation and hypersensitive response (HR). Upon treatment with virulent Pst, TaADF3-knockdown plants exhibited reduced susceptibility, which was accompanied by increased ROS production and HR. Interestingly, the silencing of TaADF3 resulted in hindered pathogen penetration and haustoria formation for both avirulent and virulent Pst. Moreover, the array and distribution of actin filaments was transformed in TaADF3-knockdown epidermal cells, which possibly facilitated attenuating the fungus penetration. Thus, our findings suggest that TaADF3 positively regulates wheat tolerance to abiotic stresses and negatively regulates wheat resistance to Pst in an ROS-dependent manner, possibly underlying the mechanism of impeding fungal penetration dependent on the actin architecture dynamics.The actin cytoskeleton has been implicated in plant defense against pathogenic fungi, oomycetes, and bacteria. Actin depolymerizing factors (ADFs) are stimulus responsive actin cytoskeleton modulators. However, there is limited evidence linking ADFs with plant defense against pathogens. In this study, we have isolated and functionally characterized a stress-responsive ADF gene ( As Pst is an obligate biotrophic basidiomycete, which could not be cultured in vitro, the wheat-Pst interaction mechanism has been largely hindered. The expanded understanding of the profound regulation of ADF/cofilins and the multifaceted functions of these ADF/cofilins in physiological changes has led to the conclusion that ADF/cofilin proteins are a functional node in cell biology  signaling pathway. Further silencing analyses revealed that TaADF3 negatively regulated wheat resistance to Pst, most likely by hindering fungus entry in an reactive oxygen species (ROS)-dependent manner. These findings provide new insight into the role of ADFs in host immunity to biotrophic fungal pathogens.As one of the top 10 plant-pathogenic fungi, Triticum aestivum L.) genotype Suwon 11 and Pst pathotypes CYR23 and CYR31 were used for this study. Wheat cv. Suwon 11 contains the stripe rust resistance gene YrSu .Wheat , 100 mM methyl jasmonate (MeJA), 100 mM ethepon, and 100 mM abscisic acid (ABA) dissolved in 0.1% (v/v) ethanol. Mock control plants were treated with 0.1% ethanol. The first leaves that were treated with chemicals along with the control plants were sampled at 0, 0.5, 2, 6, 12, and 24 h post-treatment (hpt). For various abiotic stresses, the roots of wheat seedlings were soaked in 200 mM NaCl or 20% PEG6000 for high salinity or drought treatment. To cause wounding, the first wheat leaves of 2-week-old seedlings were scraped with a sterilized needle. Low-temperature treatment was performed by transferring the wheat seedlings to a 4°C chamber. The first leaves of the treated plants and mock control plants were collected at 0, 2, 6, 12, 24, and 48 hpt. Intact tissues of different wheat organs from 2-week-old seedlings were collected for tissue-specific expression analysis, except for glume, which was collected at the adult stage of wheat seedlings.All the freshly collected samples were immediately frozen into liquid nitrogen and stored at −80°C prior to the extraction of total RNA or DNA. For each time point, three independent biological replications were performed.Pst, and different wheat tissues were extracted using the RNeasy Plant Mini Kit (Qiagen) and treated with DNase I to remove the contaminating DNA. First strand cDNA was synthesized from 2 μg of total RNA using the SuperScript First-strand Synthesis System . The expression of the TaADF3 gene was controlled using the wheat elongation factor TaEF-1α gene (GenBank accession no. Q03033). Quantitative RT-PCR was performed on a 7500 Real-time PCR system , and the relative gene expression was quantified using the comparative 2−ΔΔCT method . Total RNA from wheat leaves treated with chemicals, challenged by abiotic stresses and TaADF3 from the cDNA of wheat leaves. The DNA sequence was obtained by genomic PCR using the total DNA of wheat leaves as the template. The physical characteristics of the deduced protein encoded by the obtained cDNA were computed using the Compute pI/MW Tool. Multiple sequence alignments and phylogenetic analysis were conducted using DNAMAN and MEGA (version 4.0) software, respectively. The phylogram was constructed using the neighbor-joining method, in which bootstrap support values were based on 1000 replicates.Based on the EST sequence (TA54178_4565) in the TIGR Wheat Genome Database, a set of primers TaADF3-cDNA-F and TaADF3-cDNA-R were designed to amplify HindIII and NcoI sites of the pCaMV35S::GFP vector to generate the pCaMV35S::TaADF3-GFP fusion vector.For subcellular localization in onion cells, the TaADF3 protein-encoding sequence was amplified and inserted into the TaADF3 in the barley stripe mosaic virus (BSMV)—mediated virus-induced gene silencing (VIGS) experiment were constructed as described previously  gene fragment of γ:PDS and generate the recombinant γ:TaADF3. To guarantee the specificity of gene silencing, the cDNA sequence of TaADF3 was aligned with the T. aestivum cv. Chinese Spring (CS) genome using the service provided by the International Wheat Genome Sequencing Consortium (http://wheat-urgi.versailles.inra.fr/Seq-Repository/BLAST). The fragments that showed the highest polymorphism within the gene family and the lowest sequence similarity to other genes were chosen for constructing γRNA-based derivative plasmids.The plasmids used for the silencing of pCaMV35S::TaADF3-GFP construct and the control plasmid pCaMV35S::GFP were transformed into onion epidermal cells by particle bombardment at a helium pressure of 1100 psi using the PDS-1000/He system . The transformed onion epidermal cells were cultured on MS medium plates at 28°C for 18–24 h in a dark chamber. Fluorescent signals were observed using a Zeiss LSM 510 confocal laser microscope  with a 480-nm filter.The fusion in vitro transcription using a high-yield capped RNA transcription kit , BSMV RNAs were prepared from linearized plasmids. For inoculation, the RNA transcripts were diluted four times, and 2.5 μL of each transcript, including the BSMV RNA α, β, and γ  transcripts, were mixed with 42.5 μL of FES buffer  with CYR23 or CYR31, as well as the mock control plants. The relative expression of TaADF3 was analyzed by qRT-PCR in each assay to assess the silencing efficiency compared to the control plants. The relative transcription levels of pathogenesis-related protein (PR) genes TaPR1 (AAK60565), TaPR2 (DQ090946), and TaPR5 (FG618781) in the TaADF3-silenced leaves were confirmed by qRT-PCR.The fourth leaves inoculated with BSMV:00 or BSMV:TaADF3-knockdown plants were observed microscopically. For histological observation, leaf segments (1.5 cm in length) were fixed and decolorized in ethanol/ acetic acid (1:1 v/v). The specimens were cleared in saturated chloral hydrate until leaf tissue became translucent. The autofluorescence of the attacked mesophyll cells was observed under a fluorescence microscope  and measured using DP-BSW software to determine the necrotic cell area. The H2O2 that accumulated in the infection sites was stained using 3,3′-diaminobenzidine  at room temperature for 1 h. After washing in 25 mM PIPES and 25 mM phosphate buffer , leaf segments were treated with 0.5% Triton X-100 in 25 mM PBS (pH 6.8) at room temperature for 1 h. The specimens were washed with 25 mM PBS (pH 6.8), then with 25 mM PBS (pH 7.4) for three times. Then leaf segments were stained with Alexa-Fluor 488 phalloidin . Vacuum infiltration was performed three times for 20 s at 27 mm Hg to promote uptake of the dye. Subsequently, samples were stored at room temperature for 3 h in darkness. Finally, leaves were rinsed with 25 mM PBS (pH 7.4), mounted in 25 mM PBS (pH 7.4) on glass slides and observed by fluorescence microscopy. Three biological replications were performed and approximately five leaf segments were observed for each repliation.Actin microfilaments were stained as described previously  of T. aestivum. PCR amplification using the same primers obtained a genomic sequence of 1858 bp, consisting of three exons split by two introns with lengths of 972 bp and 91 bp. The first exon exclusively encodes the first start codon (methionine), typically found in ADF genes  genome sequence showed that there are three copies of this gene in the wheat genome, located on the long arms of chromosomes 5A, 5B, and 5D  in the wheat TIGR genome database, a cDNA fragment of 795 bp in length was obtained with an open reading frame (ORF) of 417 bp, which shows the highest similarity (97.83%) to the actin-depolymerizing factor 3  of 5.65. Multi-alignment of TaADF3 and ADFs from other higher plants revealed a preserved Ser6 in plant ADFs  that could be phosphorylated. TaADF3 contained a bipartite NLS—Lys22 and Arg28 close to the amino terminus; Ser6, Gly7, Arg97, Lys99, Asp124, and Glu127 could bind to actin monomers (G-actin); Lys81, Arg83, Glu135, and Arg136 could specifically bind to microfilaments (F-actin). A CAM combining region  and a PIP2  binding domain (Trp89—Met100) were also included in the sequence  and peaked at 24 hpt with approximate 7-fold expression. These results suggested that TaADF3 may be related to the ABA-dependent signaling pathway.Considering the involvement of ADFs in the response to various abiotic stresses, we investigated the effects of exogenous hormone chemicals and abiotic stresses on the expression of TaADF3 were also induced by some abiotic elicitors  and had the lowest expression level (~0.4-fold) at 12 hpi. The significant difference between compatible and incompatible interactions  suggested that TaADF3 may be a negative regulator in wheat defense against stripe rust fungus.To investigate the role of s Figure . In wheaTaADF3 in the wheat defense response to stripe rust fungus, BSMV-mediated VIGS was used to silence