Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.The  Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Clinical conditions can vary from febrile or hemorrhagic diseases for Dengue virus (DENV) and Yellow fever virus (YFV), encephalitis for Saint Louis encephalitis virus (SLEV), Japanese encephalitis virus (JEV), Tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Rocio virus (ROCV) to hepatitis for Human hepatitis C virus (HCV) and Human Pegivirus (HPgV). Currently, preventative vaccines for humans are available only for YFV, TBEV, and JEV and specific antiviral treatment only for HCV The Flaviviridae family, dengue is a major threat to public health. It is estimated that 390 million dengue infections occur per year, with 100 million manifesting some type of symptoms Of the diseases caused by viruses of the Flaviviridae family include viruses with a positive-sense, single-stranded RNA genome of approximately 11,000 nucleotides, surrounded by a nucleocapsid and covered by a lipid envelope in which viral glycoproteins are anchored. The RNA genome encodes a single polyprotein that is proteolytically cleaved into three structural proteins (C-prM/M-E) and seven non-structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) Members of the Chondrodendron tomentosum, was the first naturally occurring toxin used in medicine 2s, which can provide clues for designing therapeutically useful molecules et al.Bothrops jararaca) venom that were used to develop an inhibitor (captopril) of the angiotensin-converting enzyme that is widely used as an anti-hypertensive agent Measles virusSendai virusDengue virusHuman immunodeficiency virus (HIV) 2 (sPLA2), which has shown systemic toxicities that include myotoxicity, cardiotoxicity, neurotoxicity, nephrotoxicity, hepatotoxicity, reprotoxicity, and systemic hemorrhage 2 isolated from snake venoms and other sources has also shown antiviral activity against HIV Newcastle virusRous virus2  from Crotalus durissus terrificus venom against DENV and YFV 2-CB against dengue virus and three other enveloped viruses.Natural products offer a huge amount of compounds with a great diversity of chemical structures, the result of biosynthetic processes that have been modulated over millennia through evolution. Natural products have served as important sources of drugs for medical purposes. Tubocurarine, a toxic alkaloid with skeletal muscle relaxant properties and obtained from the bark of the South American plant Aedes albopictus mosquito cell line) Rocio virus (SPH 34675 strain), Mayaro virus (BeAn 243 strain), and Oropouche virus (BeAn 19991 strain) from the virus collection of the Virology Research Center of the Medical School of Ribeirao Preto, University of Sao Paulo, were used in this study. Enterovirus Coxsackie-B5 virus was kindly provided by Prof. Dr. Eurico Arruda Neto from the Viral Pathogenesis laboratory at the Virology Research Center, Medical School of Ribeirao Preto, University of Sao Paulo. Virus titration was performed in the Vero E6 cell line using the plaque assay, and the titer was expressed in plaque-forming units per milliliters (PFU/mL), as previously described VERO E6 (African green monkeys kidney epithelium cell line) Taq DNA polymerase activation, followed by 45 cycles of PCR amplification with denaturation at 95°C for 15 sec, annealing at 60°C for 40 sec, and extension at 72°C for 30 sec. Finally, to verify the specificity of the PCR products, a melting curve was constructed by incubating the amplification products from 60°C to 90°C with an increase of 0.2°C/sec. The melting temperature (Tm) values of the specific amplicons were in the range of 80.57°C-81.73°C. For viral load determination, a standard curve was constructed using RNA transcribed in vitro from a plasmidial clone containing a fragment of 2500 base pairs (bp) corresponding to the 5′ end of DENV-3 strain D3BR/RP1/2003 Escherichia coli. An aliquot of 250 µL of the bacteria was inoculated into 10 mL of LB+ampicillin medium, followed by incubation at 37°C for 14 h with shaking. The plasmid was purified using the QIAGEN Plasmid Mini Kit  following the manufacturer's recommendations. The plasmid was linearized by digestion with Bam HI, subjected to electrophoresis through a 1% agarose gel, and purified from the gel using the QIAquick Gel Extraction Kit  following the manufacturer's specifications. For RNA preparation, 1 µg of the linearized plasmid was transcribed using RNAMaxx High Yield Transcription Kit  following the manufacturer's specifications. The cDNA was digested using RQ DNase  at 37°C for 20 min. After this, the RNA was purified using the QIAamp Viral RNA Kit , and the concentration was determined by spectrophotometry at 260 nm. Based on the concentration and size of the RNA product (2500 bp), the copy number equivalent to the genomic RNA was calculated. Serial decimal dilutions of this RNA were prepared to construct the standard curve by real-time RT-PCR for determination of the virus titer, which was expressed in copy numbers per milliliters.Viral RNA detection was carried out by real-time RT-PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit , as previously described Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis, IBAMA: 1/35/1998/000846-1). Purification and evaluation of the enzymatic activity of PLA2-CB and crotoxin were carried out as previously described Snake venom was obtained from the serpentarium of the Medical School of Ribeirao