Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease. Arenaviridae virus family includes several life-threatening human pathogens that cause meningitis or hemorrhagic fever. These RNA viruses replicate and transcribe their genome using an RNA synthesis machinery for which no structural data currently exist. They synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism thought to involve the large L protein, which carries RNA-dependent RNA polymerase signature sequences. Here, we report the crystal structure and functional characterization of an isolated N-terminal domain of the L protein (NL1) from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures and mutagenesis studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. Reverse genetic studies show that mutation of active site residues selectively abolish transcription, not replication. We show that this endonuclease domain is conserved and active across the virus families: Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.The  Arenaviridae family includes 22 viral species into a single genus Arenavirus, with new species awaiting classification Lymphocytic choriomeningitis virus (LCMV) is the prototypic species and first arenavirus isolated in 1933. Because its natural host is the common house mouse (Mus musculus), LCMV is the only known arenavirus presumably exhibiting a worldwide distribution. LCMV is a human pathogen of significant clinical relevance, causing central nervous system disease, congenital malformation, choriomeningitis, and systemic and highly fatal infection in immuno-compromised, organt transplant recipient patients The Arenaviruses are enveloped viruses with a bisegmented negative single-strand RNA genome. Each RNA segment, called large  and short , contains two open reading frames in mutually opposite orientations and use an ambisense coding strategy to direct the synthesis of two polypeptides Arenaviridae L protein domain at 2.13 Å resolution, that of the N-terminus domain of the LCMV L protein. We show that this domain is able to bind nucleotides, with a preference for UTP, and RNA. Structural comparison with the N-terminal part of the influenza virus PA protein characterizes unambiguously the domain as an endonuclease. Sequence and secondary structure analysis of L proteins from various Bunyaviridae family members predict that their N-terminal end carries a similar endonuclease activity, that we demonstrate for Toscana virus (TOSV) . Activity assays and mutagenesis show that the arenavirus endonuclease exhibits sequence-specificity with a preference for uracil-containing substrates. Lastly, reverse genetics studies correlate expression of endonuclease activity with the selective production of mRNA, making the N-terminus domain of the L protein a likely candidate to be involved in the cap-snatching mechanism of arenaviruses.We report here the first crystal structure of an E.coli and purified, but yielded crystals diffracting to 8 Å. However, the homologous 196 residues domain of LCMV yielded well-diffracting crystals. The atomic structure of NL1 was first determined by the SAD technique with seleno-methionylated crystals that diffracted to 3.4 Å. The structure was refined using a native data set at 2.13 Å resolution , Lassa fever virus (LASV), Parana virus (PARV), and LCMV) L proteins. All four domains were expressed as soluble recombinant proteins. We observed a self-limited proteolysis of the Parana arenavirus N-terminus L domain which prompted us to refine boundaries into a shorter 196 aa form, hereafter named “NL1”, fully included in the previously predicted arenavirus L1 domain (1–250 aa) solution . Two NL1N) returns a Z-score of 5.7 and an r.s.m.d. of 3.9 Å for 121 superposed aa (PDB code 3EBJ) and Z-score 5.2, r.m.s.d. 4 Å for 122 aa (PDB code 2W69). As was the case for PAN, other type II endonuclease proteins are also recovered: the Tt1808 hypothetical protein from Thermus Thermophilus HB88 , and the restriction endonuclease SdaI .The LCMV NL1 monomer structure has approximate dimensions of 59 Å ×37 Å ×27 Å. It features four mixed β-strands forming a twisted plane surrounded by seven α-helices . The twoN hosts the RNA cap-snatching endonuclease activity N share a similar core structure. Except for the absence of a fifth β-strand in NL1, all other secondary structure elements are conserved (N active site (discussed below). At the aa sequence level, NL1 shares the conserved active site sequence motif characteristic of type II endonucleases: PD…(D/E)XK. In NL1, the corresponding residues are P88, D89…E102, and either K115 or K122  closely superimpose with P107, D108 and E119 of the influenza virus PAN protein, pointing to a common function for these residues  and one genus of plant virus (Tospovirus) Bunyaviridae are also thought to use a cap-snatching mechanism to prime mRNA synthesis Bunyaviridae share a conserved RdRp motif within their large L protein, as well as a conserved N-terminus domain Bunyaviridae L protein reveal that the latter also possesses the conserved active site motifs characteristic of type II endonucleases  than the L protein of other members of the Bunyaviridae family (∼2200 aa). The putative endonuclease catalytic motif was located after aa ∼700, the N-terminal of Nairovirus L protein being assigned as a so-called OTU-like domain In addition to ucleases . HoweverN. Finally, the structural organization of the putative CCGV endonuclease domain seems to diverge even further from the others. Indeed, whereas the conserved lysine is shared by the same helix for all the domains, that of Nairovirus may be located at the end of the β4 strand  virus endonuclease domain was prepared according to bio-informatic predictions described above. Its endonuclease activity was compared to both that of arenavirus NL1 and the influenza H5N1 endonuclease 2+ than Mg2+, and shows background activity with Ca2+ and Zn2+, as well as two important lysine residues K115 and K122 neighboring D119 -E-X12-K-X3-D-X2-K. The most obvious difference with the only known related RNA endonuclease, that of influenza virus PAN, is a divergence upstream the PD motif in structural elements carrying the E51 residue  pLysS (Novagen), at 17°C in 2YT medium overnight after induction with 500 µM IPTG. Cell pellets from harvested cultures were resuspended in 50 mM Tris buffer, pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1% Triton, 5% Glycerol. Lysozyme (0.25 mg/ml), PMSF (1 mM), DNase I (2 µg/ml), and EDTA free protease cocktail (Roche) were added before sonication. IMAC chromatography of clarified lysates was performed on a 5 ml His prep column  eluted with imidazole. Size exclusion chromatography was performed on preparative Superdex 200 column  pre-equilibrated in 10 mM Imidazole, pH 8.0, 50 mM NaCl, 2 mM DTT. Protein was concentrated (28 mg/ml) using a centrifugal concentrator. For enzymatic studies, WT and mutants were express in the E.coli BL21 star strain (Invitrogen) and further purified on HiTrap Q sepharose 1 ml column  to remove E. coli RNase contaminants. Proteins eluted in a linear gradient from 50 mM to 1 M NaCl in 10 mM Hepes buffer, pH 7.5, 2 mM DTT. A synthetic gene of the H5N1 PAN endonuclease was designed as described The LCMV NL1 cDNA  was cloned into pDest14 with a N-terminus hexa-histidine tag and expressed in 4 250 mM, citrate 50 mM, isopropanol 5.5%, using the hanging drop vapor diffusion method in Linbro plates by mixing 1 µl of protein solution with 1 µl of reservoir solution. Crystals were cryoprotected by dipping in a solution containing 65% of crystallization buffer and 35% of a buffer made of size exclusion chromatography buffer/glycerol (50/50). Crystals were cryo-cooled in liquid N2. The crystals belong to space group C2221 and have two molecules per asymmetric unit. Despite repeated attempts, crystal soaked into the above buffer supplemented with various concentrations of MnCl2 yielded crystals diffracting to >4 Å.Crystals grew in LiSODiffraction intensities were recorded on the ID14-4 beamline at the European Synchrotron Radiation facility, Grenoble, France. Data were processed and integrated with MOSFLM Arenaviridae endonuclease motif i.e. P-D-x-E-x(12)-K-x(3)-D-x(2)-K ; was performed against non-redundant databases Orthomyxoviridae, appears in the section with an E-Value below threshold. A fourth iteration including these two sequences allows retrieving the entire family of orthomyxoviruses, with E-value comprised between 3e−18 and 2e−4.A PHI-BLAST search using the sequence corresponding to the L1 domain and the signature of the Bunyaviridae family hitting the pfam 04196 A standard CDD search from the sequence of Tensaw virus allows retrieving all the L of the http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi). Using the secondary structure prediction of the endonuclease domain of L proteins, the putative conserved active site residues were identified and placed correctly in the alignment.A multiple sequence alignment of the N-terminal end of the L protein from LCMV, HLCV, BUNV, HANV, PUMV, RVFV, TOSV, TOMV, WTMV, CCGV, DUGV, was first performed with the T-coffee algorithm (32P NTP (0.4 µCi/µl) in 10 µl of reaction buffer containing 10 mM Imidazole, pH 8.0, 50 mM NaCl, 2 mM DTT. The reaction mixtures were then exposed to UV light (254 nm) for 6 min at 5 mm distance. The crosslinked species were separated in a 15% polyacrylamide denaturing gel and visualized by autoradiography using photo-stimulated plates and a Fujilmager (Fuji).7 µg of purified protein were incubated for 15 min at 25°C, with 0.5 µl of the various α-5′-AUUUUGUUUUUAAUAUUUC-3′ (Ambion) was [32P] 5′-end labeled, and 0.4 µM of radiolabelled RNA was incubated 20 min at 25°C without and with 1.4 µg, 4.2 µg and 7 µg of protein in 10 µl of 10 mM Imidazole, pH 8.0, 50 mM NaCl, 2 mM DTT. Reaction mixtures was analyzed by PAGE and visualized by autoradiography.The RNA 2, MnCl2, MgCl2 and ZnCl2 were performed at 1 mg/ml protein in gel filtration buffer using thermal shift assay. Technical details can be be found in Titration curves with CaCl5′-UGAGGCCCGGAAACCGGGGCC-3′ (Ambion), ΔG = −14.7 Kcal/mole), a 22 mer moderately stable hairpin (5′- CGCAGUUAGCUCCUAAUCGCCC-3′ (Ambion), ΔG = −3.4 Kcal/mole), and a long 264 mer RNA corresponding to the SARS-CoV 5′-genome sequence. The latter was radiolabelled with a cap structure at its 5′-end using the ScriptCap m7G Capping System (Epicentre Biotechnologies) with [α32P]GTP. Endonuclease assays were carried out using 3.3 µM of radio-labeled RNA in a buffer containing 40 mM Tris-base, pH 7.5, 100 mM NaCl, 10 mM β-Mercaptoethanol and 2 mM MnCl2. Reactions were initiated by the addition of 1 µM of protein and incubated at 37°C, and stopped by the addition of EDTA/formamide. Reactions products were analyzed using denaturing polyacrylamide gel electrophoresis  and analyzed by autoradiography.Endonuclease activity was assayed using 4 different heteromeric RNA substrates: an unstructured 19 mer as described above, a 21 mer stable hairpin (32P-labeled riboprobe targeting the Renilla luciferase gene. Autoradiography was quantified on a PhosphorImager (Amersham Biosciences). To verify protein expression, hemagglutinin (HA)-tagged L protein was expressed in BSR T7/5 cells inoculated with modified vaccinia virus Ankara expressing T7 RNA polymerase (MVA- T7) The LCMV replicon system is based on strain Armstrong clone 13 and has been established in analogy to the Lassa virus replicon described previously Figure S1A, Structure-based sequence alignment of NL1 with the two influenza PAN , Tt1808 (1WDJ) and SdaI (2IXS), showing the structurally-conserved endonuclease motif (Highlighted in red). B, Sequence alignment of arenavirus NL1: Lymphocytic choriomeningitis virus (LCMV), Dandenong virus (DANV), Mopeia virus (MOPV), Morogoro virus (MORV), Mobala virus (MOBV), Ippy virus (IPPV), Lassa virus (LASV), Lujo Virus (LUJV), Parana virus (PARV), Pichinde virus (PICV), Allpahuayo virus (ALLV), Chapare virus (CHAV), Tamiami virus (TAMV), Whitewater Arroyo virus (WWAV), Bear Canyon virus (BCNV), Flexal virus (FLEV), Pirital virus (PIRV), Amapari virus (AMAV), Guanarito virus (GTOV), Cupixi virus (CPXV), Machupo virus (MACV), Junin virus (JUNV), Tacaribe virus (TCRV), Sabia virus (SABV), Oliveros virus (OLVV), Latino virus (LATV). Residues in a solid red background are strictly conserved. The blue point indicates the key active site residues.Sequence Alignment of Viral Endonuclease Domains. (4.29 MB TIF)Click here for additional data file.Figure S2A, Sequence alignment showing endonuclease motif (highlighted in red) of the NL1 domain from LCMV and the five genus of the Bunyaviridae families. Each genera is represented by two viruses: Orthobunyavirus: Human La Cross Virus (HLCV) and Bunyamwera virus (BUNV), Hantavirus: Hantaan virus (HANV) and Puumala virus (PUMV), Phlebovirus: Rift valley fever virus (RVFV) and Toscana virus (TOSV), Tospovirus: Tomato virus (TOMV) and Watermelon silver mottle virus (WTMV), Nairovirus: Crimean-Congo hemorrhagic fever virus (CCGV) and Dugbe virus (DUGV). Position of the start of the motif is labelled in blue for each virus. B, Based on secondary structure predictions topology diagrams were drawn for the NL1 domains from PUMV, RVFV, TOMV, HANV, CCGV. Colours are the same as in Conservation of NL1 Between Arenaviruses and Bunyaviruses. (8.97 MB TIF)Click here for additional data file.Figure S3Verification of L protein expression by immunoblotting. L protein used for analysis in replicon assay was tagged with HA tag, expressed under T7 promoter control in cells inoculated with modified vaccinia virus Ankara expressing T7 RNA polymerase (MVA-T7), and detected in immunoblot using anti-HA antibody. MVA, cells inoculated with MVA-T7 but not transfected; neg. ctrl., L mutant containing a mutation in the catalytic site of the RdRp.(1.13 MB TIF)Click here for additional data file.Text S1Supplementary Methods.(0.08 MB DOC)Click here for additional data file.


In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible.[FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library.[FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy. In IVC, extremely small aqueous droplets suspended in a continuous oil phase isolate individual mutant DNA molecules, forming independent emulsion cell-free protein synthesis (eCFPS) reactors. Analogous to cells in an in vivo screen, the droplets co-localize the gene, the mutant protein it encodes, and the products of the desired enzymatic activity in vitro methods such as ribosome display in vivo platforms. However, IVC is unique among high-throughput in vitro methods in its ability to screen for multiple-turnover catalytic activity Depending on the target of the directed evolution project, IVC can be configured as a selection  or as a high-throughput screen in which fluorescence-activated cell sorting (FACS) is used to analyze and sort microbeads C. pasteurianum, a complex metalloenzyme containing multiple iron-sulfur electron transport clusters and an H-cluster catalytic prosthetic group composed of [4Fe-4S] and [2Fe-2S] clusters augmented by cyanide, carbon monoxide, and dithiolate ligands 12-resazurin, allowing detection by FACS. We demonstrate the system by enriching a mock library for beads bound to CpI DNA.Recently, our laboratory demonstrated production of active [FeFe] hydrogenases in the cell-free protein synthesis reactions at the heart of IVC E. coli BL21 DE3 containing the three [FeFe] hydrogenase maturases Shewanella oneidensis, and emulsified the mixtures by stirring, vortexing, or extruding the aqueous phase into various oil/surfactant mixtures. Methyl viologen assays Previously we demonstrated the ability to produce and activate [FeFe] hydrogenases in bulk CFPS reactions. However, many proteins have been found to be incompatible with CFPS expression within emulsion droplets Isolation of oxygen-tolerant mutants by FACS requires that a fluorescent signal develop in the presence of an active hydrogenase surviving oxygen exposure. We evaluated the ability of a number of redox-sensitive fluorophores to exchange electrons directly with hydrogenase. The only compatible fluorogenic molecule we identified was resazurin  by elect12-resazurin  tag antibodies were mixed into a CFPS reaction solution and emulsified into a continuous oil phase. Within the droplets, triply HA-tagged hydrogenase was synthesized, matured, and bound to the antibodies. The beads could then be broken out of the emulsion, washed, and re-emulsified for a separate fluorescence-generation reaction step. We delivered C12-resazurin to pre-formed emulsion droplets C12-resorufin nonspecifically adsorbs to the surfaces of the polystyrene beads, allowing beads recovered from fluorescence-generation emulsions to be directly sorted by FACS. Beads coated with C12-resorufin can be washed and stored for weeks with little decay of the fluorescent signal. Switching from the 1 µm diameter beads used in previous reports to 5.6 µm diameter beads increased the surface area available to bind C12-resorufin, and dramatically improved signal resolution.C12-resorufin fluorescence, we mixed beads coated with anti-HA antibodies and ∼1000 molecules of CpI DNA per bead with a 50∶1 excess of beads coated with antibodies but no DNA. The bead mixture was suspended in CFPS reaction solution, emulsified into oil, and incubated. Following protein synthesis the beads were recovered, resuspended in assay solution, re-emulsified, and incubated to allow antibody-bound hydrogenase to reduce C12-resazurin to C12-resorufin. Finally, the beads were recovered and analyzed for C12-resorufin fluorescence by FACS. In the fluorescence histogram of the single sample, two separate populations are easily distinguished . Both templates contained the same homoprimer Amplification of the DNA on the sorted low-fluorescence beads gave rise to a bright CAT gene band and a very light CpI gene band, while the high-fluorescence beads yielded a bright CpI gene band and a light CAT gene band . This enin vitro compartmentalization using C12-resazurin to link enzyme activity to the development of a florescent signal. However, further work will be required before screening of true mutant libraries can begin. The enrichment experiment described above used two populations of beads that were incubated with DNA separately and then mixed. A more realistic scenario is one in which the DNA is mixed before incubation with beads. Enrichment of hydrogenase DNA from mixtures with excesses of decoy DNA much larger than 20∶1 must also be demonstrated.The success of the enrichment experiment indicates that [FeFe] hydrogenase mutants can be screened in an extremely high-throughput fashion by Chlamydomonas reinhardtii, and confirmed its compatibility with both eCFPS and C12-resazurin. While this IVC screen was developed with the goal of discovering oxygen-tolerant hydrogenase mutants, it is actually a screen for hydrogenase activity, useful for screening for mutants with any of a number of industrially important characteristics, such as thermostability (by incubating beads at high temperatures between the eCFPS and C12-resazurin emulsion steps), tolerance for conditions such as might