the expression of TaADF3. Ten days after BSMV inoculation, mild chlorotic mosaic symptoms appeared on the fourth leaves of infected wheat seedlings, and the BSMV:TaPDS inoculated plants exhibited strong photo-bleaching . In incompatible interaction, the infection incidence of Pst CYR23 was also reduced by 1.51% compared to the controls  contributes to wheat cold acclimation regulated by genes located on chromosome 5A that are associated with cold hardiness  and late (96–120 h) infection stages of Pst (Wang et al., TaADF3 expression at 120 hpi in compatible interaction may be responsible for the suppression of ROS production. Thus, silencing of TaADF3 led to increased ROS accumulation in TaADF3-kncokdown plants. It seemed that TaADF3 negatively regulated wheat resistance in an ROS-dependent manner. Nevertheless, upon avirulent Pst infection, TaADF3-kncokdown plants retained complete resistance, but with less HR and ROS, which is closer to the immune response. The Arabidopsis AtADF1-4 RNAi lines exhibited suppressed HR mediated by AvrPphB but retained the disease resistance phenotype (Tian et al., TaADF3 may function in a dose-dependent manner to amplify defense signals. According to the hypothesis described by Jones and Dangl (TaADF3 was sufficient to sustain disease resistance but insufficient to attain the threshold for eliciting strong HR, as observed for AtADF4 (Tian et al., ADFs have been implicated to play an important role in determining plant resistance against pathogenic microbes (Hardham et al., nd Dangl , effectiHvADF3 in barley leaf epidermal cells confers enhanced fungal entry of the powdery mildew fungus by interfering with the integrity of the plant actin cytoskeleton (Miklis et al., TaADF3 resulted in transformed array of the actin filaments, which presumably serve as tracks for deposition of callose, secretion of antimicrobial products and delivery of vectorial vesicle (Henty-Ridilla et al., Pst germinates on the leaf surface and penetrate through the stoma, it is tempting to speculate that the altered actin architecture partially attenuated Pst infection. The observed decreased haustoria number may be partially attributed to the impeded fungal entry. As the unique infection structure of biotrophic pathogens in host cells, the haustorium makes intimate contact with the host cell membrane and allows nutrient uptake and effector release into host cells (Voegele and Mendgen, Pst in TaADF3-knockdown plants would lead to a repressed nutrient supply, further limiting the growth and expansion of Pst. Whether the hindered Pst entry was due to the role of TaADF3 on actin architecture is still unclear, although it seems that no detectable change was observed in mesophyll cells actin filaments.It has been documented that the actin cytoskeleton plays a crucial role in resistance during early stages of fungal penetration (Hardham et al., TaADF3 can positively modulate plant acclimation to abiotic stresses, possibly as a downstream component of ABA signaling pathway. Moreover, it suggested that TaADF3 functions as a negative regulator in wheat resistance to Pst dependent on interfering the actin architecture, via limiting ROS release and hindering pathogen penetration. Nevertheless, the exact functional mechanism of TaADF3 still needs further exploration.In conclusion, this study demonstrated that CT, LD, and ZK designed the experiment. CT, LD, DC and SC performed the experiments and analyzed the data. XW helped with data interpretation and article editing. CT wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


High consequence human pathogenic viruses must be handled at biosafety level 2, 3 or 4 and must be rendered non-infectious before they can be utilized for molecular or immunological applications at lower biosafety levels. Here we evaluate psoralen-inactivated Arena-, Bunya-, Corona-, Filo-, Flavi- and Orthomyxoviruses for their suitability as antigen in immunological processes and as template for reverse transcription PCR and sequencing. The method of virus inactivation using a psoralen molecule appears to have broad applicability to RNA viruses and to leave both the particle and RNA of the treated virus intact, while rendering the virus non-infectious. The media and assays used to detect viable organisms will vary depending on the target agents. For human pathogenic viruses, sterility testing is an involved process that begins with inoculation of a portion of the sample of interest into a permissive cell culture line. Depending on the sample, which may contain materials toxic to the cell culture line or require an extended incubation to ensure detectable quantities of virus are present, additional rounds of cell culture amplification may be employed. Once sufficient time has passed to allow amplification of surviving virus, a detection assay such as a plaque assay, immunomicrotitration, or immunofluorescence is performed. The detection of viable agent indicates the process of inactivation was insufficient, and the sample may not be moved to a lower biocontainment area.One of the challenges facing researchers in biocontainment laboratories is that all materials moved from a higher level of biocontainment to a lower biocontainment level  must be proven to be sterile, or undergo a valid and verified method of agent inactivation. This involves inoculating a portion of the material or biological agent containing the sample to be moved to a medium capable of supporting growth of the target organism and evaluating for the presence of agent after sufficient incubation time has passed to allow any viable organisms present to amplify to a detectable level , and researchers have sought inactivation methods that can be validated to support a reduced time before a sample can be moved to a lower biocontainment level. In this sense, inactivation refers to the use of a tested and validated method known to sufficiently and repeatedly render a sample sterile. Materials inactivated using a well-documented and validated inactivation procedure may be moved to a lower biocontainment level, transported, and disposed of without a sterility test. An example of a widely accepted inactivation method is autoclaving; with the process performed correctly and the equipment maintained and operating within normal parameters, and with suitable verification of a full, effective cycle such as biological indicators or machine read-outs, the material subjected to the process is considered sterile. Sterility tests are not performed on every sample that undergoes autoclaving as a method of disposal.Various formulations of formaldehyde have been widely utilized for the inactivation of virus preparations ,2,3,4. HAnother widely accepted method of agent inactivation is the use of reagents containing phenol and a chaotropic salt  for the inactivation of viral samples ,9. The uTRIzol LS is comprised of phenol, an extremely caustic organic solvent, and guanidine isothiocyanate, a chaotropic salt that denatures macromolecules such as DNA, RNA and proteins. Conventional methods to confirm sterility begin with placing a portion of the inactivated sample on a cell culture layer to provide an opportunity for any surviving virus to infect and grow in sufficient quantity to be detectable by observation of cytopathic effects (CPE) or other detection methods. The toxicity of the TRIzol LS in undiluted, treated viral samples causes almost immediate death of the cell culture layer used for sterility testing. Previous studies have diluted the TRIzol LS-inactivated samples 100- and 1000-fold and still observed tissue culture cell death . While tAttempts have been made to remove TRIzol LS prior to sterility testing. Methods such as dialysis and spin column filtration were investigated, but again the caustic nature of the reagent was problematic. Dialysis material is rated only for very small percentages of phenol (well below the concentration present in TRIzol LS-inactivated samples), and membrane filters were not resistant to phenol. Nevertheless, there is wide acceptance of the TRIzol LS method as a biocontainment laboratory standard for the inactivation of viruses based on historical data, precedent, and the known mechanism of action of the inactivating agent.Investigation into the properties of psoralens led to the realization that this group of compounds might provide a method for inactivating viral agents while preserving structures needed for antigenic activity and nucleic acids for molecular or genomic analysis. Psoralens are small, photoreactive compounds that freely penetrate phospholipid bilayers and intercalate between nucleic acid pyrimidine residues, causing cross-linking and inactivation via inhibition of replication following exposure to UV-A radiation. Psoralens do not appear to interact with proteins and inactivation of viruses with psoralen may leave immunogenic surface epitopes intact ,11,12. T2. Six to ten days (depending on the virus) post-inoculation, the culture supernatant was collected, centrifuged at low speed to remove cell debris, and used as virus preparation.Orthomyxoviruses were propagated in MDCK cells; Crimean-Congo Hemorrhagic Fever virus (CCHFV) in HepG2 cells and Lassa virus (LASV) was grown in both RK13 and Vero cells. All other viruses were propagated in Vero cells. Eighty to ninety-five percent confluent cultures were inoculated at a multiplicity of infection (MOI) of 0.001 and propagated in Minimal Essential Medium with 10% fetal bovine serum and 1% antibiotic-antimycotic  at 37 °C and 5% CO6.4 50% tissue culture infectious dose (TCID50) per milliliter. Five-milliliter aliquots of the mixture were transferred into the wells of 6-well plates. The plates were exposed to UV-A radiation  for 0, 2, 5, 10, 20, 30, 40, 50, or 60 min at a measured energy level of 200 µW/cm2 . Other viruses inactivated in a similar manner included CCHF