Preto, University of Sao Paulo  was incubated with non-cytotoxic concentrations  of PLA2-CB and crotoxin in a final volume of 100 µL for 1 h at 37°C 5 cells/well). After dilution, two concentrations (0.008 and 0.004 ng/µL) were above and one bellow (0.000004 ng/µL) the effective concentration that inhibits 50% (EC50) of cells infection found in our previous study (EC50 of 0.00003 for PLA2-CB and 0.001 for crotoxin) To evaluate the direct effect of PLA5 cells/well) in a 24-wellsplate and incubated at 37°C for 24 h. The medium was then removed, and the cells were incubated with several concentrations of PLA2-CB or crotoxin . After 1 h of incubation, the supernatant was removed, and cells were washed five times with PBS at room temperature. The cells were infected with 25–100 PFU of DENV-2 in 400 µL of L-15 for 1 h. The supernatant was removed, and 1 mL of L-15m supplemented with 2% FBS was added to the cells. After 72 h of incubation at 37°C, the cell supernatants were collected for viral RNA extraction using AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit  following the manufacturer's recommendations. Virus inhibition was evaluated by qRT-PCR, as mentioned above.This assay was carried out to evaluate whether the toxins could protect VERO E6 cells against viral infection. VERO E6 cells were seeded (2×103 PFU) was incubated with 8 ng/µL of PLA2-CB, 8 ng/µL of crotoxin, 1 µg/µL of proteinase K , 1% Triton X-100 , or PBS in a total volume of 100 µL for 1 h and 2 h at 37°C. Then, the virus was treated with PBS or RNase A  according to the manufacturer's recommendation. RNA purified from DENV-2 (2.0×103 PFU) was used as a control for RNA degradation induced by RNase A. After 1 h at 37°C, the viral RNA was extracted using AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit , and RNA degradation was evaluated by qRT-PCR as mentioned above.This assay was carried out as previously proposed Dengue virus type 2, Rocio virus, Mayaro virus, and Oropouche virus and the non-enveloped Coxsackie B5 virus were used in this experiment. The viruses (25–100 PFU) were incubated with serial decimal dilutions of PLA2-CB and crotoxin  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate for 1 h. The supernatant was removed, and the cells were overlaid with 1 mL of L-15 supplemented with 2% FBS and 1.8% carboxymethylcellulose. After 7 days at 37°C, the semisolid overlay medium was removed, and the cells were fixed and stained with naphthol blue-black in 5% acetic acid for plaque counting. The treatment of each virus was repeated three times, and the concentration that inhibited 50% of plaque formation  was calculated by comparison with the number of plaques observed in the PBS-treated cells.The enveloped viruses et al.et al.2-CB crystal structure was obtained under PDB ID: 2QOG 2+ ion) with software Visual Molecular Dynamics – VMD 3kJ mol−1 nm−1. Maxwell-Boltzmann distribution assigns velocities to all atoms in the system according to the chosen initial temperature, and a position restriction was applied to the protein portion, allowing full solvation with the newly introduced water. After this step, we removed all restrains from the heavy atoms of the protein and used the P-LINCS algorithm 2-CB plus the extracted 3-fold pore from DENV were generated with the APBS (Adaptive Poisson-Boltzmann Solver) plugin 2-CB and the DENV 3-fold pore were attributed with aid of the pdb2pqr online server editconf (-mead option) in the package GROMACS 4.5.5. In all cases, we set as default the solvent and protein/lipid dielectric constants to 78.0 and 2.0, respectively.The DENV external structural model was obtained from RCSB Protein Data Bank under the PDB ID: 3J35 P values of <0.05 were considered to indicate statistically significant differences. The statistical analyses were carried out using GraphPad Prism version 5.03 .A Mann–Whitney U test was used to compare the differences between the treated and untreated control groups. 2-CB and crotoxin have a direct effect on the virus particle, DENV-2 (2.0×103 PFU) was treated with 8, 4, and 0.0004 ng/µL of each toxin or PBS. One hour after treatment, the mixture was diluted 100 times  and then used to infect Vero cells. The purpose of the dilution was to reduce the concentration of toxins in contact with the cells and to use concentrations above (0.08 and 0.04 ng/µL) and below (0.000004 ng/µL) the EC50 found in our previous study 2-CB and crotoxin in a dose-dependent manner, suggesting a direct action of the toxins on the virus particle , before infection. One hour after treatment, the cells were washed and infected with the same amount of DENV-2 used in the virucidal assay (20 PFU). The qRT-PCR analysis of the cell culture supernatant after 72 h of infection showed that virus replication was significantly inhibited only at the higher toxin concentrations, 1 and 2 ng/µL , Oropouche virus (Bunyaviridae family), and Mayaro virus (Togaviridae family) and the non-enveloped virus Coxsackie B5 virus (Picornaviridae family). As expected, the toxins showed a high antiviral effect (very low EC50) only against the enveloped viruses , Dengue virus assumes an expanded and mostly spherical configuration, whereby E protein sub-units are arranged in such a conformation that leads to an enhanced area of lipid envelope exposure 2, as opposed to the highest value of ∼264 Å2), indicating that these regions concentrate the deepest cavities on the virus surface. Combining