exist in a fuel cell, and so on. Hydrogenases with any of these phenotypes can be screened for with only minimal modifications to the platform described here. The throughput of IVC (thousands of beads per second on modern FACS machines) represents a dramatic improvement over traditional 96-well microtiter plate-based screens, and the methods described here provide a way forward towards the discovery of hydrogenase mutants with the potential to revolutionize the world's energy economy.We also tested the platform described here with a second [FeFe] hydrogenase, HydA1 from 5′-CCATTCATTAATGCACATAACTAT-3′) and the 3xHA tag: 5′-CCA TTC ATT AAT GCA CAT AAC TAT TCC AAC GAG TTC GCG GCC GCT TAG GCA CCC CAG GCT TTA C-3′, 5′-GGC ATA GTC TGG GAC GTC ATA TGG ATA TGC ATA GTC CGG AAC ATC GTA TGG GTA TTT TTT ATA TTT AAA GTG CAG GAT TTC-3′, 5′-CCA TTC ATT AAT GCA CAT AAC TAT TCC AAC GAG TTC GAC GAG CGT CAG CTT GCA TGC CCT GCA GCT-3′, 5′-TAT CCA TAT GAC GTC CCA GAC TAT GCC TAC CCG TAT GAC GTG CCA GAT TAC GCG TAA TAA TAA TTT TTT TAA GGC AGT TAT TGG TG-3′. The CAT linear template was created from the pK7-CAT plasmid in the same way. The linear templates were re-amplified with biotinylated TR homoprimer to yield constructs with biotin groups at both ends.The DNA template encoding C-terminally triply HA-tagged templates was created by overlap extension PCR using the pK7-CpI plasmid 7 molecules/µL.Following purification with PCR cleanup kits , DNA concentrations were measured with the Quant-It dsDNA broad-range assay on a Qubit fluorimeter  following the manufacturer's protocol. The solutions were then diluted in water or TE buffer  to 107 5.6 micron diameter streptavidin-coated beads  were washed with DNA binding buffer , resuspended in the same, and incubated with 0.3–1 molecule of biotinylated linear DNA template per bead for 16 hours at room temperature with rotation to prevent settling. The beads were then incubated in DNA binding buffer with 3 million biotinylated reverse primers per bead for 2 hours, and washed in PBS.2×10http://thebigone.stanford.edu/foundry/index.html). The chip was bonded to a glass substrate by PDMS-PDMS bonding. The device was pre-treated with Aquapel A PDMS microfluidic flow-focusing emulsification device with the design shown in 4/µL.Following incubation with DNA templates and biotinylated ePCR primers as described above, the beads were resuspended in a mixture of 1X Pfu buffer, 0.2 mM each dNTP, 0.1 µM TR homoprimer, 0.1% Tween 20, and 0.2 U/µL Pfu Turbo polymerase  at a bead concentration of 5×10The PCR mixture and an oil phase consisting of Fluorinert FC-40  with 2% w/w PFPE-PEG block copolymer surfactant shown to be compatible with both PDMS chips and eCFPS The emulsion was distributed in 50 µL aliquots into thin-walled PCR tubes and heated at 95°C for 1 minute, followed by 50 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 2 minutes. The thermocycled emulsions were collected into one tube and broken by addition of a volume of A104 emulsion destabilizer  equal to 10% of the volume of emulsified aqueous phase, followed by gentle stirring with a pipette tip and recovery of the upper aqueous phase. Beads were then incubated in PBS with 200,000 biotin-conjugated rabbit anti-HA antibodies per bead  for two hours at room temperature.2S, and then spun at 10,000 g until all precipitates were removed. The following two solutions were prepared : Solution A consisted of a small molecule substrate mixture E. coli cell extract. Gam protein and T7 RNA polymerase were expressed and purified in house.Inside an anaerobic glove box the cell-free extract harboring the three [FeFe] hydrogenase helper proteins HydE, F, and G was reconstituted 5 beads/µL. Solutions A and B were loaded into separate Hamilton gastight syringes. The FC-40/surfactant oil phase described above, having been stored in the glove box to remove oxygen, was loaded into a third, larger syringe.Solution A was added to washed, pelleted beads and vortexed to give a suspension of 10The microfluidic chip described above was used to generate monodisperse emulsions. Flow rates of 3 µL/min of each of the two aqueous phases and 50 µL/min of the oil phase produced monodisperse droplets approximately 30 µm in diameter. Emulsification was carried out outside the glove box on an inverted microscope. The emulsified mixture was collected into an airtight vial (containing 500 µL buffer to dilute and thus prevent protein synthesis from taking place in any cell-free mixture that avoids emulsification) and returned to the glove box immediately upon completion. The emulsified reactions were incubated at room temperature for 6–16 hours. Following this incubation the emulsion was broken with A104 emulsion destabilizer as before.When emulsions were formed by bulk methods, 60 µL of the combined CFPS mixture above (omitting Tween 20) was added to the pelleted beads, mixed, and immediately emulsified into 500 µL cold microbiology grade light mineral oil containing 1.8% v/v Span 80 and 0.2% v/v Triton X-100  surfactants. Emulsions were formed by stirring the mixture in the bottom of a 2 mL round-bottom cryovial with a 3 mm×8 mm magnetic stir bar at 600 rpm for 7 minutes, briefly vortexing at maximum speed after each minute. Alternatively, eCPFS emulsions were prepared by extrusion, with an oil phase of mineral oil with 1.8% v/v Span 80 and 0.2% v/v Triton X-100, or of decane with 1% w/v Span 60 and 1% w/v cholesterol . The beads were suspended in 50 µL of CFPS solution and extruded through a 19 mm track-etch polycarbonate membrane with 14 µm pores  into 200 µL of oil using a hand extruder . After a total of 15 passes through the membrane the emulsion was dispensed into a 1.5 mL tube. The emulsified CFPS reaction droplets were incubated at room temperature for 16 hours. After the incubation, the emulsion was centrifuged to settle the droplets to the bottom of the tube, and the clarified oil phase was removed. 400 µL of 10 mM Tris-HCl pH 8 with 0.5% v/v Tween 20  was added, and the emulsion was centrifuged at 10000 g for 3 minutes to break the beads out of the destabilized droplets, vortexed, and centrifuged for another three minutes. This resulted in a pellet of beads at the bottom of the tube, buffer in the middle, and a thick oil phase at the top. The pelleted beads were aspirated from the bottom of the tube with a pipette and washed in a new tube with 200 µL 50 mM Tris-HCl pH 9, then resuspended in the same buffer.For oxygen exposure experiments, a 1.5 mL tube completely filled with 10 mM Tris-HCl pH 8 equilibrated with air was moved into the anaerobic chamber. Suspended beads were added to the tube and incubated for 60–90 minutes before being pelleted by centrifugation and washed with anaerobic buffer.12-resazurin  in DMSO was added to the emulsion, which was vortexed for 30 seconds to deliver the C12-resazurin to the droplets. The emulsion was then incubated anaerobically in the dark at room temperature for 2–16 hours. Following the incubation, the emulsion was removed from the anaerobic chamber and centrifuged at 20,000 g for 1 minute, and the clarified excess oil phase was removed. 400 µL of 10 mM Tris-HCl pH 8 with 0.5% v/v Tween 20  was added to disrupt the emulsion, which was then centrifuged at 20,000 g for three minutes, vortexed, and centrifuged for another three minutes. The pelleted beads were aspirated from the bottom of the tube with a pipette and washed in a new tube with 200 µL 50 mM Tris-HCl pH 9 , then resuspended in the same buffer and analyzed by FACS.Oxygen-exposed beads were suspended in 30 µL of 50 mM Tris-HCl pH 9 and re-emulsified by extrusion as described above into 300 µL of mineral oil mixed with 3% v/v Abil EM90 surfactant , which has been reported to perform well in redox-sensitive emulsion applications Beads were analyzed and sorted using various flow cytometers at the Stanford Shared FACS Facility. A dye laser tuned to 570 nm was used for excitation, and emission was collected through a 605/40 nm filter. Beads were analyzed at rates of 500–1000 events per second.Unsorted and sorted beads were amplified with Phusion DNA polymerase  using a single-molecule PCR protocol described previously Note on proprietary reagents: it is the policy of Raindance Technologies to provide commercially unavailable reagents to academic researchers at no cost.


Because the composition of faeces modulates colorectal carcinogenesis, promotional effects of the secondary bile salt sodium deoxycholate (SDC) were compared with those of dilute homogenised faeces (12.5% w/v) or saline alone in rat colon isolated from the faecal stream as a Thiry-Vella fistula (TVF). Each fluid was used to irrigate a group of TVFs 3 times per week for 12 weeks. Other rats had TVF without irrigation or colonic transection and reanastomosis (sham TVF). Operations followed a 6-week course of azoxymethane injections. At sacrifice 15 weeks postoperatively crypt depth and tumour yield were reduced to the same extent in both the non-irrigated TVFs and the SDC-irrigated TVFs, when compared to shams. Irrigation with faeces and saline completely restored crypt depth and partly restored tumour yields to the levels in shams. Tumours were smaller in the SDC group than in the other 4 groups. While tumours developed mainly in the left colon of shams, there was significantly more even distribution in the TVFs. Exclusion of the colon from the faecal stream leads to mucosal hypoplasia and impaired carcinogenesis. Irrigation with faeces or saline partly reverses these changes. Deoxycholate has no such effect and clearly is not co-carcinogenic in this model.