IbAr10200 (105.1 TCID50/mL), Lassa Josiah (105.0 TCID50/mL) (LASV), and Middle East Respiratory Syndrome Coronavirus Jordan (106.9 TCID50/mL) (MERS-CoV). A UV-A radiation energy level of 1000 µW/cm2 and 20 µg/mL AMT was required to fully inactivate Venezuelan Equine Encephalitis TC83 (106.7 TCID50/mL) (VEEV), Junin Candid #1 (104.2 TCID50/mL) (JUNV), Rift Valley Fever ZH-501 (107.8 TCID50/mL) (RVFV), and Ebola Zaire (107.3 TCID50/mL) (EBOV). JUNV and RVFV required 90 min UV-A exposure for full inactivation. EBOV required 120 min UV-A exposure for full inactivation. At each time point, an aliquot was removed from the plates and the titer was determined by TCID50. Each aliquot was assayed for viability by virus isolation in Vero cells using indirect immunofluorescent antibody (IFA) assay.DENV-2 was inactivated according to a previously described method . AMT  received injections of viral antigen on days 0, 30, and 60 followed by exsanguination on day 90. The day 0 injection consisted of 250 µL of antigen emulsified with 250 µL of complete Freund’s adjuvant (Sigma-Aldrich). The remaining injections consisted of 250 µL of antigen emulsified with 250 µL of incomplete Freund’s adjuvant (Sigma-Aldrich). The injected virus antigen was clarified supernatant centrifuged at 2000 RPM at 4°C for 15 min. The minimum pre-inactivation or live titer for injected viral antigen was 1.0 × 102 for 10 days and periodically observed for CPE. Cultures were allowed to incubate for the maximum possible time to allow any viable virus to have a chance to replicate to detectable levels. After 10 days, a portion of each culture supernatant was placed into new cell culture flasks and incubated under the same conditions for another 10 days and observed for CPE. The resulting cell monolayers were scraped and applied to microscope slides following the method of Hsiung [Viruses were tested by IFA ,26. Aliq7 pfu/mL were added to a V-bottom 96-well plate  containing 1 × 105 per 50 µL of Raji-DC-SIGN cells. After 4 h of incubation in a humidified 37 °C CO2 incubator, the cells were washed, fixed, and permeabilized using the BD Cytofix/Cytoperm™ kit  following the manufacturer’s instructions. The fixed cells were stained with a monoclonal antibody to DENV envelope protein (3H5) or an IgG1 isotype control (BD Biosciences) followed by a FITC (fluorescein isothiocyanate) labeled secondary goat-anti-mouse IgG antibody (NBACC). Cells not treated with virus served as the negative control. After 30 min of staining for each step, the cells were sorted on a FACSCANTO II (BD Biosciences). The percent of 3H5 positive cells represented the number of cells that captured and internalized DENV using the DC-SIGN receptor.The amount of DENV internalization by host cells was determined in the Raji B-cell leukemic cell line stably expressing the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule . Brieflyhttp://www.ncbi.nlm.nih.gov/genbank). Primers and probes were designed using Integrated DNA Technologies PrimerQuest primer and probe design tool (http://www.idtdna.com/PrimerQuest/Home/Index). All oligonucleotide primers and probes used in this study are listed in Sequences for virus genes were obtained from Genbank (® 2.0 Fluorometer (Life Technologies) and the Qubit® RNA HS Assay Kit (Life Technologies) according to manufacturer’s instructions. RNA was stored at −80 °C.Total RNA was extracted from psoralen-treated cell supernatants using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. All extracted RNA samples were eluted in a volume of 120 µL. RNA purity was assessed using a Qubit® III Platinum One-Step qRT-PCR Kit w/ROX (Life Technologies) according to manufacturer’s instructions, using a final concentration of 0.2 µM/reaction for each of the primers, and 0.1 µM/reaction for each probe. Five microliters of sample were assayed per 50 µL reaction. Each reaction consisted of 1 cycle of 50 °C for 15 min for reverse transcription, followed by 1 cycle of 95 °C for 2 min for reverse transcriptase inactivation, then 45 cycles of 95 °C for 15 s and 60 °C for 75 s. Reverse transcription real-time PCR assays for each target included no-template controls and test samples. All reverse transcription real-time PCR reactions were run on an Applied Biosystems 7900HT or 7500 Fast (Life Technologies) in standard mode.Reverse Transcription Real-Time PCR assays were performed using the SuperScript® Terminator v3.1 Cycle Sequencing Kit and POP-7™ Polymer (Life Technologies) following the manufacturer’s instructions. The reactions were processed on an Applied Biosystems 3130 Genetic Analyzer (Life Technologies). Raw sequences were analyzed using Sequencher 5.3 software .PCR amplicons were sequenced using the Applied Biosystems BigDye® III Reverse Transcriptase (Life Technologies) following the manufacturer’s instructions. cDNA was amplified with the Ready-To-Go GenomiPhi V3 Kit , and a Nextera® XT library  was prepared for sequencing on the Illumina HiSeq platform following the manufacturer’s instructions in protocol “Paired-end 2 × 150 bp” (Illumina), which uses joined fragments of known length to provide more information in regions of repetitive sequence [Total RNA prepared from psoralen-inactivated DENV was reverse transcribed using random primers and Superscriptsequence ,34.50, repeat TCID50 after subculture, and IFA. Again, thirty minutes of exposure to UV-A light in the presence of AMT was sufficient to reduce viability to a level undetectable by TCID50 after 10 days of culture. However, sequentially passaged subcultures displayed visible CPE after a second 10 days of culture and virus was detectable by IFA. This is a significant change from the earlier plaque assay data and illustrates that rigorous testing methods must be used to confirm inactivation. Forty minutes of exposure to UV-A light in the presence of AMT was required to fully inactivate DENV as determined by CPE and IFA after two ten-day sequential cultures (Dengue virus (a representative ssRNA(+) virus) was previously inactivated by 30 min of UV-A exposure in the presence of a psoralen compound, AMT ,22. Treacultures . Viral cTo assess the general applicability of psoralen-mediated virus inactivation, representative viruses from seven families were exposed to UV-A in the presence of AMT . VirusesArenaviridae), Rift Valley fever virus (Bunyaviridae), and Marburg and Ebola viruses (Filoviridae) were each successfully amplified using well-characterized and previously published assays . Five microliters of fully inactivated JUNV produced a cycle threshold (Ct) of 32.13, LASV 19.48, RVFV 32.07, MARV 18.08, and EBOV 21.03. Each inactivated virus produced an amplicon of the expected size. Amplicons from LASV, JUNV, RVFV, and EBOV were successfully sequenced and produced identical sequence to untreated virus.To determine whether the viral RNA isolated from AMT-inactivated viruses was useful for molecular analyses, five representative viruses were tested for competence in reverse-transcription real-time PCR after inactivation with AMT. Junin virus and Lassa virus  to 961 bp in length were chosen to represent multiple genes and the 3′ untranslated region of the genome. All five targets were successfully amplified from AMT-inactivated DENV. Any photoadducts present in the genome after AMT treatment were not inhibitory to reverse transcription. The cycle in which the fluorescent signal was detectable above background (Ct) of reactions using inactivated virus was similar to the Ct of reactions using live, untreated virus . Each DEDENV RNA isolated from psoralen-inactivated preparations was reverse-transcribed, amplified, and subjected to whole genome sequencing on the Illumina HiSeq using a paired-end strategy of two 150 bp reads. Approximately 98% of the DENV genome was covered in 13,909 of 203,040,312 total reads. Sequencing coverage varied with the majority of the genome falling between 25- and 400-fold .AMT-inactivated DENV was previously shown to bind to a panel of monoclonal antibodies specific for DENV-2 envelope protein and to be highly immunogenic in mice and monkeys ,20. In tIFA is frequently used to detect viral infection of cultured cells, and has been used in this study to demonstrate that viruses treated with psoralen were fully inactivated. After inactivation with AMT and UV-A, EBOV preparations in cell culture were incubated for 10 days at 37 °C before visualization of viruses using IFA. Some fluorescence was visible within the cells, indicating that the virus had been recognized by cell-surface receptors and internalized. The level of fluorescence was significantly lower than the level observed in the positive control cultures containing live virus , even thDengue virus enters the cell via specific interaction with clathrin-coated pits  and the The methods of virus inactivation widely in use rely on intense heat or toxic chemicals to render the virus non-infectious. The treatments also denature or crosslink proteins and nucleic acids, preventing inactivated virus preparations from being fully effective as antigens and diminishing or preventing amplification of the nucleic acids for PCR or sequencing. DENV had previously been shown to be inactivated by exposure to UV-A light in the presence of AMT . In thisThe method was effective at inactivating Alpha-, Arena-, Bunya-, Corona-, Filo-, Flavi- and Orthomyxoviruses. Among the families represented were spherical and flexuous viruses, as well as those with positive- and negative-sense RNA genomes, which suggests that the inactivation method will be successful for all RNA viruses. Each virus tested required a different amount of UV-A exposure in the presence of AMT for inactivation, and an increasing proportion of the virus particles became inactivated with increasing exposure to UV-A, indicating a dose-dependent response to the treatment. Rod-shaped viruses that use a polyprotein