the properties of this pronounced cavity with its aforementioned large diameter, it becomes clear that the 3-fold pore is indeed a remarkable site of exposure of the buried DENV lipid envelope. The planar surface that surrounds the active site (i-face) from the bovine group-IB (bGIB) PLA2 has an area of approximately 1500 Å22-CB shares a very similar structure with (bGIB) PLA2, we assume the same i-face area for both. By structural analysis, we estimated that the 3-fold pore has an area of at least approximately 2500 Å2, being therefore at least 60% wider than a PLA2-CB monomer by superimposition at the planar orientation  with the hydrophobic matrix just below the level of the polar phospholipid heads in the zwitterionic DENV bilayer. Electrostatic analyses of the complex PLA2-CB/DENV 3-fold pore showed that the prominently positive inner surface of the 3-fold pore facing the negative charges of the membrane tends to establish a perpendicular electrical field to the plane of membrane around its periphery, whereas the centermost region displays a weaker electrical field ; crotapotin act as a chaperon that enhances PLA2-CB activity To further analyze if the virus external structure influence the antiviral activity of toxins, and considering that DENV acquire a “bumpy,” expanded conformation when incubated at 37°C as opposed to its smooth structure at room temperature 2s isolated from the venom of Crotalus durissus terrificus have a potent antiviral action against DENV-2 and YFV, especially at the early stage of the multiplication cycle 2 can occur by direct action on the pathogen or as alternative processes on the host or surrounding medium 2-CB in a virucidal assay, providing strong evidence of the direct action of these toxins on the virus particle. Crotoxin is a heterodimeric protein composed of the basic PLA2-CB and the acidic nontoxic catalytically inactive protein crotapotin, which act as a chaperon enhancing PLA2-CB activity 2-CB enzymatic activity, which relies on the hydrolysis of the sn-2 acyl ester linkage of sn-3-glycero-phospholipids, producing fatty acids and lysophospholipids as reaction products 2-CB lost its antiviral activity in the absence of Ca+2, which is an essential cofactor for its enzymatic activity, or by the inhibition of the enzymatic activity, suggesting that its antiviral action is due to the glycero-phospholipids cleavage on the virus envelope 2-CB treatment strongly supports this hypothesis. As degradation was considerably lower when compared to that induced by detergent (Triton X-100) and protease (proteinase K) treatments, these results suggest that PLA2-CB induces a partial envelope destabilization rather than full virus disassembly, in accordance with the PLA2-CB enzymatic function. This hypothesis is in agreement with the results obtained when Newcastle disease virus (NDV), an enveloped virus of the Paramyxoviridae family, was treated with PLA2s from different snakes In a previous study, we showed that PLA2-CB inactivates DENV-2 via the cleavage of envelope glycerophospholipids, which are derived from host cell membranes Yellow fever virus2-CB was able to inhibit other enveloped viruses such as Rocio virus from the same Flaviviridae family as DENV and YFV, Oropouche virus from the Bunyaviridae family, and Mayaro virus from the Alphaviridae family. Further support was revealed by the failure of the enzyme to inactivate the non-enveloped Coxsackie B5 virus from the Picornaviridae family.The fact that PLA2-CB inhibits virus infection via glycerol-phospholipid cleavage prompted us to determine how PLA2-CB gains access to the viral lipid-bilayer envelope if multiple copies of the E glycoprotein form a shell around it. A recent study about the extended configuration that DENV assumes at physiological temperatures revealed a series of gaps among the E proteins that provide an enhanced area of direct exposure of the virus lipid bilayer 2-CB monomers, which have been shown to be enzymatically active against glycerol-phospholipid substrates 2-CB and crotoxin. The crotoxin complex (PLA2-CB and crotapotin) dissociates upon interaction with the membrane, and only the PLA2-CB unit binds to the substrate 2-CB, which explains their similar efficiencies in virus inhibition.The evidence that PLA2s show similar activity to phospholipids composed of different fatty acids, they do have particular affinities with regard to the phospholipid headgroup 2s containing tryptophan in the lipid-binding surface display the highest activity toward zwitterionic lipids 2-CB  can reportedly act upon both phosphatidylethanolamine (PE) and phosphatidylcholine (lecithin - PC), two major components of the DENV envelope, approximately 30 and 60% molar, respectively 2-CB has a high affinity for lecithin-rich vesicles 2 hydrolytic efficiency: the higher the curvature, the higher the enzymatic rate. Ahyayauch et al.2 changes from 50% to 71% as the size of unilamellar vesicles decreased. The results appear to be similar for venom phospholipases: Kensil and Dennis Naja naja naja PLA2 on small unilamellar PC vesicles (SUVs), with faster initial rates than those observed for large (LUVs) and multilamellar vesicles (MLVs). Interestingly, these authors detected distinctly higher catalytic rates on LUVs and MLVs at a small temperature range around the thermotropic phase transition of PC, at which a mixture of both gel and liquid crystalline phases are detected in vesicle lipid organization. Such