A species barrier may protect humans from this disease. Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy, or prion disease, that affects deer, elk, and moose. Human susceptibility to CWD remains unproven despite likely exposure to CWD-infected cervids. We used 2 nonhuman primate species, cynomolgus macaques and squirrel monkeys, as human models for CWD susceptibility. CWD was inoculated into these 2 species by intracerebral and oral routes. After intracerebral inoculation of squirrel monkeys, 7 of 8 CWD isolates induced a clinical wasting syndrome within 33–53 months. The monkeys’ brains showed spongiform encephalopathy and protease-resistant prion protein (PrPres) diagnostic of prion disease. After oral exposure, 2 squirrel monkeys had PrPres in brain, spleen, and lymph nodes at 69 months postinfection. In contrast, cynomolgus macaques have not shown evidence of clinical disease as of 70 months postinfection. Thus, these 2 species differed in susceptibility to CWD. Because humans are evolutionarily closer to macaques than to squirrel monkeys, they may also be resistant to CWD. Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurodegenerative diseases that affect many mammalian species. Some examples include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, Creutzfeldt-Jakob disease (CJD) in humans, and chronic wasting disease (CWD) in cervids. CWD was first found in captive deer in Colorado in 1967 , by direct infection into new species, or by in vitro conversion assays. The most sensitive method for testing susceptibility to TSE agents is intracerebral injection. Unfortunately, this route does not mimic most natural situations and only enables assessment of whether the possibility of transmission exists. Hamir et al. infected cattle and sheep with CWD by the intracerebral route and found protease-resistant PrP (PrPres) in 5 of 13 cattle and 2 of 8 sheep, which indicated that these ruminant species can propagate CWD (Saimiri sciureus) infected by the intracerebral route with brain homogenate from a single CWD-affected mule deer became clinically sick at 31 and 34 months postinfection, and both were positive for PrPres  and squirrel monkeys, each of which has previously shown susceptibility to various human prion diseases . Experimentation followed protocols approved by the National Institutes of Health Rocky Mountain Laboratories Animal Care and Use Committee.,CWD-positive brain homogenates were provided by E.S.W. and M.W.M. Contents of each pool were as follows: MD-1, 6 free-ranging mule deer from Wyoming  were generated by using a transgene, cosSHa.HumPrP, which was created by ligating the human PrP open reading frame into the cosSHa.Tet vector (Tissues were prepared by making a 20% (wt/vol) homogenate in 0.01 M Tris buffer, pH 7.4. Samples to be analyzed for protease-sensitive PrP (PrPsen) contained the following protease inhibitors: 10 μmol/L leupeptin, 1 μmol/L pepstatin A, and 1 μg/mL aprotinin. Samples were sonicated for 1 min and centrifuged at 5,000 rpm for 10 min. Supernatants were mixed 1:1 in 2× sample buffer and boiled for 3 min before electrophoresis.Preparation of samples for PrPres analysis has been described , and PrP bands were detected with antibodies 3F4 (residues 109–112) . Anti-PrP antibodies D13 and 3F4 were used for PrPres immunostaining as described . Two primers from the extreme outer ends of the open reading frame, including the previously published forward primer HM-1 (50) titers ranging from 6.3 × 107 to 5.0 × 108 ID50/g of brain homogenate (When the 8 pools of CWD (representing both wild and captive deer and elk) used as inocula were analyzed by immunoblot, PrPres in the 8 pools showed similar electrophoretic mobilities and glycoform patterns , panel Amogenate , panel Cmogenate , panel CTo test susceptibility to CWD, we inoculated squirrel monkeys with each of the 8 CWD pools described above. Of 13 squirrel monkeys, 11 became symptomatic (33–53 mo postinfection [mpi]) . The mosNo clear correlation between incubation period and amount of agent inoculated was noted . For exaIn all monkeys with clinical signs, CWD was confirmed by Western blot detection of PrPres in brain , panel BTissues from squirrel monkeys euthanized after intracerebral injection with CWD  were alsTo test a more natural route of infection, we exposed squirrel monkeys orally to CWD. Of the 15 exposed squirrel monkeys, 1 (no. 345) was found dead in its cage at 69 mpi; it had shown no neurologic signs or weakness. Western blot results indicated PrPres in brain, spleen, and lymph nodes , panel DTwo other orally infected monkeys were euthanized during the first 69 mpi . Monkey We inoculated cynomolgus macaques both orally and intracerebrally with 3 CWD inocula representing elk, mule deer, and white-tailed deer . Of the Amino acid substitutions in PrP can alter susceptibility to TSE agents, including CWD  was causTo determine whether passage of CWD in squirrel monkeys altered the tropism of the infectious agent, we inoculated tgDeerPrP mice and tg mice expressing human PrP (lines 66 and RM) intracerebrally with tissue homogenates from 3 CWD-positive squirrel monkeys  with PrPres and from an intracerebrally inoculated cynomolgus macaque (no. 609). Clinical disease did not develop in any tgDeerPrP, tg66, or tgRM mice during 600–700 days . The lacAs new CWD foci continue to emerge among cervid populations, the risk for CWD transmission to humans needs to be assessed. We used 2 monkey species and 2 routes of inoculation to test the susceptibility of primates to 8 different pools of CWD. To date, we have verified CWD in 11 of 13 intracerebrally inoculated squirrel monkeys; average incubation period was 41 months (range 33–53 months). Using a single CWD pool, Marsh et al. noted infection in 2 of 2 squirrel monkeys 31–34 months after intracerebral inoculation (The signs of wasting syndrome in CWD-infected monkeys were similar to those of CWD infection in cervids, in which loss of body condition is nearly always a major component of infection and neurologic deficits vary (We did not observe a strong correlation between infectivity titer inoculated and attack incidence or incubation period . One potFor humans, eating infected or contaminated tissue is a likely route of CWD exposure. In other animal models, oral transmission of TSE is generally 1,000-fold less effective than direct intracerebral challenge and results in longer incubation periods and lower efficiency of disease transmission. In our oral transmission experiments, we found evidence of CWD infection in 3 monkeys; 2 at 69 mpi had abundant PrPres in brain and lower levels in spleen and lymph nodes, and 1 euthanized at 39 mpi had PrPres in lymphatic tissues only. Thus, transmission seems to be slower by the oral route than by the intracerebral route, and other orally infected monkeys may be affected in the future.Cynomolgus macaques are evolutionarily closer to humans than are squirrel monkeys (The PrP gene sequence can influence cross-species transmission of prion disease. Therefore, we compared squirrel monkey and cynomolgus macaque PrP gene sequences to look for differences that might account for different susceptibilities of these monkeys to CWD. In the PrP gene excluding the signal peptide, deer differed from squirrel monkeys at 17 residues and from cynomolgus macaques at 16 residues, but 14 of these differing residues were identical in squirrel monkeys and macaques . TherefoHuman exposure to CWD-infected cervids in past decades is likely. The highest levels of prion infectivity are present in the central nervous system and lymphatic tissues of CWD-infected cervids; contamination of knives, saws, and muscles with these tissues can easy occur when processing game. Despite the likelihood of exposures, epidemiologic studies of humans living in CWD-endemic areas of Colorado and Wyoming during 1979–2001 have not shown any increases in human TSE cases (Susceptibilities of Nonhuman Primates to Chronic Wasting Disease


To determine circulation of West Nile virus (WNV) during nonepidemic times, we serosurveyed wild birds of Morocco in 2008. We found antibodies against WNV in 12 (3.5%) birds, against Usutu virus in 1 (0.3%), and against both in 2 (0.6%). High WNV prevalence among juvenile birds suggests local virus circulation among resident birds. This finding has led to the hypothesis that the virus is absent from Europe and North Africa and periodically seeded into different places by infected migratory birds. An alternative hypothesis is that the virus can remain silent, circulating in a sylvatic enzootic bird–mosquito cycle and only under appropriate conditions causing new outbreaks in humans and horses  causes sporadic disease outbreaks, which usually affect a low number of humans and animals, after which long periods without virus circulation occur. This pattern has occurred in France , Italy (1998 and 2008), Algeria (1994), Tunisia (1997 and 2003), Morocco (1996 and 2003), Romania (1996–2000), and Israel (1998–2000) , ≈40 km northeast of Kenitra. The area is dominated by rice fields flooded from regulated channels from the Sebou River. Each captured bird was marked with a numbered metal ring; when possible, age was determined according to plumage characteristics. Blood was taken from the jugular vein and allowed to clot at ambient temperature. The blood was then centrifuged , and the serum was stored in liquid nitrogen and transported to a deep freezer (–80°C) in the laboratory.4 cells/mL) of Vero cells plus fetal calf serum was added to the same medium to reach a final concentration of 5%. The mixture was further incubated for 6–7 days at 37°C until cytopathic effects were observed in control wells containing ten 50% tissue culture infective doses of virus. Samples were titrated by analyzing serial serum dilutions from 1:10 to 1:640. Only samples that showed neutralization (absence of cytopathic effect) at dilutions 1:>20 were considered positive. Samples that showed neutralization at the same dilutions were scored as positive for flavivirus but not conclusive for WNV and USUV. Controls for cytotoxicity in the absence of virus were included for every sample at a dilution of 1:10. Cytotoxicity prevented determination of neutralizing antibodies in 1 sample, which was therefore excluded from the analysis. A bird was considered to have seroconverted if it was seronegative at the time of first capture and seropositive (titer increase by at least 4-fold) at the time of recapture  and Usutu virus (USUV) (SAAR1776) were determined by using a micro virus-neutralization test as described previously (Turdus merula); neutralizing antibodies against WNV were found in 6 (19.3%) blackbirds, against flavivirus in 2 (6.5%), and against USUV in 1 (3.5%). Prevalence of WNV neutralizing antibodies among house sparrows (Passer domesticus) was much lower (2.2%). Additionally, 1 Cetti’s warbler (Cettia cetti) was negative for WNV neutralizing antibodies in June but had seroconverted by the time of recapture in July. Of the 13 additional birds sampled twice, 10 were negative for antibodies in both samples and 3 were positive in both samples. Prevalence of antibodies was significantly higher among adult than among juvenile (<1 year of age) blackbirds  but not among house sparrows .We analyzed 360 samples from 346 birds . NeutralAntibodies in juvenile birds provide evidence of circulation of WNV and USUV in 2008 in the study area . Because antibodies are maternally transmitted for only 2–4 weeks after birth  accumulates, the need to understand the ecologic factors related to virus circulation and the conditions leading to disease outbreaks increases (