strategy required orders of magnitude more microwatts of UV-A than most segmented viruses for full inactivation. It is not known why such a large range of susceptibility to AMT + UV-A inactivation was observed. There does not seem to be a consistent set of virus characteristics that leads to the requirement for more exposure to AMT + UV-A for full inactivation. For example some enveloped, spherical viruses tested required only low levels of UV-A in the presence of AMT for full inactivation , while others required quite high levels . Virus concentration in the inactivation reaction, the type of virus, and the light conditions or UV-A intensity are all parameters that could conceivably alter the inactivation process. It is likely that each individual investigator will have to perform a titration experiment to define the appropriate conditions that consistently produce inactivation in their chosen virus system.The method of virus inactivation via UV-A exposure in the presence of AMT has multiple benefits. We were able to use AMT-inactivated viruses as targets for antibody detection in IFA and FACS and as antigen for production of new polyclonal antibodies. The polyclonal antibodies produced against inactivated virus worked equally well for detecting untreated virus compared to antibodies produced against live virus preparations. These data taken together suggest that the surface proteins of the inactivated viruses are intact and correctly oriented, allowing full interactions with cells and protein complexes. This will allow viruses inactivated by this method to be used in vaccine development, in antibody generation for research, as positive controls for antibody-based detection assays, or studied by microscopy. The nucleic acids extracted from AMT-inactivated viruses were non-infectious and were sufficiently undamaged in EBOV, JUNV, LASV, MARV and RVFV to achieve successful detection by reverse-transcription real time PCR. The viral nucleic acids were non-infectious and sufficiently undamaged in DENV to be appropriate substrates for real time PCR and both Sanger and next-generation sequencing, thus supporting these methods at lower levels of biocontainment. Library-preparation attempts for next-generation sequencing using standard methods for AMT-inactivated LASV and VEEV were unsuccessful, which may indicate that the crosslinking of these viruses in our hands was too great to allow next-generation sequencing. However, amplicons of up to 971 base pairs were successfully detected by PCR, suggesting that number of photoadducts per genome is less than 1 per 1000 bases, at least in the case of DENV. The 3′ end of the DENV genome was amplified as readily as an internal target, which suggests that the photoadducts are not concentrated near the ends of the virus. Most importantly, the method provides inactive, non-infectious virus preparations that may be reliably sterility tested without attempting to remove toxic chemicals from the preparations.If this method of viral inactivation is to become a generally accepted method of inactivation, it is necessary to perform multiple site-specific and agent-specific validation studies rigorously testing the method under multiple scenarios to ensure that preparations inactivated by this method are truly non-infectious. The UV-A exposure time and AMT concentration required to inactivate each virus is affected by viral titer and is species-specific, and may even be strain-specific. More data are needed to determine if all members of families of viruses react the same to the treatment. Investigators should work closely with their Health and Safety Offices to plan studies with multiple methods of analysis and a statistically significant number of replicates. In time, this method could provide rapid access to virus preparations and genomic material from pathogenic samples quickly and safely.


To the Editor: Zika virus is an emerging virus that has spread to most countries in Latin America and the Caribbean  pathogen in the European Union, except for the United Kingdom (where it is BSL-3), requires specific safety precautions (50/mL)  of the Zika virus MR766 strain  radiation inactivates viruses by chemically modifying the genome. We exposed 200 μL of Zika virus preparations containing increasing concentrations of serum to UV light of a laminar flow for up to 60 minutes. Exposure for 10 minutes entirely inactivated Zika virus in the presence of 2.5% FCS serum; increasing concentrations of serum reduced the antiviral effects of UV light , panel B50 was reduced by ≈3 orders of magnitude (Next, we evaluated environmental stability by drying 100 μL of Zika virus stock for 18 hours. Thereafter, dried virus was reconstituted in the same volume of medium or disinfectants. Endpoint titrations showed that the reconstituted virus remained infectious, although TCIDagnitude , panel Cagnitude , panel Cagnitude , panel D>60°C (<pH 4 abrogated infectivity (We also assessed the environmental stability of Zika virus to heat and change in pH. The virus was stable at temperatures up to 50°C but lost all infectivity at temperatures of >60°C , panel E>60°C , panel Fectivity , panel FFinally, we analyzed whether gloves routinely used in BSL-2 laboratories protect against Zika virus. For this, we cut off fingertips of nitrile and latex gloves, filled tips with a Zika virus suspension, and placed them into cell culture plates containing medium. Virus-containing fingertips were inserted in such a way that diffusion would only occur if the virus passed through the nitrile/latex barrier. As a control, we made a hole of <1 mm in the fingertips. All 3 tested gloves prevented virus diffusion , panel GWe demonstrated that Zika virus is destroyed by classical disinfectants and inactivation methods and that nitrile and latex gloves are protective. We also showed that UV light of a laminar flow hood inactivates Zika virus, but particularly if the virus is in a protein-rich environment, the exposure time range should be in hours rather than in minutes. Although we expected that Zika virus would be inactivated by alcohol and disinfectants, we conducted a thorough experimental verification to exclude uncertainties surrounding work with this emerging pathogen.Methods of testing susceptibility of Zika virus to various disinfectants and inactivation methods.


Robert Kissling , a pillaThe fondest of memories return; they remind the authors, his colleagues, and friends that it was with this attitude that the viral disease programs of CDC were established and have been maintained through the years. Bob, as an eminent virologist and a leader of CDC’s viral disease programs, left a permanent legacy.Born in Toledo, Ohio, Bob attended Otterbein College, was awarded a DVM and an MSc from the College of Veterinary Medicine, Ohio State University, and then spent a year at the graduate school of the University of California at Davis. In 1947, the year after CDC was founded, he joined the US Public Health Service and began a long and distinguished career with CDC, then known as the Communicable Disease Center.The 1950s was a period of great progress in field and laboratory research in virology and at CDC’s Virus and Rickettsia Section in Montgomery, Alabama. Bob began collaborative studies of the natural history, epidemiology, and pathogenesis of vectorborne viral encephalitides and began a career-long interest in rabies research; CDC developed an outstanding reputation in these areas, not the least because of Bob’s contributions. While in Montgomery, Bob married Martha Eidson, his laboratory technician, and they had 3 children.In 1958, Bob and guest researcher Robert Goldwasser succeeded in adapting the then-new immunofluorescence methodology to detect rabies viral antigen in brain specimens of animals suspected of being rabid  and the emergence of increasingly sophisticated approaches to virus research, virus detection and identification, and the means to guide CDC’s mission of disease prevention and control. Two new diseases are exemplary of the importance of his work, Marburg hemorrhagic fever and Lassa fever.In 1967, cases of hemorrhagic fever occurred among laboratory workers in Germany and Yugoslavia who were processing kidneys for cell culture; the kidneys were from African green monkeys that had been imported from Uganda. A new virus, Marburg virus, was isolated from patients and monkeys, first in Germany but shortly thereafter at CDC. From the beginning, it was realized CDC must have the capacity to deal with this virus and that Marburg virus was dangerous, calling for the best biocontainment of the day. CDC borrowed a mobile containment laboratory from the National Institutes of Health; the containment level provided was about what would be called Biosafety Level 2+ today. The work was led by Bob (As understood clearly by Bob, the Marburg virus episode had to be the impetus for the development of permanent biocontainment facilities at CDC. A glove port cabinet line laboratory was constructed, which opened in 1968. Completion of the laboratory coincided with the emergence of Lassa fever in West Africa. Early work on Lassa virus elsewhere was terminated after 1 person died and another nearly died; all virus stocks were sent to CDC. Under Bob’s leadership, Tom Monath and others set to work to develop diagnostic systems and uncover the natural history and reservoir host of Lassa virus (Retiring to a farm north of Atlanta in 1973, Bob and Martha were at last able to bird-watch, farm a bit, and enjoy the wildflowers. Martha died in 1999 and Bob, whose health was declining, moved to North Carolina to enjoy his children, grandchildren, and great-grandchildren. A gentleman of common sense, practical sense, he was always polite, always helpful, always willing to teach, and greatly respected by his peers. Bob Kissling was one of the giants of CDC.