a coexistence of two or more states triggers uneven phase boundary effects, which are loosely considered defects et al.2s, as opposed to more homogeneous surface areas. Such preferential sites of action for PLA2s were also detected at monolayer borders, sites where the pronounced curvature provides effects similar to those of a disordered lipid patch et al.2 catalytic-enhancing conditions. Indeed, its diminutive size and spherical shape confer a relatively high curvature to its membrane (radius of curvature ∼232 Å) 2 activity. Because of the described points of access to the bilayer on the DENV surface, PLA2-CB is able to hydrolyze the outer phospholipid layer, most likely accumulating fatty acids (FAs) and lysophospholipids (LPLs) on the membrane; this, in turn, destabilizes the membrane to a point at which fundamental protein conformational changes should be impaired. Additionally, it is known that the accumulation of LPLs in a bilayer causes an enhancement on its permeability 2Although sPLA2-CB have a direct effect on Dengue virus particles, most provably by glycerophospholipid cleavage on the virus envelope, which would lead to disruption of the lipid bilayer and destabilization of the E proteins on the virus surface, with the consequence of inactivation. Crotoxin and PLA2-CB most likely gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices in the E protein shell on the virus surface. In addition, PLA2-CB was able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for the development of broad-spectrum antiviral drugs.In summary, we provide strong evidence that crotoxin and PLAFigure S150 of crotoxin against DENV-2 in the virucidal assay.EC DENV-2 (25–100 PFU) was incubated with serial decimal dilutions of crotoxin  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate. The percentage of inhibition was calculated by the reduction of the number of plaques found in the infected cells in comparison with the number of plaques in the cells infected with the untreated virus.(TIF)Click here for additional data file.Figure S250 of PLA2-CB against DENV-2 in the virucidal assay.EC DENV-2 (25–100 PFU) was incubated with serial decimal dilutions of PLA2-CB  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate. The percentage of inhibition was calculated by the reduction of the number of plaques found in the infected cells in comparison with the number of plaques in the cells infected with the untreated virus.(TIF)Click here for additional data file.Figure S350 of crotoxin against ROCV in the virucidal assay.EC ROCV (25–100 PFU) was incubated with serial decimal dilutions of crotoxin  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate. The percentage of inhibition was calculated by the reduction of the number of plaques found in the infected cells in comparison with the number of plaques in the cells infected with the untreated virus.(TIF)Click here for additional data file.Figure S450 of PLA2-CB against ROCV in the virucidal assay.EC ROCV (25–100 PFU) was incubated with serial decimal dilutions of PLA2-CB  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate. The percentage of inhibition was calculated by the reduction of the number of plaques found in the infected cells in comparison with the number of plaques in the cells infected with the untreated virus.(TIF)Click here for additional data file.Figure S550 of crotoxin against MAYV in the virucidal assay.EC MAYV (25–100 PFU) was incubated with serial decimal dilutions of crotoxin  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate. The percentage of inhibition was calculated by the reduction of the number of plaques found in the infected cells in comparison with the number of plaques in the cells infected with the untreated virus.(TIF)Click here for additional data file.Figure S650 of PLA2-CB against MAYV in the virucidal assay.EC MAYV (25–100 PFU) was incubated with serial decimal dilutions of PLA2-CB  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate. The percentage of inhibition was calculated by the reduction of the number of plaques found in the infected cells in comparison with the number of plaques in the cells infected with the untreated virus.(TIF)Click here for additional data file.Figure S750 of crotoxin against OROV in the virucidal assay.EC OROV (25–100 PFU) was incubated with serial decimal dilutions of crotoxin  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate. The percentage of inhibition was calculated by the reduction of the number of plaques found in the infected cells in comparison with the number of plaques in the cells infected with the untreated virus.(TIF)Click here for additional data file.Figure S850 of PLA2-CB against OROV in the virucidal assay.EC OROV (25–100 PFU) was incubated with serial decimal dilutions of PLA2-CB  for 1 h at 37°C. The treated viruses were used to infect VERO E6 cells contained in a 24-well plate. The percentage of inhibition was calculated by the reduction of the number of plaques found in the infected cells in comparison with the number of plaques in the cells infected with the untreated virus.(TIF)Click here for additional data file.