Laboratory strains of the mouse polyoma virus differ markedly in their abilities to replicate and induce tumors in newborn mice. Major determinants of pathogenicity lie in the sialic binding pocket of the major capsid protein Vp1 and dictate receptor-binding properties of the virus. Substitutions at two sites in Vp1 define three prototype strains, which vary greatly in pathogenicity. These strains replicate in a limited fashion and induce few or no tumors, cause a disseminated infection leading to the development of multiple solid tumors, or replicate and spread acutely causing early death. This investigation was undertaken to determine the Vp1 type(s) of new virus isolates from naturally infected mice. Compared with laboratory strains, truly wild-type viruses are constrained with respect to their selectivity and avidity of binding to cell receptors. Fifteen of 15 new isolates carried the Vp1 type identical to that of highly tumorigenic laboratory strains. Upon injection into newborn laboratory mice, the new isolates induced a broad spectrum of tumors, including ones of epithelial as well as mesenchymal origin. Though invariant in their Vp1 coding sequences, these isolates showed considerable variation in their regulatory sequences. The common Vp1 type has two essential features: 1) failure to recognize “pseudoreceptors” with branched chain sialic acids binding to which would attenuate virus spread, and 2) maintenance of a hydrophobic contact with true receptors bearing a single sialic acid, which retards virus spread and avoids acute and potentially lethal infection of the host. Conservation of these receptor-binding properties under natural selection preserves the oncogenic potential of the virus. These findings emphasize the importance of immune protection of neonates under conditions of natural transmission. Strains of the mouse polyoma virus adapted to growth in cell culture vary greatly in their abilities to cause disease. Pathogenicities of these laboratory strains range from “attenuated” to “highly virulent” when tested in animals. The biological differences are based in large part on variations in the outer capsid protein, which dictate the manner in which the virus recognizes and binds to cell receptors. In contrast, strains of virus newly isolated from wild mice are uniform in their receptor-binding properties. Naturally occurring strains avoid binding to pseudoreceptors, which would severely limit their ability to spread. At the same time, their avidity of binding to true receptors is sufficiently strong to avoid rapid dissociation and potentially lethal spread. They are therefore neither attenuated nor virulent. The new isolates do, however, retain the ability to induce a broad spectrum of tumors in the laboratory. These findings emphasize the importance of neonatal and maternal immune responses in allowing a potentially highly oncogenic virus to disseminate without causing disease. Interest in the murine polyoma virus (Py) grew out of its discovery as a tumor-inducing agent in its natural host under experimental conditions . The ori, may differ greatly in their abilities to replicate and induce tumors in the intact host  in which the GalNAc in the third position may be linked via an α2,6 linkage to another sialic acid, forming a branched di-sialic acid chain [NeuNAc--Gal---GalNAc]. Small plaque strains such as RA bind to both the branched and straight chain oligosaccharides, while large plaque strains bind only to the straight chain ,25. The The valine to alanine substitution at position 296 introduces a subtle alteration in a hydrophobic surface forming part of the sialic acid binding groove. Valine at this position (PTA) makes a van der Waals contact with the sialic acid ring, while the shorter chain alanine (LID) does not. The alanine substitution thus weakens the hydrophobic interaction with the receptor ). Thus, To determine the Vp1 type(s) of Py under natural conditions, feral mice were trapped and screened serologically for ones that carried the virus. A total of 71 mice were trapped alive from 12 locations in and around Boston and as far away as Cuttyhunk Island, MA, and New York City. Mice were humanely killed, sera collected, and kidneys frozen for attempts at virus isolation. Screening for Py was carried out on the serum from each mouse using a hemagglutination inhibition (HA-I) assay. Results are shown in 6–107 pfu/ml. These titers were lower by 1–2 orders of magnitude compared with those of laboratory-adapted strains. Several isolates were tested and shown to be neutralized by anti-Vp1 antibody or by sera from laboratory mice infected by Py (unpublished data). The feral mouse virus isolates were further amplified and analyzed by DNA sequencing of selected regions of the viral genome. Six isolates from mice trapped at three locations were tested for their abilities to induce tumors in newborn mice (see further below).Py was isolated and amplified from kidney homogenates of HA-I–positive mice. Primary kidney epithelial cells prepared from virus-free baby mice were used for amplification. Cytopathic effects typical of Py developed over a period of 7–12 d in every inoculated culture. First-round lysates gave plaque titers in the range of 10One virus isolate from each of the eight locations was chosen initially for sequence analysis . To deteRegulatory sequences flanking the replication origins of the prototype wild-type strains are variable compared with their highly conserved coding regions . It was Insertions, deletions, and single base changes distinguish the different isolates. The S and Z2 isolates were identical to each other but different from all three prototype strains. Differences in the new isolates were found dispersed throughout the non-coding regions in both the “A” and “B” enhancers and on the early side of the origin. PTA carries two copies of a 40-bp sequence around the Bgl1 site, while the related LID strain carries a single copy . Two of 5 pfu/animal or higher kills C3H/BiDa within roughly two weeks. At much lower doses, animals survive and develop a tumor profile similar to that induced by PTA.PTA, RA, and LID have been compared for their abilities to induce tumors following inoculation into newborn mice of the highly susceptible C3H/BiDa strain ,9. PTA iThe finding of a uniform Vp1 type identical to PTA in the feral mouse isolates makes several predictions about the biological behavior of the new strains. First, they should induce tumors at high frequency and these should include epithelial as well as mesenchymal types. Second, because the isolates lack the virulence determinant of LID (296A), they should not cause early deaths (≤ 3 wk). To test these predictions, two isolates from each of three locations were inoculated into newborn C3H/BiDa mice. The results clearly support the predictions based on the importance of Vp1 type. All six isolates induced epithelial as well as mesenchymal tumors . There wPy establishes a silent persistent infection in natural populations of mice, yet behaves in the laboratory as a powerful oncogenic agent (reviewed in ). This dGlutamic acid at position 91 and valine at position 296 define critical features of this common Vp1. Glutamic acid–91 is critical for discriminating between true receptors carrying a terminal unbranched sialic acid and pseudoreceptors with branched chain sialic acids ,11,12. AValine at position 296 establishes a hydrophobic contact with the receptor, decreasing the rate of dissociation and spread of the virus. Substitution of alanine at this position results in loss of the hydrophobic contact and a decreased overall avidity of binding . No variVpu and nef proteins of HIV and simian immunodeficiency virus which act directly or indirectly to destroy cell receptors. For Py and perhaps other viruses that lack the means to destroy their own receptors, it appears to be critical that they regulate their receptor-binding properties within narrow limits. Affinity of binding must be high enough to promote efficient cell attachment and entry, yet not so high as to inhibit dissociation from cell debris and prevent virus spread. At the same time, receptor affinity must not be so low as to allow rapid dissociation and spread, which could endanger the life of the host. The properties of Py strains RA and LID, lying outside these limits, serve to illustrate the constraints of natural selection.Some viruses promote their release and spread through mechanisms that effectively destroy host cell receptors. Prominent examples are the neuraminidase of influenza viruses and the While Vp1-coding sequences were entirely conserved, regulatory sequences showed considerable variability among the new virus isolates. This variability was evident comparing isolates from different locations. In a limited sampling of multiple virus isolates from the same location, regulatory sequences were conserved, indicating relative stability of virus circulating at a given time within the same host breeding population. This finding is consistent with the epidemiology and natural history of infection in wild mice  from 12 locations in and around Boston and as far away as Cuttyhunk Island, MA, and New York City were trapped and recovered alive using Sherman Traps and mouse chow coated with peanut butter as bait. Mice were humanely killed, sera collected for serological testing, and kidneys frozen at −80 °C. One kidney from each animal was sent to the Laboratory of Comparative Medicine at Yale University School of Medicine for testing of common mouse pathogens before attempting virus isolation in our laboratory. Results on all mice were negative for all agents tested, including: hantavirus; mouse parvovirus; lymphocytic choriomeningitis virus; Theiler's mouse encephalomyelitis virus; Sendai virus; minute virus of mice; mouse hepatitis virus; ectromeila; reovirus; and mycoplasma.Feral mice (7/ml). Further incubation was at 4 °C for a minimum of 6 h. HA-I titer was scored as the reciprocal of the highest dilution of serum that prevented agglutination.Serological testing for Py was performed in our laboratory using a standard HA-I assay in V-shaped 96-well microtiter plates. Serial 2-fold dilutions of mouse sera were incubated with an equal volume of 2–4 HA units of wild-type polyoma (PTA strain) for 30 min at 37 °C, followed by addition of a suspension of guinea pig erythrocytes  and 5′ AGGTGCTGGACCTTGTGACAGGG3′ (anti-sense)] and then sequenced using primers 5′ AACAGTGAGCCAGAGCCCACCACC3′ for the 91 region and 5′ GAGGGAGGCCATGGGATAGGG3′ for the 296 region. Additional sequencing was carried out to confirm and extend coverage of the entire Vp1. The origin regions were amplified with primers 5′ GTACCGCTGTATTCCTAG3′ and 5′ CATTCTCAGATTGTATACTTCAG3′ giving a fragment of 1061 bp encompassing all of the non-coding sequences between the ATG of Vp2 on the late side and the ATG for the T antigens on the early side of the origin. Sequencing of this amplicon was carried out with primers 5′ CTCATTACACCCTCCAAAGTC3′ and 5′ CATTCTCAGATTGTATACTTCAG3′. Amplicons were sequenced using cycle sequencing with BigDye v3.1 dideoxy fluorescent terminators and a 3730xL DNA Analyzer from Applied Biosystems.5 pfu/animal. Six to 12 animals were used for each virus. Animals were followed for tumor development grossly and were necropsied when moribund. Tumors were confirmed by histological examination and grouped as epithelial or mesenchymal. The former includes tumors arising from mammary glands, thymus, skin, and salivary glands and the latter includes fibrosarcoma, osteosarcoma, and renal sarcoma. Results with the six new isolates are compared with previously published results for PTA and RA http://ww


This report describes a new hamster model for West Nile (WN) virus encephalitis. Following intraperitoneal inoculation of a New York isolate of WN virus, hamsters had moderate viremia of 5 to 6 days in duration, followed by the development of humoral antibodies. Encephalitic symptoms began 6 days after infection; about half the animals died between the seventh and 14th days. The appearance of viral antigen in the brain and neuronal degeneration also began on the sixth day. WN virus was cultured from the brains of convalescent hamsters up to 53 days after initial infection, suggesting that persistent virus infection occurs. Hamsters offer an inexpensive model for studying the pathogenesis and treatment of WN virus encephalitis.