Several orphan drugs have been approved by the European Medicines Agency (EMA) over the past two decades. However, the drugs are expensive, and in some instances, the evidence for effectiveness is not convincing at the time of regulatory approval. Our objective was to evaluate the clinical effectiveness of orphan drugs that have been granted marketing licenses in Europe, determine the annual costs of each drug, compare the costs of branded orphan drugs against their generic equivalents, and explore any relationships between orphan drug disease prevalence and annual costs.We searched the EMA database to identify orphan drugs granted marketing authorisation up to April 2014. Electronic searches were also conducted in PubMed, EMBASE and Google Scholar, to assess data on effectiveness, safety and annual costs. 2 reviewers independently evaluated the levels and quality of evidence, and extracted data.We identified 74 orphan drugs, with 54 (73%) demonstrating moderate quality of evidence. 85% showed significant clinical effects, but serious adverse events were reported in 86.5%. Their annual costs were between £726 and £378 000. There was a significant inverse relationship between disease prevalence and annual costs (p=0.01); this was largely due to the influence of the ultra-orphan diseases. We could not determine whether the balance between effectiveness and safety influenced annual costs. For 10 drugs where generic alternatives were available, the branded drugs were 1.4 to 82 000 times more expensive.The available evidence suggests that there is inconsistency in the quality of evidence of approved orphan drugs, and there is no clear mechanism for determining their prices. In some cases, far cheaper generic agents appear to be available. A more robust, transparent and standard mechanism for determining annual costs is imperative. We employed a robust strategy to search for the best quality evidence for effectiveness.We used standardised methods to rate the quality and level of evidence for each orphan drug.We also used reliable data to document the prevalence of each orphan disease and identify the annual cost of each orphan drug.Because we could not document the research and development costs associated with the approval of each orphan drug, the influence of this variable on their annual costs could not be ascertained.The inconsistencies in the quality of evidence for some orphan drugs limit the conclusions that could be drawn regarding their effectiveness and safety.Orphan drugs are therapeutic agents designed for the management of orphan diseases4Because low patient volumes make it unfavourable for commercial investment in orphan drug research, drug regulatory authorities have incentives in place to encourage their development and manufacture by pharmaceutical companies. In the USA, these include exclusive licensing to market such drugs for 7 years, faster assessment procedures, and tax incentives.10A previous systematic review of 11 orphan drugs approved in the Netherlands concluded that there is scarcity of information on the cost-effectiveness of the drugs.The objective of this review was to evaluate the effectiveness and safety of all orphan drugs that have been granted marketing licenses in Europe, determine the annual costs of each drug based on UK estimates, compare the costs of branded orphan drugs against their generic equivalents, and explore the relationship between prevalence of orphan disease and annual costs.http://www.ema.europa.eu/ema/>find medicine>human medicines>Browse by type>Orphan medicine: “Include authorised medicine, withdrawn post-approval and suspended” >SUBMIT’. Orphan drugs granted marketing authorisation were identified up to 30 April 2014. Drugs that have been designated ‘orphan status’, but have not received EMA marketing authorisation, were excluded. The EMA Orphanet Report Series (April 2014)We searched the European Medicines Agency (EMA) database for orphan drugs and their approved indications using the search modality ‘For each identified drug, electronic searches were then conducted in the following databases: PubMed, EMBASE, the Clinical Trials database and National Electronic Library for Medicines. The search terms used included: “orphan drug name” AND “meta-analysis”, systematic review”, “randomised”, “clinical trial”, “placebo”, “follow-up study”, “retrospective studies”, “case-series”, “cohort”, “follow-up studies”, “adverse events”. The prevalence of orphan diseases was documented using the reference values reported in the Orphanet Report Series Bibliographic data,16http://www.pharmaletter.com) and PharmaTimes (http://www.pharmatimes.com) websites, and Google Scholar, for the most recent data on annual costs. The costs of treatment with drugs not used on an annual basis/duration were computed as annual costs. Where orphan drugs were approved for two or more indications, we documented the annual costs of treatment separately for each indication.We estimated the annual average cost in the UK for each orphan drug. Costs of orphan drug regimens might vary according to the individual patient's needs including body size, disease progression, or complications of disease. However, we did not have information on these factors, and so we used the most recent data to estimate the average annual cost in the UK. We searched the UK Medicines Information, the National Electronic Library for Medicines, North East Treatment Advisory Group (NETAG), Scottish Medicines Consortium, and All Wales Medicines Strategy Group databases, for the most recent evidence relating to the annual costs of each drug. Where these were inadequate to compute the annual costs of a specific drug, we searched The Pharma Letter  levels of Evidence,The quality and strength of the overall body of evidence for each orphan drug was then evaluated using a checklist adapted from the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) criteria,19Two reviewers (IJO and EAS) independently evaluated the level, quality and strength of the evidence, and extracted data. These were then cross-checked by two other reviewers (MJT and CJH). Disagreements were resolved through consensus.The EMA database searches identified a total of 74 approved drugs  for managing 63 orphan conditions, with approval dates ranging from 15 May 2002 to 04 April 2014 . Twenty-Using the OCEBM criteria, 25 (33.8%) orphan drugs had level 1 evidence, 35 (47.3%) were level 2, and 14 (18.9%) were level 3 A. BasingOf the 29 drugs approved for management of cancer, 6 (20.7%) showed evidence of significant benefits for both progression-free survival and overall survival; 7 (24.1%) showed evidence of significant improvement only in progression-free survival, while 9 (31.0%) had evidence of only significantly increasing overall survival (see web appendix 2 and table S1). Of the 24 orphan drugs approved for treating inborn errors of metabolism, 21 (75%) showed evidence of beneficial effect on at least one outcome measure (see web appendix 2 and table S2). Of the remaining 21 drugs used for managing other orphan diseases, 20 (95.2%) showed evidence of significant beneficial effects (see web appendix 2 and table S3).Twenty-eight (96.6%) of the orphan drugs approved for treating cancerous conditions had evidence of serious adverse events (see web appendix 2 and table S1). The most common events were bone marrow suppression (58.6%) and hepatotoxicity (20.7%). Adverse events with Dacogen and Evoltra were severe enough to warrant premature termination of clinical trials.Six of 24 drugs (25%) approved for treating inborn errors of metabolism had no evidence of serious adverse events, while another (Cholic acid) did not have any preclinical safety studies prior to approval (see web appendix 2 and table S2). The most common events involved the gastrointestinal and respiratory systems .Eighteen of the 21 drugs (85.7%) approved for treating other orphan conditions had evidence of serious adverse events (see web appendix 2 and table S3). Gastrointestinal adverse events were the most common (23.8%). Other notable adverse events included cardiotoxicity (9.5%), metabolic abnormalities (9.5%) and possible risk of suicide with Prialt (ziconotide).The annual cost ranged between £726 and £378 000 (median £31 012) . Twenty-four per cent of the drugs cost less than £10 000 annually, 58% cost between £10 000 and £100 000, while 18% cost ≥£100 000 annually. For cancer drugs, the range was £1800 to £92 000, compared with £726 to £378 000 for inborn errors of metabolism.A scatter plot of prevalence against annual cost  revealedBecause of discrepancies in outcome measures and time points for outcome measurements, we could not use scatter plots to explore associations between clinical effectiveness and annual costs. All orphan drugs approved for managing pulmonary arterial hypertension showed comparative level of effectiveness at improving 6 min work distance, and decreasing clinical worsening irrespective of annual cost (see web appendix 4). Similar findings were observed for progression-free survival and overall survival for orphan drugs approved for treating cancers. The annual costs of two drugs approved for treating Pseudomonas in cystic fibrosis were comparable. We could not determine whether the risk or occurrence of serious adverse events played a role in the annual costs of approved orphan drugs.Two or more orphan drugs were approved by the EMA for treating seven orphan conditions (see web appendix 4). Scatter plot of annual cost against year of approval for the five drugs approved for pulmonary arterial hypertension suggested a trend towards a significant relationship for higher annual costs with more recent approvals (p=0.06). A significant relationship between annual cost and year of approval was observed with four drugs approved for the management of chronic myeloid leukaemia (p=0.03). There was no significant relationship between annual cost and year of approval for drugs approved for treatment of acute lymphoblastic leukaemia (p=0.38); however, exclusion of the most recently approved drug of which approval was based on a historical perspective  resulted in a significant relationship being observed (p=0.01).We found 15 approved drugs with generic versions, of which data on annual cost of generic or unlicensed versions for 10 (13.5%) were available. Our results show that, of the 74 EMA-approved orphan drugs over a 12-year period, none has shown an overall high quality in available evidence for their effectiveness; there is a moderate level of evidence for three-quarters, while a fifth is low in evidence. Our analyses of annual drug costs revealed several interesting and potentially concerning findings. We found a significant inverse relationship between annual costs of orphan drugs and the prevalence of orphan diseases; in other words, the drugs for the most rare diseases are cheaper than those for more common diseases, largely driven by the drug costs for the ultra-orphan diseases. We also found that drugs approved more recently cost more than those approved over the previous decade. Finally, the annual costs of the drugs did not appear to be related to their clinical effectiveness, and for the subset of the branded orphan drugs that had generic equivalents, we found several extremely large differences in cost.While the clinical trial evidence for over two-thirds of EMA-approved orphan drugs suggests that they have clinically significant beneficial effects, the evidence for about one-fifth indicates that either they do not have clinically beneficial effects, or, more importantly, they may do harm. The results of systematic reviews for several of the drugs are also inconclusive, either because of deficiency in trial reporting or due to a lack of available clinical trials. Our inability to determine whether the level of clinical effectiveness influences annual costs for orphan diseases with two or more approved indications indicates uncertainty about how this variable determines price setting.The findings from our review show that except for the very rare (ultra-orphan) diseases, there is no significant inverse relationship between disease prevalence and annual cost. There were inconsistencies in the strengths of correlation between year of approval and annual costs when three or more drugs used for the same orphan condition were compared. This indicates that there is no standardised process for setting the prices of orphan drugs. It is important to realise that many orphan drugs target very small patient numbers, and are for conditions with few treatment alternatives, thereby allowing their prices to be relatively higher than non-orphan drugs.The results of our review confirm a previous research report, which concluded that prevalence of orphan disease is inversely associated with annual costs for only very rare diseases.We employed a robust strategy to search for the best quality evidence for effectiveness, safety and annual costs of each orphan drug, and we used standardised methods to rate the quality and level of evidence for each identified drug. We also used reliable data to document the prevalence of each orphan disease and identify the annual cost of each orphan drug.However, we do recognise several limitations. Because we could not document the R&D costs associated with the approval of each orphan drug, the influence of this variable on their annual costs could not be ascertained. The heterogeneity in clinical outcomes across different diseases, discrepancies in time points for outcome measurements, and the inconsistency in the levels of outcome reporting, also limited the type of analyses and, therefore, the conclusions that could be drawn regarding the effectiveness and safety of some orphan drugs.Our finding that the trial evidence for the effectiveness and safety of 65% of drugs has not been systematically reviewed suggests an urgent need for prioritising reviews for these drugs. Moreover, systematic reviews evaluating the effectiveness and safety of some EMA-approved orphan drugs are now outdated because more recent clinical trials have become available, and, consequently, such reviews need to be updated. Given the value of systematic reviews for clinicians and health policymakers, we suggest the formation of a Cochrane Collaboration review group focused on orphan drugs as one way of reducing these gaps in knowledge.In addition, further independent and industry-funded trials are also warranted, especially for drugs that currently have poor quality and low levels of evidence. Clinical trial investigators should adequately report any suspected adverse events observed in the course of trials; and statements made in some primary studies that such events, if any, were not related to the drug in question (or not serious enough) may be premature. The importance of close postapproval monitoring cannot be overemphasised. Finally, given the inconsistencies we found in drug costs, a more detailed and transparent analysis of the relationship between R&D costs, and annual costs of orphan drugs, is now imperative.The use of novel therapeutic agents has resulted in improvements in clinical outcome for several orphan diseases. It is also rational that drug companies should aim to recoup investment in R&D costs through sales and reimbursements for these drugs, even though the size of the market for such drugs is quite small.The high costs of orphan drugs are reportedly due to the large amounts of funds invested in R&D by the drug manufacturers.30The marketing authorisations granted to some orphan drugs, such as Ceplene, Firdapse and Xaluprine, were based on a historical perspective, that is, these compounds have previously been used in patient management prior to being designated orphan status.The postapproval withdrawal of some orphan drugs suggests that premarketing and postmarketing surveillance is not stringently assessed  during the process of clinical trial and drug approval. For example, Onsenal (celecoxib) was also withdrawn 8 years postapproval after it was discovered that the risks outweighed its benefits in its use for managing familial adenomatous polyposis.There is inconsistency in the level and quality of evidence for approved orphan drugs. While some orphan drugs have demonstrated evidence of significant benefits, evidence of effectiveness is lacking for several others, and some are associated with serious unwanted adverse effects. The available evidence suggests that, except for the ultra-orphan diseases, the annual costs of orphan drugs approved in Europe are not influenced by disease prevalence. There is inadequate data to determine whether clinical effectiveness influences the price setting of orphan drugs. Further research into the effectiveness and safety of orphan drugs is required, and a standard, transparent and robust mechanism for determining their prices should be a priority.


The entropy-driven binding between the V domain of RAGE and Ca+2-bound S100P was found to lie in the micromolar range (Kd of ∼6 µM). NMR data-driven HADDOCK modeling revealed the putative sites that interact to yield a proposed heterotetrameric model of the S100P-RAGE V domain complex. Our study on the spatial structural information of the proposed protein-protein complex has pharmaceutical relevance and will significantly contribute toward drug development for the prevention of RAGE-related multifarious diseases.The S100P protein is a member of the S100 family of calcium-binding proteins and possesses both intracellular and extracellular functions. Extracellular S100P binds to the cell surface receptor for advanced glycation end products (RAGE) and activates its downstream signaling cascade to meditate tumor growth, drug resistance and metastasis. Preventing the formation of this S100P-RAGE complex is an effective strategy to treat various disease conditions. Despite its importance, the detailed structural characterization of the S100P-RAGE complex has not yet been reported. In this study, we report that S100P preferentially binds to the V domain of RAGE. Furthermore, we characterized the interactions between the RAGE V domain and Ca The receptor for advanced glycation end products (RAGE) is a cell surface signaling receptor and a member of the immunoglobulin superfamily 2+, Mg2+ and Zn2+ have been characterized using circular dichroism (CD), fluorescence spectroscopy, size exclusion chromatography and equilibrium analytical ultracentrifugation S100P is a member of the S100 family of small calcium-binding proteins and has been reported to possess both intracellular and extracellular functions S100 proteins are known to bind to diverse targets and utilize the hydrophobic patch present on the S100 linker region (between helices 2 and 3) and either helix-4 or the C-terminus. However, the sequence variability in the linker region between helix-2 and helix-3, helix-4, and the extended C-terminal region of S100 proteins with an altered net charge and polarity of their binding interface allows for the interaction with a wide range of target proteins In this study, we demonstrate that two RAGE V domains bind to two symmetrical interfaces in the calcium-bound S100P homodimer, forming a heterotetrameric complex. This complex further leads to RAGE homodimerization and activation of downstream signaling pathways, such as ERK, NF-κB, and MAPK pathways. The binding affinity for the interaction between S100P and the V domain of RAGE was determined by fluorescence spectroscopy and isothermal titration calorimetry (ITC). A HADDOCK approach was used with the interaction sites as constraints to model the structure of the S100P-RAGE V domain complex using a previously determined X-ray crystallographic structure of calcium-bound S100P 15NH4Cl, 13C-labeled glucose, and D2O were purchased from Cambridge Isotope Laboratories, and β-mercaptoethanol was obtained from Sigma. SW-480 cells were purchased from the American Type Culture Collection .The reagents for Luria broth were obtained from AMRESCO. 