We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional  and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1.Mutations in the photoreceptor protein peripherin-2  cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification,  The OS neration , 4, and neration . Multiplneration .rds-/- background) [In the disc rim, RDS is tightly associated with ROM-1 (rod outer segment protein 1) a non-glycosylated homolog with high structural similarity to RDS . After skground) , 14.Both our past research and the widely varying disease phenotypes in patients suggest that RDS may function differently in rod vs. cone photoreceptors , 13, 15.Here, our goal was to identify novel RDS/ROM-1 interacting partners. Three members of the SNARE family were identified, of which two (Syn3B and SNAP-25) were subsequently validated. These SNARE proteins are known to be involved in the trafficking of other OS integral membrane proteins such as rhodopsin , so it ird8 allele. Retinas from the following mouse strains were used: WT , rds-/- , rom1-/- , nrl-/- , knock-in lines carrying a single point mutation in RDS , and previously characterized transgenic mouse lines expressing mutant RDS on the rds-/- or rds-/-/nrl-/- background . Animal/nrl-/- . For RDS, we used RDS-CT . We also used anti-SNAP-25  at 1:1,000, anti-flag DDK  at 1:1,000, anti-flag DYKDDDDK  at 1:1,000, anti-VAMP2  at 1:10,000 for WB and 1:1,000 for IF, and anti-Cytochrome C  at 1:100 for IF, anti-Na+K+ATPase  used at 1:500 for IF, and anti-prominin-1  used at 1:1,000 for IF. The a5 monoclonal antibody was developed by D.M. Fambrough, and was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Anti-rhodopsin  and anti-CNGB1/GARP  kind gifts from Dr. Robert Molday, University of British Columbia, Vancouver, Canada, and anti-arrestin 1  were used at 1:1,000 for WB. Anti-β-actin HRP antibody  was used at 1:50,000 for WB. Secondary antibodies were goat anti-mouse HRP and goat anti-rabbit HRP , used at 1:25,000 for WB, and donkey anti-rabbit Alexafluor 555, donkey anti-mouse Alexafluor 488, donkey anti-sheep Alexafluor 488 and donkey anti-rat Alexafluor 594 , both used at 1:1,000 for IF.Multiple antibodies for each protein were used and are indicated in the figure legends. Antibodies are summarized in lyclonal ) at 1:1,lyclonal ), both ulyclonal ) at 1:5 lyclonal ) at 1:1,rds-/- mice were homogenized by passing through a series of needles ranging from 18G-26G, after suspending in homogenization buffer containing 20 mM Tris-HCl, pH 7.2, 100 mM NaCl, 1 mM MgCl2 and protease inhibitor . Sucrose was added to a final concentration of 12%. The extract was layered on top of a 50%/60% sucrose gradient and centrifuged at 43,000 x g for 40 min. The pellet was resuspended in homogenization buffer to dilute the sucrose and subsequently centrifuged at 32,000 x g. The pellet was then solubilized in buffer containing 20mM HEPES, pH 7.5, 150mM NaCl, 5mM CHAPS, 0.55mM DTT and protease inhibitor  for 1h at 4°C. The protein extract was subjected to immunoprecipitation (IP) with antibodies against RDS, ROM-1, Syn3B, or IgG (control). For RDS (2E7) and ROM-1 (2H5), antibodies were chemically linked to sepharose 2B beads and extracts were incubated with beads for 1 h at 4°C. For the Syn3B and IgG, the protein was incubated with the antibody  and protein A beads overnight at 4°C. Elution was performed with a 1% SDS solution and was separated on SDS-PAGE. The proteins were subjected to trypsin digestion in-gel (250 ng), extracted from the gel and freeze dried. Analysis of the protein fragments was carried out by LC-MS (ESI-TRAP) by the University of Rochester Proteomics Center. Analysis of the peptide fragments was performed using Scaffold 4 Proteomics software . The peptide fragments were identified by using a subset of the NCBInr_20130830 database restricted to Mus musculus and search engines Mascot and X!Tandem. Proteins were included in subsequent analysis if their identity was established at a probability level of 95% or higher and at least one peptide had been identified (Probability model Peptide prophet with Delta mass correction) [For LC-MS, 40 mouse retinas from either wild-type (WT) or rection) , 25. In 6 cells per 60.1 cm2 petri dish, covered with 2ml of medium). The DNA was added to 500 μl of a 250 mM CaCl2 solution and an equal amount of 2x BBS  was added while vortexing at 1700 rpm. The transfection mixture was incubated at room temperature for 20 min and then added to the cells. The medium was replaced after an overnight incubation, and cells were harvested after 48 h.Vectors containing WT ROM-1 cDNA, WT RDS cDNA, or RDS cDNA with the C150S mutation under the control of the CMV promoter were generated by site-specific mutagenesis of the previously characterized pCDNA3.1-RDS/ROM-1 vectors . We acqu2PO4, 8.1 Na2HPO4, pH 7.0) with 5 mM EDTA, 5 mg/ml NEM  and protease inhibitor . Either 2% Triton X-100 and 2.5% glycerol or 10 mM CHAPS was added as indicated in the results. The retinal extract was solubilized at 4°C for 1 hour on a rocker and then centrifuged at 18,000 xg for 10 minutes. 