Isolation of Novel Adenovirus from Fruit Bat To the Editor: Bats are thought to be one of the most important reservoirs for viruses such as Nipah virus, severe acute respiratory syndrome (SARS) coronavirus, and Ebola virus  appeared without any visible microbe, indicating that the cell culture contained a virus. The virus, tentatively named Ryukyu virus 1 (RV1), caused apparent CPE on primary kidney cells derived from a Ryukyu flying fox and on our established bat kidney T1 (BKT1) cells, which were derived from the kidney of a horseshoe bat (Rhinolophus ferrumequinum) and transformed with expression plasmid DNA encoding the large T antigen of replication origin-defective simian virus 40.–Adenoviridae.To identify the virus, RV1, we applied the rapid determination of viral RNA (RDV) system version 1.0  to viruses in other genuses, Siadenovirus, Aviadenovirus, and Atadenovirus. In addition, a phylogenic tree based on amino acid sequences indicated that RV1 belongs to family Adenoviridae, genus Mastadenovirus  of a nested PCR method, targeting the viral DNA polymerase gene with highly degenerate consensus primers that have been described recently (enovirus .Electron microscopy of RV1-infected BKT1 cells indicated that RV1 accumulated in the nucleus and that the size of capsids was 60–70 nm (data not shown). Restriction endonuclease analysis of the RV1 genome indicated that the genome was ≈20–30 kbp (data not shown). These features are consistent with RV1 being an adenovirus.Until now, a number of RNA viruses have been isolated from bats, but isolation of DNA virus is rare (In conclusion, we isolated a novel virus from a fruit bat. This virus was isolated from a healthy bat, which suggests that the virus may persistently infect fruit bats. Although its pathogenicity for humans is still unknown, knowledge of RV1 will be useful in epidemiologic studies of infectious diseases emerging from bats because persistently infecting viruses might be isolated together with primary pathogens. We are planning to establish cell lines from bats and isolate more viruses from persistently infected bats.


From July to September 1999, a widespread outbreak of meningoencephalitis associated with West Nile virus  occurred in southern Russia, with hundreds of cases and dozens of deaths. Two strains of West Nile virus isolated from patient serum and brain-tissue samples reacted in hemagglutination-inhibition and neutralization tests with patients' convalescent-phase sera and immune ascites fluid from other strains of West Nile virus.


Aedes aegypti mosquitoes, has now repeatedly been associated with a new vector, Ae. Albopictus. Analysis of full-length viral sequences reveals three independent events of virus exposure to Ae. Albopictus, each followed by the acquisition of a single adaptive mutation providing selective advantage for transmission by this mosquito. This disconcerting and current unique example of "evolutionary convergence" occurring in nature illustrates rapid pathogen adaptation to ecological perturbation, driven directly as a consequence of human activities.Since 2004, several million indigenous cases of Chikungunya virus disease occurred in Africa, the Indian Ocean, India, Asia and, recently, Europe. The virus, usually transmitted by  Mosquito-transmitted Chikungunya virus (CHIKV) is responsible for explosive outbreaks of febrile arthralgia in humans ,2. SeverAe. aegypti. However, CHIKV has been recently associated with an alternative vector, Ae. Albopictus (the "Asian Tiger Mosquito"), which has spread in areas previously occupied predominantly by Ae. aegypti, and dispersed globally via commercial transportation of, for example scrap car tyres [Until recently, during human outbreaks, the principal identified vector of CHIKV was ar tyres .Ae. aegypti. Previous studies showed that Ae. Aegypti-associated CHIKV isolates from Comoros and Seychelles, as well as early isolates from other islands in this region, had an Alanine residue at position 226 of the E1 gene [Ae. aegypti is absent or scarce and Ae. albopictus predominates. Within one year, a new mutation  was identified in some CHIKV samples [Ae. aegypti and Ae. albopictus are common. The A226V mutation was identified in all sequenced 2006 Mayotte isolates [Ae. Albopictus and, indeed, we have recently shown that it improves virus replication and transmission efficiency in this mosquito [Ae. albopictus is present, the A226V adaptive mutation was observed 1 or 2 years after the introduction of CHIKV. Whether this mutation was acquired several times independently or if an "Ae. albopictus-adapted" strain evolved in one island and then dispersed to neighbouring islands is unknown.During the 2004 epidemic in Kenya and the subsequent outbreaks, when CHIKV was introduced into Comoros and Seychelles, CHIKV was transmitted by  E1 gene . However samples . The virisolates  and in aEU244823) has the A226V mutation, acquired in Italy  or, more probably, in India . Since 2006 Indian isolates originate from an ancestor with an Alanine at position 226 , but, in contrast to original Central-African strains (transmitted by Ae. aegypti) both the Cameroon  and Gabon (this study) isolates have the A226V mutation. This implies an independent adaptive mutation in response to a similar requirement of transmission by Ae. albopictus.The situation is different in India. Our phylogenetic analyses suggest that CHIKV originating from East-Africa or Comoros was introduced into India in 2006 fig-&2. In 20 226 fig-, the A22Ae. albopictus from Asia to Europe and the Americas is largely the result of human commercial activities, the adaptation of CHIKV to Ae. albopictus provides a fascinating demonstration of how viruses can readily circumvent the impact of human interference on the ecosystem. Our observations also have very serious implications for future emerging arboviruses that infect humans. Aedes albopictus, which has dispersed into central Africa is also becoming widespread in Europe and North-America. Thus, CHIKV, and possibly other tropical arboviruses, have the potential to invade more northerly geographic regions.It is extremely rare for this phenomenon, known as "evolutionary convergence", to be observed in nature. In virology, convergent mutations have been reported under the extreme selective pressure of antiviral therapy during the treatment of acute (eg neuraminidase mutations of influenza virus) or chronic (eg reverse-transcriptase/protease mutations of HIV) viral diseases. Our results demonstrate that the selective pressure exerted on CHIKV through the constraint of having to replicate in a new vector, is similar to that cited for antiviral therapy. Since the dispersal of CHIKV: Chikungunya virus.The authors declare that they have no competing interests.XdL led and coordinated the project and the manuscript redaction, realised phylogenetic analysis. EL isolated and characterised Gabon strains , was involved in data analysis and manuscript redaction. RNC isolated and characterised Madagascar strains , was involved in data analysis and manuscript redaction. KT characterised Reunion strains, made substantial contribution to analysis, have been involved in manuscript redaction. SH made substantial contribution to analysis and interpretation of data, was involved in manuscript redaction. EAG substantially contributed to phylogenetic analysis and interpretation of data, was involved in manuscript redaction. All authors approved final version of the manuscript.