2. The cDNA encoding the recombinant RAGE V domain (residues 24–121) was subcloned into the pET-15b(+) expression vector, transformed into BL21(DE3) Codon Plus host cells and expressed and purified as previously described 2. The eluted fractions were concentrated using Ultra-Centricon filters (Millipore) to a protein concentration of 0.7–1.0 mM. The purity was estimated to be approximately 95% by Coomassie-stained SDS-PAGE and HPLC analyses. The molecular weight was confirmed by ESI-TOF mass spectrometry.Recombinant wild-type S100P (residues 1–95), the single mutants S100P E5A and S100P D13A and the triple mutant S100P F44G/Y89G/F89G were cloned into the pET-20b(+) expression vector, overexpressed in BL21(DE3) host cells and purified as previously reported 2 using 1H-15N HSQC, 1H-13C HSQC, HNCA 15N-edited TOCSY All NMR experiments were performed at 298 K on a Varian 700 MHz spectrometer equipped with a cryogenic triple-resonance probe. The backbone and side chain chemical shifts of calcium-bound S100P have been previously assigned and have been deposited in the BMRB 2 for 36 h with three buffer exchanges using a dialysis membrane with a molecular weight cut-off of approximately 3500 Da. All proteins samples were centrifuged and degassed under vacuum for 15 min to remove air bubbles prior to the titration experiment. ITC experiments were performed using a MicroCal VP-ITC calorimeter. A 2.0 mM solution of S100P was placed in a syringe as the protein ligand and titrated into a sample cell containing a 0.06 mM solution of the RAGE V domain. All titrations were performed at 25°C, and an injection volume of 10 µL was used for all subsequent titrations, with a 60-s initial equilibration delay and a 280 s delay in the intervals between the injections. The titration curves were corrected using buffer controls and analyzed using Origin software . A similar approach was used for subsequent experiments to characterize the interactions between mutant S100P proteins and the V domain of RAGE.For the preparation of protein samples, respective samples of S100P and the V domain of RAGE were dialyzed in 20 mM Tris-HCl (pH 7.0), 100 mM NaCl and 4 mM CaCl2 at 25°C. After each addition of S100P, the sample was stirred for 2 min prior to scanning. The changes in the fluorescence intensities (ΔF) were monitored at 348.5 nm. S100P does not contain tryptophan residues, and the tyrosine and phenylalanine residues in S100P do not interfere with the excitation wavelength used here. The ΔF versus [S100P] plots were then fitted using the Origin program with d is the dissociation constant and ΔFmax is the maximal fluorescence change.An emission spectrum for the intrinsic tryptophan fluorescence of the RAGE V domain was obtained using a Hitachi F-2500 fluorescence spectrophotometer. The tryptophan in the RAGE V domain was excited at a wavelength of 295 nm, and subsequent changes in the emission spectra were monitored by scanning from 305 to 400 nm with a slit width of 5 nm. A solution of S100P was added in 2-µL increments to a 2 µM solution of the RAGE V domain in 20 mM Tris-HCl (pH 7.0), 100 mM NaCl, and 4 mM CaCl1H-15N HSQC spectra at 25°C on a Varian 700 MHz spectrometer equipped with a cryoprobe. For HSQC characterization, all protein samples were prepared at a concentration of 0.3 mM in 20 mM Tris-HCl  containing 100 mM KCl and 4 mM CaCl2. Unlabeled calcium-loaded S100P was added to uniformly 15N-labeled RAGE V domain at molar ratios of 1∶0.2, 1∶0.4, 1∶0.6, 1∶0.8, 1∶1, 1∶2, 1∶3 and 1∶4. Similar mixtures of unlabeled RAGE V domain and uniformly 15N-labeled S100P were also prepared. The shift and/or missing peaks were identified by overlaying the HSQC spectra. The observed changes in the cross-peak intensities of the 15N-labeled S100P-RAGE V domain complex were compared to free S100P and plotted in a bar graph. Furthermore, 3D 13C(ω2)-edited, 12C(ω3)-filtered NOESY-HSQC spectra were acquired with a mixing time of 120 ms using doubly labeled  RAGE V domain mixed with unlabeled  S100P at a 1∶1 molar ratio 2. The final sample was lyophilized and then dissolved in >99% D2O.The NMR HSQC titrations were monitored by recording 2D 15N-1H HSQC analysis observed upon complex formation were used to define the ambiguous interaction constraints for residues at the interface. Active residues were defined as residues that exhibited the maximum decrease in the intensity of a cross-peak and a relative residue accessible surface area larger than 30% for either side chain or backbone atoms, as calculated by NACCESS 13C(ω2)-edited, 12C(ω3)-filtered NOESY-HSQC analysis, and these structural data were used as input for the HADDOCK calculations. That is, unambiguous restraints derived from 10 assigned NOEs were defined as intermolecular constraints between S100P and the V domain of RAGE during the docking calculations. A total of 2000 rigid-body docking trials were performed using the standard HADDOCK protocol with optimized potentials for liquid simulations (OPLS) parameters The model of the S100P-RAGE V domain complex was generated using the HADDOCK2.0 web server with CNS 1.1 2 at 37°C.The human colorectal adenocarcinoma cell line SW-480 (ATCC: CCL-228) was cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator with 5% CO4 cells/well in a 96-well cell culture plate. Subsequently, the cells were treated with 100 nM protein  and 10 µM FPS-ZM1 for 48 h. MTT solution was then added to each well to a final concentration of 0.5 mg/ml, and cells were incubated at 37°C for an additional 3 h. After removal of the medium, formazan crystals were dissolved in 100 µL of DMSO (dimethyl sulfoxide) by gentle agitation on a shaker for 10 min. The absorbance was measured at 570 nm using a Synergy 2 microplate reader . The cell counts were determined from the relative absorbance of the experimental treatment compared with the control treatment.Cell proliferation was measured using an MTT  assay. The day before the experiment, SW-480 cells were seeded at a density of 1×10d = 6.2±0.1 µM with additional parameters of N = 1.07±0.03, ΔH = 4.094±0.02 kcal/mol, and ΔS = 37.6 cal/mol/deg. The 1∶1 stoichiometry of the binding interaction is evident from the existence of two identical binding sites between two V domains of RAGE and the S100P homodimer. Additionally, the formation of a symmetrical heterotetrameric S100P-RAGE V domain complex was inferred from the single-binding site model, as evident from the ITC binding isotherm. The thermodynamic factors ascertained for the interaction of S100P with the RAGE V domain signified that their binding is stabilized by entropic factors and counter affected by a positive change in the enthalpy of binding. The positive change in entropy, ΔS (favorable), and positive change in the heat capacity of the system, ΔH (unfavorable), suggested that the binding was driven by entropy and supports the role of the hydrophobic residues present at the interface between the RAGE V domain and S100P in complex formation. The favorable entropic contribution to the stability of the S100P-RAGE V domain complex is likely due to the entropy gain from the burial of the hydrophobic patches at the interface and the displacement of water from the nonpolar surfaces of both S100P and the V domain of RAGE. Overall, our findings from ITC analysis of the interaction between S100P and the V domain of RAGE are consistent with previous studies Isothermal titration calorimetry (ITC) experiments were performed to assess the binding affinity and stoichiometry of the complex by determining the heat changes that occur during protein-protein binding d, of approximately 6.8 µM. Overall, our fluorescence results are consistent with our ITC analysis and suggest a modestly strong binding affinity of S100P for the RAGE V domain that is in the lower micromolar range, confirming the formation and stability of this protein complex under physiological conditions.The intrinsic tryptophan fluorescence of proteins is sensitive to the polarity of the local environment and conformational changes in the protein associated with substrate binding and can be monitored 1H-15N HSQC NMR is a useful technique to identify the protein-protein or protein-ligand binding interface 1H-15N HSQC spectra of free S100P compared with S100P in complex with the RAGE V domain. Overlaid HSQC spectra of 15N-labeled free S100P and S100P in complex with the unlabeled RAGE V domain are shown in 1H-15N HSQC spectrum. Previous NMR studies on S100 proteins with their target proteins have also reported that the residues most affected by complex formation will experience substantial changes in the NMR resonance line width and forms the basis for the identification of the interfacial features of the protein-protein complex 0) were plotted in a bar graph, as shown in 15N-labeled RAGE V domain with unlabeled S100P at a 1∶1 molar ratio. These experiments were performed to identify the RAGE V domain residues that interact with S100P. A subset of cross peaks that exhibited altered intensities due to affected nuclear signals of RAGE V domain residues upon S100P