100 μg of retinal extract (diluted to 0.5 μg/μl in 200 μl) (or 300 μg for rdsY141C/Y141C and R172W/rds-/- and 500 μg for rdsC150/C150S and rds-/- to adjust for low levels of RDS/ROM-1 in these lines) or 50 μg cell extract (diluted to 0.25 μg/μl in 200 μl) was incubated for 2 h at 4°C in the presence of RDS-CT, ROM-1-CT, or Syn3B-478. Subsequently, protein A sepharose beads  were added for 1 h. The beads were washed and eluted with 50 μl of a 1:1 mixture of the extraction buffer and 4x Laemmli buffer containing 400mM DTT. For non-reducing gels, the same buffer was used, but without DTT. For IPs carried out using retinal extract, 7.5 μg (15 μl) of protein was loaded for the input and an equal volume of the unbound fraction . We loaded 65% of the bound fraction. Western blots were performed using standard protocols and detection was performed with HRP substrate .Retinas or cell extracts were quickly sonicated in 1x phosphate buffered saline  on a Chrom1-/- retinal extracts as previously described [rdsC150S/C150S) or gradient fractions (9 μl/fraction) were denatured for ten minutes at 100°C using the denaturing buffer provided with the endoglycosidase H (EndoH)/PNGase F  in a total reaction volume of 30 μl. This was then separated into 3 aliquots of 10 μl each and an additional 10 μl of enzyme master mix was added to each sample for a final reaction volume of 20 μl. One of the three samples received EndoH master mix (0.1 μl of EndoH plus manufacturer indicated buffer and water), the second sample received PNGaseF master mix  and the third sample received mock master mix (EndoH buffer and water without enzyme). All samples were then incubated at 37°C for two hours. Experiments were repeated on 3–10 independent retinas/gradient fractions per group.Non-reducing sucrose gradient velocity sedimentation was performed on WT and escribed , 13. RetTransfected HEK293 cells were fixed with cold methanol:acetone (80%:20%) for 20 minutes. Cryosections were prepared from whole eyes fixed for 2h with 4% paraformaldehyde at 4°C collected at post-natal day 30 and underwent IF as described previously . BrieflyPLA assay was carried out using the Duolink® In Situ kit  on retinal cryosections. Antigen retrieval was carried out with methanol at -20°C for 20 minutes followed by several washing steps with water. The sections were treated with 1% sodium borohydride for 2 minutes to quench tissue autofluorescence, washed with water and 1x PBS and blocked for 90 minutes in blocking buffer  at room temperature. Primary antibodies were applied on sections in blocking buffer and incubated overnight at 4°C. After washing with 1x PBS, the secondary antibodies (anti-rabbit and anti-mouse) provided with the kit were diluted 1:5 in blocking buffer and incubated on the sections for one hour at 37°C. After washing with 1x PBS, ligation stock from the kit was diluted 1:5 in water, ligase was added (1:40 dilution) and incubated on the sections at 37°C for 30 minutes. Amplification stock from the kit was diluted 1:5 with water and polymerase was added at a dilution of 1:160 before application on the sections (washed with 1x Buffer A supplied with the kit after the ligation step) for 75 minutes at 37°C. After the final incubation step, sections are washed with 1x and 0.01x Buffer B from the kit subsequently. Sections were mounted and analyzed as described above for IF using filters for Texas Red. In each PLA experiment, paired sections underwent regular IF labeling using the identical protocol (except with standard fluorescent secondary antibodies rather than the PLA reagents) to confirm specificity of primary antibody labeling.S2 Fig). LC-MS analysis was performed using sepharose columns cross-linked to RDS mAB 2E7 or ROM-1 mAB 2H5. As controls for the RDS and ROM-1 pulldowns, retinal extracts from rds-/- animals were used. As expected, RDS was identified in the ROM-1 IP and vice-versa (with high mascot scores). We also detected another known RDS binding partner, CNGβ [rds-/- group from the RDS IP  were considered non-specific and were excluded. Although ROM-1 levels are very low in rds-/- retinas, there is some ROM-1 present, so proteins which appeared in the ROM-1 IP from rds-/- retinas  are not necessarily non-specific, but could be ROM-1 binding partners which do not require RDS. However, they were excluded from further analysis in this study as we were interested in components which interact with RDS or the RDS/ROM-1 complex. After manually removing proteins found in the negative controls by cross referencing the two lists, a substantial number of RDS and ROM-1 potential interacting partners remained , and of these, the majority (54 proteins) were common to the RDS and ROM-1 groups .Our goal was to isolate RDS complexes and identify novel RDS/ROM-1 interacting partners. For these studies, adult  WT mouse  retinal extracts were solubilized using CHAPS since, of the four detergents we tested, CHAPS was the mildest  that still provided good RDS/ROM-1 solubilization . SNAREs are known to play a role in targeting other proteins from the inner segment to the OS, so for this study, we narrowed our focus to this category. Specifically, two proteins identified by mass spectrometry (MS), Syntaxin 3 B (Syn3B) and synaptosomal-associated protein 25 (SNAP-25) are known to be involved in OS targeting of rhodopsin [Of the 54 proteins common to the RDS and ROM-1 groups, several were either SNARE or SNARE-associated proteins or other proteins potentially involved in protein trafficking . The IP with the Syn3B antibody pulled down a numerous proteins including several expected trafficking proteins as well as ROM-1 and RDS, thus providing initial confirmation of an interaction between RDS/ROM-1 and Syn3B. Multiple proteins overlapped between RDS/Syn3B and ROM-1/Syn3B pull-downs  and a few proteins were present in all three . All trafficking proteins found in one of the IPs (RDS/ROM-1/Syn3B) are summarized in Table 2.Syn3B was selected as the first candidate for further study. We performed an IP on CHAPS-extracted WT retinas using an antibody against Syn3B followed by LC-MS analysis as described