Enzyme entrapped polyacrylamide and agar/agarose gels were fragile and could not withstand repeated use whereas enzyme entrapped in large calcium alginate beads was used successfully for 50 cycles for the conversion of starch into product without much damage to the beads under stirring conditions. Amylase preparation was also mixed with urease, lysozyme and coimmobilized in large sized calcium alginate beads. These beads were used for 10 repeated cycles to check the conversion of substrates into their products by their respective enzymes and we concluded that an enzyme or mixture of two or three enzymes can be immobilized in the same large sized calcium alginate beads. This will save the additional cost of bioreactor, manpower, maintenance conditions required for the conversion of one drug into another using enzyme/s entrapped in large sized beads.To check the suitability of enzyme entrapped beads for use in pharmaceutical industry, amylase enzyme was entrapped in agar/agarose, polyacrylamide gels and calcium alginate beads. Sodium alginate of 1% concentration was found to be best with respect to immobilization efficiency and calcium alginate beads so obtained were not much susceptible to breakage. When sodium alginate- amylase mixture was added from a height of about 20-30 cm. into CaCl Knowledge of immobilization of enzymes has led to exploit more fully the potential of enzymes for industrial purposes. The cost of producing the enzymes is often high so immobilized enzyme preparations may be more effective, since they are recoverable and can be used repeatedly in bioreactor or for other purposes, remain separated during the reaction by physical entrapment or other methods and more stable than free enzymes. Many instances of successful application of immobilized cells/ enzymes from bacterial, fungal, higher plant, mammalian cells and subcellular organelles in/onto different matrices by different immobilization methods have been reported–11.Various methods used for immobilization of enzymes are adsorption, covalent binding, entrapment and membrane confinement. The cost of immobilization by covalent binding and membrane confinement is high as compared to entrapment methods. Various types of gels are used for entrapment of enzymes. The enzyme may be entrapped within polymeric mesh such as agar, polyacrylamide gel or calcium alginate by carrying out the polymerization reaction and /or cross – linking reaction in the presence of enzyme–14.Polyacrylamide, the most commonly used matrix for the entrapment of enzymes, has the property of being non- ionic. A number of procedures for the entrapment of enzymes in polyacrylamide gels have been published16. TrevaGelidium, Gracilaria, Gigartino and pterocladia. It liquifies on heating to 96° and hardens into a jelly on cooling to 40-45°. Agar/ agarose have been used for the entrapment of cells in the form of spherical beads, blocks and membranes. Matsunaga et al, have described entrapment of Clostridium butyricum in agar, gelation occurs. Since gel formation can take place under mild conditions, entrapment in this matrix is very suitable for immobilization of enzymes, drugs, cells, proteins[Alginate, the major structural polysaccharide of marine brown algae, contains β-D-mannopyranosyl uronate and α-L-gulopyranosyl uronate in regular (1-4)-linked sequences. Both homopolymeric sequences are found together, although to different extents, in all alginate molecules. In the presence of monovalent cations, the polysaccharide forms a viscous solution even at high concentrations, whereas in the presence of divalent cations, especially Ca proteins–25. Incr proteins.Immobilization of enzyme by entrapment method can be done by very simple procedure and moreover, the cost of immobilization is low as compared to other methods. Our main aim of investigation was to find out the gel matrix suitable for immobilization of enzymes using various entrapment methods and to check the viability of enzyme entrapped beads for use in pharmaceutical industry.2 (fused) was purchased from Ranbaxy Laboratories Ltd. India. Tetramethylethylenediamine (TEMED) was from Sigma, St. Louis, USA. All other chemicals used for the assay of enzymes were of analytical grade. Standard lysozyme and Micrococcus luteus were from Banglore Genei, Banglore, India. Amylase enzyme was used for all the entrapment methods. Same batch of amylase was used for comparison of entrapment in different gels.Agar, agarose, bisacrylamide were purchased from Himedia laboratories Ltd. Mumbai, India. Sodium alginate and acrylamide were from Loba Chemie, Mumbai, India. CaCl2  were used for entrapment of enzyme and entrapped enzyme was checked for the conversion of substrate into product up to 50 repeated cycles. A mixture of enzymes  was entrapped in the same calcium alginate beads and checked for the ability of these enzymes to convert their respective substrates into their products.Amylase enzyme was immobilized by gel entrapment method in calcium alginate, polyacrylamide, agar/agarose gels. Agar/agarose , polyacrylamide , sodium alginate  and CaClThe solution of agar/agarose of concentrations 0.5, 1, 2, 3 and 4% were heated separately to liquify the agar. Gel was allowed to cool to 45-50° and then equal amount of amylase in phosphate buffer (25 ml) was added to it so that the final concentration of agar was 0.25, 0.5, 1, 1.5 and 2%, respectively. The agar-amylase mixture was then allowed to solidify onto the glass plate and cut into small blocks and used for the conversion of starch into maltose for 50 repeated cycles.Polyacrylamide gel is the most commonly used material for entrapment. Method given by Freeman and Aharonowitz for the entrapment of cells by polyacrylamide gel was modified. For the2 concentrations  were used to optimize the best concentration. Different concentrations of sodium alginate  mixed with enzyme solution were added separately from a height of nearly 1-2 cm and 20-30 cm, into excess of CaCl2 solution. For 50 ml sodium alginate- enzyme mixture, 500 ml of CaCl2 solution was used.For calcium alginate beads, 25 ml of 0.5, 1, 2, 3 and 4% solutions of sodium alginate were prepared and mixed with equal volume of amylase solution to get the final concentration of sodium alginate 0.25, 0.5, 1, 1.5 and 2%, respectively. Entrapment of enzyme in calcium alginate gel was done by modifying the method of Kierstan and Bucke. DiffereAll these gels were kept at 4° after proper washings till further use. For the estimation of enzyme activity, the gels were dried in the air. The beads prepared by entrapment of enzyme amylase in polyacrylamide gel, agar/agarose and calcium alginate were checked for the conversion of starch into maltose for 50 repeated cycles. Overhead stirrer was finally used for mixing the reaction mixture instead of magnetic stirrer.A mixture of enzymes  was entrapped in calcium alginate beads and checked the ability of these enzymes to convert their respective substrates into their products. Urease was taken from jackbean meal and lysozyme from egg white. Amylase, urease and lysozyme enzymes were filtered separately, concentrated by ultrafiltration and then phosphate buffer of pH 7.0 was added to this preparation to give final concentration of 0.1 M.Micrococcus luteus was used to check the lysis by lysozyme. The activity was calculated using the standard lysozyme with known activity. Absorbance of Micrococcus luteus solution was taken at 450 nm before and after the addition of lysozyme to check the lysis[Amylase activity was measured by digesting 5 ml of 1% starch solution to achromic point. The time required for the conversion of starch into maltose was noted by change in colour of iodine solution from blue to colourless. Time ofthe lysis. Urease the lysis.2 was also checked with respect to immobilization efficiency and 0.5 M concentration of CaCl2 was found to be optimum. Different concentrations of sodium alginate showed difference in the diffusion of substrate into the beads and with increase in the concentration of sodium alginate, diffusion ability of the substrate into the beads decreased. This is due to more cross-linking at higher concentrations with more calcium binding sites that is why beads at high concentration appear bright white in colour[In case of agar/agarose, polyacrylamide and sodium alginate, the different concentrations were tried to choose the best concentration of gel with respect to immobilization efficiency as well as working efficiency of the beads with respect to diffusion of substrate into the beads Tables  and 2. Ain colour. At 0.5%in colour–25.2 solution, all beads  were spherical in shape , they were light white and transparent in colour. The beads at all concentrations were small and nearly of same size. When the sodium alginate-amylase preparation was added from the height of 20-30 cm into the CaCl2 solution, size of beads was larger at higher concentration  were resistant to breakage but the main problem was of diffusion of substrate . Sodium In order to check the immobilization efficiency of different gels, amylase enzyme entrapped gels were washed three times with 0.1 M phosphate buffer, pH 7.0 and immobilization efficiency was calculated on the basis of activity of enzyme in the supernatant. Although the efficiency was highest in calcium alginate beads but the conversion of substrate into product decreased at higher concentrations of sodium alginate. This is due to the decrease in the diffusion of the substrate into the beads.Entrapped enzyme in polyacrylamide gel, agar/ agarose gel took nearly 16-19 min to convert starch into maltose as compared to the free enzyme, where the time required for conversion was nearly 11 min. In case of calcium alginate entrapped enzyme, the time required to convert starch was nearly 13-15 min. This indicates that the time for the conversion of starch into maltose has increased in case of entrapped enzyme as compared to free enzyme and further the conversion time in case of calcium alginate entrapped enzyme was less as compared to enzyme in polyacrylamide, agar/agarose gel.The different gels entrapping amylase enzyme were used to check their susceptibility to breakage by stirring with overhead stirrer and magnetic stirrer for 10 repeated cycles. Stirring of the reaction mixture with overhead stirrer caused less breakage of the beads as compared to stirring with magnetic stirrer. The activity of amylase enzyme entrapped in polyacrylamide, agar/agarose gel and calcium alginate beads left after 10 repeated cycles was 56, 44, 95% and 29, 08, 86% in the presence of overhead and magnetic stirring, respectively. The gels of polyacrylamide and agar were fragile and can not be used for repeated conversion of substrate into product. The gels of polyacrylamide and agar have been used for immobilizing enzymes–17.Amylase enzyme immobilized in different gels was checked for 50 repeated cycles for the conversion of starch into maltose. Amylase enzyme activity left after 25 cycles in polyacrylamide, agar/agarose gel and calcium alginate beads was 18, 12 and 88%, respectively. The activity of amylase enzyme in calcium alginate beads after 50 cycles was 78% where as in polyacrylamide and agar/agarose gels, activity was less than 5% after 30 cycles. In case of polyacrylamide/agar/agarose gels, the activity of amylase was decreased, as these gels were fragile. So 1% concentration of sodium alginate under all conditions was found to be suitable with respect to immobilization efficiency and conversion of substrate into product.When all the enzyme entrapped gels were packed in separate columns and checked for the conversion efficiency, it was again highest in calcium alginate beads as compared to polyacrylamide gel and agar/agarose gels.In order to check the effect of these matrices on the entrapped enzyme, the activity of the amylase enzyme entrapped in all these gel matrices was checked by keeping these gels at 4° and 25° and compared with the free enzyme control, also kept at 4° and 25°. In all these matrices, the decrease in activity pattern was nearly same when compared with the control. This indicates that these matrices have no adverse effect on the activity of the enzyme.2. The large sized beads so obtained were used for 10 repeated cycles to check the conversion of substrates into their products by their respective enzymes (Amylase enzyme preparation was then mixed with urease and lysozyme and the mixture was immobilized in 1% sodium alginate and 0.5 M CaCl enzymes . The act