binding, such as R48, K52, N54, W61, K62, Q67, G68, L86, C99, Q100, R104, N105 and K110, are shown in 0) was used to map the critical residues involved in complex formation and is shown in 15N-labeled S100P and unlabeled RAGE V domain were performed at higher molar ratios  to monitor the rate of association-dissociation equilibrium between free and complexed S100P. During the high molar ratio titrations, a majority of the resonances exhibited significant line broadening (data not shown). Overall, the observed spectral changes reflect the intermediate exchange regime between free S100P and S100P in complex with the RAGE V domain.Two-dimensional 15N-HSQC titration experiments (Table S1 in File S1). Most of these residues are surface exposed in the free proteins and form one continuous interface in the protein-protein complex, indicating localized changes in the spatial geometries of both S100P and the RAGE V domain. These findings thus support the NMR restraint-driven HADDOCK approach utilized to generate the structure of the heterotetrameric complex of S100P and the RAGE V domain 13C-filtered experiments and were used as unambiguous distance restraints in docking . A set of 2000 complex structures was generated by rigid-body minimization and was further refined to the best 200 structures based on the total energy, selecting for torsion angle dynamics and subsequent Cartesian dynamics in an explicit solvent (water) model to flexibly optimize the contacts. The 200 structures with the lowest water-refined interaction energies were used for subsequent analysis. HADDOCK clustered these 200 structures into a single cluster, with an RMSD of 0.6±0.3 from the overall lowest energy structure . Figure S3 in File S1). PROCHECK analysis of the structure indicated good stereochemistry for the bond lengths and bond angles; 99.4% of all of the non-glycine residues fall within the allowed region of the Ramachandran plot . Mutation of E5 to alanine produced a 3.8-fold reduction (22.8 µM) in the binding affinity and the S100P D13A mutant similarly exhibited a 146-fold reduction in the dissociation constant (877.8 µM) for the interaction with the RAGE V domain. However, no interaction was observed between the S100P F44G/Y88G/F89G triple mutant and the RAGE V domain, as shown in . The substantial reduction in the binding affinity of the S100P mutants in comparison to wild-type S100P suggests that E5, D13 and the aromatic residues act as ‘hotspot’ residues that are involved in crucial interactions with the RAGE V domain and, thus, are significant for protein-protein complex stability. An additional comparative analysis of the thermodynamic parameters of the E5A and D13A S100P mutants with wild-type S100P indicates modest variations in the enthalpic factor in contrast to significant deviations in the entropic factor (Table S2 in File S1). Complete abolition of binding between the S100P triple mutant and the RAGE V domain indicates the importance of hydrophobic residues in complex formation, i.e., mutations at the interface of the protein complex affect the buried surface area (BSA) between both proteins, confirming that the binding between S100P and the RAGE V domain was predominately driven by favorable entropic factors as proposed for the D13A and E5A mutants Additional structural insights into the binding interface between calcium-bound S100P and the V domain of RAGE were obtained from ITC analysis of S100P mutants. Three mutants were designed to evaluate the contribution of the residues at the binding interface of the S100P-RAGE V domain complex. We mutated the S100P residues E5 and D13 to alanine to assess the role of crucial electrostatic interactions at the protein-protein interface. We also constructed an S100P triple mutant (F44G/Y88G/F89G) using site-directed mutagenesis of hydrophobic residues to glycine instead of the conventional alanine to completely abolish the hydrophobic side chain contributions of these residues at the binding interface, as shown in S100P-mediated RAGE receptor activation has been demonstrated to be a critical step in the overall pathway for the subsequent downstream stimulation of cell proliferation in various cancer cell lines 1H-15N HSQC spectra in the absence and presence of pentamidine, we identified the S100P residues involved in pentamidine binding from the altered or missing cross-peaks in the calcium-bound S100P 1H-15N HSQC spectra, as shown in d value of 305 µM and indicated that the interaction was driven by enthalpy  with a 1∶1 stoichiometry  estimated from ITC and fluorescence spectroscopy is approximately 6.0 µM, suggesting a moderately strong interaction. Our ITC results also suggested the presence of two identical and independent binding sites between two RAGE V domains and a calcium-bound S100P homodimer, supporting the hypothesis that hydrophobic interfaces are buried between the proteins upon complex formation. Additional NMR titration experiments identified the putative binding interface for the S100P-RAGE V domain complex. Mapping of the binding interface residues indicated the nature of the binding interfaces of S100P and the RAGE V domain. Overall, our findings reveal the extracellular role of the S100P homodimer, which symmetrically interacts with two RAGE V molecules. Based on these conclusions, we modeled the S100P-RAGE V heterotetrameric complex using HADDOCK, which revealed the putative interfaces between S100P and the RAGE V domain. An analysis of the total interface area for the HADDOCK-calculated S100P-RAGE V domain complex is approximately 4738 Å2 per two interfaces, with a ratio of the hydrophobic to hydrophilic areas of 58∶42. Interestingly, the RAGE V domain residues at the S100P binding interface, particularly R48, K52, R98 and R104, are similar to those that interact with S100B, S100A6 and AGE. Overall, we can infer that the binding interface of the RAGE V domain is well conserved for its interaction with S100P and is similar to that for other known RAGE V domain binding partners, as has been observed in the RAGE-AGE complex, the S100B-RAGE VC1 model complex, and the S100A6-RAGE V model complex S100 proteins are calcium-binding proteins that represent a subfamily of EF-hand proteins. The members of this family share a common topology that consists of two calcium binding helix-loop-helix structural domains that mediate calcium-dependent signal transduction. These S100 proteins engage in a large number of intracellular and extracellular functions File S1Combined file containing supporting figures and tables. Table S1. Active and passive residues used to define the ambiguous interaction restraints for the docking of S100P with the V domain of RAGE. Table S2. Thermodynamic parameters of the interaction between wild-type or mutant S100P and the V domain of RAGE, as determined by ITC. Kd, dissociation constant; ΔH and ΔS, changes in the enthalpy of binding and entropy of binding, respectively; ΔGbinding, Gibbs free energy of binding; T, temperature in Kelvin; and ΔGbinding = ΔH − TΔS. Figure S1. Intermolecular NOEs between the V domain of RAGE* and S100P. The intermolecular NOE peaks between the V domain of RAGE and S100P were observed in 13C(ω2)-edited, 12C(ω3)-filtered NOESY-HSQC experiments and are represented as strip plots. Figure S2. Scatter plot of the HADDOCK score versus the fraction of native contacts (FCC) for a single cluster generated by HADDOCK. Figure S3. Detailed view of Intermolecular NOEs between residues in the RAGE V domain (green) and S100P (cyan) of the modeled RAGE V domain-S100P complex. Figure S4. Secondary structure characterization of wild-type S100P and S100P mutants. Each protein was measured at a concentration of 32 µM in 20 mM Tris-HCl (pH 7.0), 100 mM NaCl, and 4 mM CaCl2. An average of three far-UV CD spectra scans were recorded for each S100P protein from 195 nm to 260 nm using a JASCO-720 spectropolarimeter.(ZIP)Click here for additional data file.


When a disease affects only a few individuals in each country (ultra-orphan disease), it can be very hard to establish the costs of illness (COI) and the cost-effectiveness (CE) of treatments (ultra-orphan drugs). Conventional methods for COI and CE of drugs for common conditions do not apply, and additional factors need to be considered. As expensive medications  show promising results, it becomes crucial to have detailed information on as many patients as possible.This international, multicenter, longitudinal observational COI and CE study will evaluate the burden of CAPS in terms of direct and indirect costs and quality of life (QoL). The COI analysis implies the identification, quantification and evaluation of resources from a societal perspective.http://www.printo.it/eurofever/), 8 Centers of reference for autoinflammatory diseases in Italy and France (sentinel countries) are currently enrolling all consenting/assenting CAPS adult patients and children with the indication for either the use of an anti IL-1 agent or other than anti IL-1 therapies , and compliant to the study. To perform the COI analysis, clinical and economic data are collected at country-specific- and general levels, both retrospectively (since disease onset) and prospectively (from study enrollment to the last visit) by mean of general questionnaires administered to patients or their proxy on direct medical and non-medical costs, and indirect costs of productivity loss. QoL is prospectively measured using EQ5D to estimate utility score and calculate QALY.In the frame of the EuroFever registry  will develop a model to evaluate costs and long-term benefits in an ultra-orphan group of diseases such as CAPS. The same model may be used in other very rare disorders.