above. Knock-out animals for Syn3B are not available, so we used pull-down with non-specific IgG as a negative control . This antibody gave a single band of the expected size in mouse and bovine retinal extracts and in transiently transfected Syn3B in HEK293 cells . No band was detected in untransfected HEK293 cells, and all bands were indistinguishable from those obtained with commercially available antibodies for Syn3B . To study RDS/ROM-1/Syn3B interactions, cells were co-transfected with vectors to express RDS and Syn3B or ROM-1 and Syn3B. Transfection efficiency was relatively high (~60–70%) and immunocytochemistry confirmed that RDS/Syn3B and ROM-1/Syn3B were usually co-expressed  in the double transfected cells. After extraction with Triton-X 100, RDS and ROM-1 were both detectable in the bound fraction of the respective Syn3B IPs  suggesting a direct interaction between RDS and Syn3B as well as ROM-1 and Syn3B. This interaction was confirmed by the reciprocal pull-down with RDS and ROM-1 .To begin our validation of interactions between RDS/ROM-1 and Syn3B, we conducted reciprocal co-IP studies using transiently transfected HEK293 cells (which do not endogenously express any of the proteins of interest). To facilitate detection of Syn3B along with RDS, we generated and characterized a new monoclonal antibody (mAB 12E5) against Syn3B , while Syn3B and RDS did interact . This suggests that RDS/Syn3B likely do not initially (i.e. at the time of synthesis) assemble into a complex, but rather that the RDS/Syn3B interaction occurs later.We have previously observed that RDS and ROM-1 do not form interactions when mixed together post-extraction; a finding we attributed to the early assembly of RDS/ROM-1 complexes in the ER and the fact that RDS/ROM-1 complexes occur within the same membrane. In contrast, stable fusogenic SNARE complexes can form even in cell free systems , 31 or pin vivo by using Triton-X 100 extracted WT retinas for the IPs. The results closely mirrored the findings from the in vitro approach; Syn3B pulls down both RDS and ROM-1 and vice-versa . Interestingly, in both in vitro and in vivo experiments, only a small fraction of RDS and ROM-1 interacts with Syn3B. Direct binding between RDS/Syn3B and ROM-1/Syn3B was confirmed in vivo using retinal extracts from rds-/- and rom1-/- animals . To confirm that this binding was not due to non-specific interactions, we assessed a panel of positive and negative control proteins. In IPs with Syn3B, only rhodopsin  proteins were detected only in the input and flow through . Similarly, rabbit and mouse IgG were used as negative controls, and no Syn3B, ROM-1, or RDS was detected in the bound fraction with IgG pull-down .The expression of proteins in cell culture to test their interaction is convenient, enabling testing of individual pairs of proteins, but the setting is artificial. Overexpression of proteins in culture can induce aggregation, and neither HEK293 cells nor any other tissue culture system form elaborate structures like the OSs. Therefore, we next confirmed the interactions between Syn3B/RDS and Syn3B/ROM-1 ody 1D4, , and knoody 1D4, ) was detFig 3E, left and middle, synaptic layer not shown) [Fig 3E and 3F yellow, arrows) in the apical IS. This region of co-localization can be observed more clearly at 100x; the right panel of Fig 3F shows single confocal plane from a section co-labeled with RDS or ROM-1 (green) and Syn3B (red). We do not observe a region of Syn3B/RDS co-labeling in all cells, suggesting the amount of overlap between RDS and Syn3B varies from cell-to-cell and may depend on how much protein trafficking is occurring in any given cell. In addition, since most RDS/ROM-1 synthesized in the IS is not detected by standard IF, other approaches are needed to assess the extent of the interaction.Consistent with previous findings, Syn3B is localized to the IS/cell body/synaptic layer  , while mt shown) , 21 within vivo: proximity ligation assay (PLA). This approach involves using secondary antibodies to which short oligonucleotides are conjugated. The subsequent ligation of a fluorescently labeled third oligonucleotide which binds only if the two labeled proteins are in very close proximity enables fluorescent detection of protein interacting partners [Fig 4, each image pair shows IF on the left  and a corresponding PLA image on the right. In the PLA image, if the proteins do not interact, no signal is detected, while red spots indicate areas in which the two interacting proteins are found in close proximity. When we labeled with RDS/Syn3B  substantial PLA signal was detected. Although PLA labeling was less prominent with ROM-1/Syn3B it was still present . No signal was detected in the negative control . Positive controls included RDS/ROM-1 and Syn3B/SNAP-25 . RDS/Syn3B PLA signal is often concentrated at the apical IS  RDS/ROM-1 , and Syn3B/SNAP-25 . This observation is consistent with the idea Syn3B may interact with newly synthesized RDS and ROM-1 which are found in the IS.Thus we turned to an alternative cell biological approach to study RDS/Syn3B and ROM-1/Syn3b interactions partners . Using cFig 5A). In preliminary IP experiments in which cell extracts were solubilized with Triton X-100, VAMP2 was not pulled down with RDS or ROM-1, but was abundant in the bound fraction after Syn3B pull-down. Syn3B binding to VAMP2 is essential for vesicle fusion in synapses so this latter result is expected [Fig 