Sensitivity analyses showed little effect on overall costs. The main trial showed de Gramont and Lokich to be equally effective in terms of survival, quality of life and response rates but Lokich had higher toxicity and hand-foot syndrome. Raltitrexed showed similar response rates and overall survival but increased toxicity and inferior quality of life making it a clinically inferior regimen despite its ease of administration and costs. For a comparable clinical outcome, Lokich can be administered for approximately half the cost of de Gramont.An economic sub-study was run alongside a large multi-centre randomised trial (MRC-CR06) comparing three chemotherapy regimens; de Gramont, Lokich and raltitrexed in patients with metastatic colorectal cancer. Patients in six of 45 centres in the main trial were approached to take part in the sub-study. Chemotherapy delivery costs were assessed in each sub-study centre with external validity verified by questionnaire to all other centres. Patient representativeness was assessed. Stochastic resource use data, including patient borne costs and non-hospital health service resource use were monitored prospectively. Mean total societal costs were de Gramont=£5051 (s.d. £1910), raltitrexed=£2616 (s.d. £991) and Lokich=£2576 (s.d. £1711). In pairwise comparisons, statistically significant mean total cost differences were shown for de Gramont British Journal of Cancer (2002) 86, 1684–1690. doi:10.1038/sj.bjc.6600273www.bjcancer.comCancer Research UK© 2002  Colorectal cancer kills about 20 000 people each year in the UK. Once the disease has spread beyond the limits of surgical cure, the aim of treatment is to improve quality of life and survival. In a meta analysis of randomised controlled trials, chemotherapy has been shown to improve median survival from 8 to 11.7 months .A large nation-wide multi-centre randomised trial (MRC-CR06) has been undertaken to compare three chemotherapy treatments for patients with advanced colorectal cancer: de Gramont bolus and infusion 5FU plus folinic acid, Lokich Protracted Venous Infusion 5FU, and raltitrexed (Tomudex™)  bolus 400 mg m−2 and infusion 600 mg m−2 day 1 and 2, q14d), Lokich (protracted venous infusion of 5FU 300 mg m−2 day−1) or raltitrexed (3 mg m−2 i.v. q21d) for an initial period of 12 weeks.Details of the design of the main trial are available elsewhere  were collected prospectively over the 12 week period following randomisation. A research nurse in each of the six centres monitored patient notes for investigations undertaken and all additional treatments received. Patient borne costs were recorded in diaries by patients, or where necessary by a relative. Weekly entries were made listing all costs incurred by patients and their families, including travel and time taken off work by others to care for the patient. Use of non-hospital health and social care resources  were also recorded in the diaries.Monetary valuation of resource use was undertaken by attaching unit costs to recorded quantity data. All costs are in 1998/99 prices. In the case of all chemotherapy drugs, costs have been calculated on the assumption that any drug remaining in partly used vials will be discarded. Unit costs for drugs are from the British National Formulary  which dot-tests (α=0.05) were used to test for differences in mean costs in pairwise comparisons of the three regimens. Representativeness of the sub-sample was assessed by comparing demographic and other characteristics with all other CR06 patients using χ2. Details of the statistical methods used on clinical outcome data are available elsewhere . In terms of serious adverse events, patients in the economics sub-study were generally representative of the whole, and although no fatal or life threatening chemotherapy related events occurred, a higher than expected proportion of patients required hospitalisation.The survival pattern of patients in the economics sub-study was similar to that of those not included . Table 2Mean total societal costs were highest for de Gramont , followed by raltitrexed  and Lokich  as shown in Table 3With regard to de Gramont, the costs shown reflect the way that chemotherapy was actually delivered within the trial i.e. with a mix of in-patient (63%) and out-patient (37%) treatment. Cost of chemotherapy drugs (£1046) represented 39% of the cost of de Gramont delivery (£2672) which in turn made up 53% of total societal costs (£5051). In-patient admissions other than for chemotherapy (£1274) represented a further 25% of total societal costs with patient borne costs (£914) representing 18%.In the case of Lokich, cost of chemotherapy drugs (£433) represented 44% of the cost of chemotherapy delivery (£983) which in turn represented 38% of total societal costs (£2576). In-patient admissions (£400) were responsible for a further 16% of total societal costs with patient borne costs (£762) representing 30%.By contrast, the cost of raltitrexed drugs (£1260) represented almost the whole (97%) of chemotherapy delivery (£1305) which in turn made up 50% of total societal costs (£2616). In-patient admissions (£446) represented a further 17% with patient borne costs (£404) lower than the other regimens and representing 15%.vs Lokich) Table 4bvs raltitrexed) and Table 4cvs raltitrexed). Differences in mean total societal costs between de Gramont and Lokich (£2475) were statistically significant (P<0.01) as were those between de Gramont and raltitrexed . Total costs of Lokich and raltitrexed were similar with a mean difference of only £40.Total societal costs are made up of 15 individual cost elements. Mean differences and confidence intervals for those variables where these differences were statistically significant (pairwise comparisons) are given in Table 4aP<0.001), pharmacy staff , hospital nurses , district nurses  and patient borne costs excluding carers (Lokich higher P<0.01).Examination of differences in the mean estimates for each element specifies where the differences in total costs occur. Although there were no significant differences in total societal costs between Lokich and raltitrexed, pairwise comparisons showed statistically significant differences in the cost of chemotherapy drugs .Full clinical results are available elsewhere , role functioning, social functioning and global quality of life. Raltitrexed was also inferior to Lokich in terms of palliation, fatigue, appetite, nausea/vomiting, constipation and emotional functioning.A number of factors were identified that could have implications for the cost of the different chemotherapy regimens. As Another important difference is the two possible methods of delivery of the de Gramont regimen. The base case analysis calculated the cost of the de Gramont regimen based on a mixture of in-patient and out-patient delivery. There are clearly cost implications if this regimen is always provided on an in-patient or always provided on an out-patient basis.By the end of the study period, many centres had changed from using Hickman lines in the delivery of the Lokich regimen, to cheaper peripherally implanted central catheters. The impact of this change needs also to be considered.In the base case analysis, the costs of in-patient stays, investigations, materials and disposables were provided by the Finance Department of one of the participating centres. To take account of possible variations in these figures, the analysis was repeated twice more using extreme values; once assuming that costs were half those in the base case analysis and once assuming they were double.One final factor worth considering is the impact on nursing time of patients receiving the Lokich regimen being trained to change the pump themselves rather than attending hospital for a nurse to change it.Tables 6aThis study has demonstrated that over a 12 week period, study patients who received the de Gramont regimen had significantly higher costs in terms of both direct chemotherapy delivery and total societal costs than those receiving either Lokich or raltitrexed.New drugs usually carry a high drug cost but this study has shown that despite the relatively high cost of raltitrexed, overall societal costs compare favourably with those of the other regimens. This is consistent with From the hospital perspective the Lokich regimen is the least costly. Although raltitrexed had the lowest costs in terms of pharmacy and nursing time, these were not sufficient to offset the higher drug costs. This study thus provides no evidence to support the results of vs Lokich, mean difference=£874, P=0.181, de Gramont vs raltitrexed, mean difference=£828, P=0.206), although this must be considered in the light of the small sample size. General practitioner visits, either at home or in the surgery, were also similar suggesting that patients on one regimen are not seeking more help in primary care than patients on any other regimen. Overall, this study provided no evidence that the cost implications of side effects and serious adverse events  differ between regimens.As regards the cost of treating chemotherapy related side effects, no significant differences were found in terms of prescribed drugs. This is in contrast to the finding of n=6) had chemotherapy delivery costs which were roughly 50% lower (P=0.011) than for in-patient delivery. The relatively low degree of statistical significance is likely to be due to the small numbers involved. Chemotherapy delivery, however, only represents a part of total societal costs and de Gramont patients receiving out-patient delivery had higher primary care costs than those receiving it as in-patient . Even on the basis of these very small numbers, use of de Gramont as an out-patient regimen is likely to have important implications for hospital costs.Clearly, these reported costs can vary by changing the way in which chemotherapy is delivered. For example, providing de Gramont on an out-patient basis avoids in-patient costs and can thus significantly reduce hospital costs. Within this study patients who received de Gramont as out-patients . Delivering modified de Gramont on an out-patient basis will have further implications for hospital costs. Patients who received de Gramont as an out-patient in the present trial still incurred mean in-patient costs for chemotherapy delivery, although these were much lower (£231 vs £2084) than for those who received this regimen as an in-patient.There is also the possibility of further reducing de Gramont drug costs by modifying the dose. In an ongoing MRC trial (CR08) the de Gramont regimen has been modified to 200 mg mThe toxicity and impaired quality of life observed with raltitrexed make it a clinically inferior regimen despite its ease of administration and costs. In making a choice between de Gramont and Lokich, a number of issues need to be taken into consideration given that their overall clinical benefit is equivalent. Lokich is inferior to de Gramont only in terms of the increased incidence of minor adverse events and central line complications. There was no evidence that the management of these events had different cost implications between the two regimens. However, hand and feet soreness, while neither affecting measurable quality of life nor resulting in costs, can be an added burden, if a manageable one. Patients have recorded no difference in terms of interference with daily living between de Gramont and Lokich. However, in current practice modified de Gramont is now administered as an out-patient regimen in nearly all centres. This has the positive effect of reducing interference with normal life and cutting hospital costs, but does mean that all patients now require central lines with the associated incidence of line complications.In conclusion, in comparison with de Gramont as administered in this study, the significant reduction in costs of Lokich with comparable clinical benefit suggest that it offers the best value for money of the three regimens.