5B). This is consistent with the proteomics results, and suggests that binding of VAMP2 to RDS and ROM-1 is of a weaker nature than the binding of RDS/ROM-1 to Syn3B. However, when we repeated the IPs using retinal extracts, no VAMP2 was pulled down by RDS or ROM-1, even with solubilization in CHAPS . Although some other proteomic studies have identified VAMP2 in OS preparations [Fig 5D, green) rather than in the IS or OS. Combined, these data suggest that VAMP2 likely does not directly bind RDS/ROM-1 in the retina.Having established that Syn3B interacts with RDS and ROM-1 we next examined other SNAREs expressed in photoreceptors and identified in our RDS proteomics screen, namely SNAP-25 and VAMP2. To validate potential interactions between RDS/ROM-1 and VAMP2, we double transfected HEK293 cells with RDS or ROM-1 and VAMP2  and WT retinas . Cell extracts were solubilized in CHAPS and after IP with RDS, ROM-1, and Syn3B, SNAP-25 was detected in the bound fractions , indicating that the interaction between RDS/ROM-1 and SNAP-25 is direct and not mediated by Syn3B (no endogenous Syn3B is detected in HEK293 cells). This interaction was also confirmed in vivo; IP of CHAPS extracted WT retinas with antibodies against RDS or ROM-1 showed SNAP-25 in the bound fraction . From this we conclude that RDS and ROM-1 interact not only with Syn3B but also with SNAP-25. As expected, Syn3B and SNAP-25 also interacted in vivo . In contrast to VAMP2, SNAP-25  co-localizes with Syn3B  in the IS of photoreceptor cells and thus gives a similar distribution to Syn3B in reference to RDS  and ROM-1 . These cell biological and biochemical data are consistent with the idea that SNAP-25 interacts with newly synthesized RDS and ROM-1 in the IS.Finally, we assessed interactions between RDS/ROM-1 and SNAP-25, using both double transfected cells . Similarly, ROM-1 eluted from Syn3B IPs was both monomeric and dimeric . To confirm covalent linkage was not required for interactions between RDS/Syn3B, we asked whether Syn3B could bind to C150S mutant RDS. This form of RDS lacks the cysteine which is responsible for the formation of disulfide bonds between RDS molecules, and as a result we and others have previously shown that it forms no covalently linked complexes, and thus no dimers are present on western blots prepared under either reducing or non-reducing conditions from either transgenic retinas or transfected cells [rdsC150S/C150S, Fig 7B) or HEK293 cells transfected with C150S-RDS . We find that C150S-RDS retains the ability to bind Syn3B  both in vivo and in tissue culture, confirming that assembly of RDS into covalently linked complexes is not required for RDS to bind to Syn3B.Having confirmed that RDS and ROM-1 interact with members of the SNARE family, we next asked what types of RDS/ROM-1 complexes are involved in these interactions. RDS and ROM-1 initially assemble into non-covalently linked homo- and hetero-tetramers with subsequent assembly into higher-order covalently linked complexes. These complexes are held together by C150-mediated disulfide bonds in the D2 loop of RDS , 9. Multed cells , 11. We Most transmembrane proteins which are synthesized in the IS traffic to the OS by a conventional secretory pathway, i.e. through the trans-Golgi. However, recent work has shown that RDS traffics through an unconventional pathway which bypasses the trans-Golgi . Using a lane E) or PNGaseF  or were mock digested  followed by western blotting. We find that 56.9% ± 5.5% of the total pool of RDS in the murine retina is EndoH sensitive, with the rest remaining resistant, suggesting that in the mouse retina some RDS traffics by both conventional and unconventional pathways. To ensure this outcome was not due to incomplete digestion, we conducted the experiments with increasing amounts of enzyme  without seeing any change in the fraction digested.First, we assessed what portion of RDS in the murine retina was trafficked via the conventional vs. unconventional pathway. WT retinal extracts underwent digest with EndoH . We found that higher order RDS oligomers were completely EndoH sensitive, while intermediate complexes exhibited mixed EndoH sensitivity and tetrameric complexes were mostly EndoH resistant .We next asked whether different types of RDS complexes had differential sensitivity to EndoH. WT retinal extracts underwent non-reducing velocity sedimentation on 5–20% sucrose gradients to separate RDS/ROM-1 complexes. Previously we have observed that covalently linked higher-order RDS homo-oligomers are found in fractions 1–3, intermediate sized RDS/ROM-1 heteromeric complexes  are found in fractions 4–5 while heterotetramers are found in fractions 6–9 . One gra bound lanes). This is consistent with our observation that Syn3B binds both covalently linked  and non-covalently linked  RDS.Finally, to determine whether Syn3B binds RDS trafficked through both the conventional and unconventional pathway, we conducted IP with Syn3B, and the eluents underwent EndoH treatment followed by western blotting for RDS. We find that Syn3B binds to both EndoH sensitive and EndoH resistant RDS .Various RDS mutations in patients lead to different forms of retinal degeneration which can affect rod- or cone-based vision (or both) and which can affect RDS/ROM-1 complex assembly. We have also previously demonstrated that rods and cones have a differential requirement for RDS and that some proteins such as the oligomerization-incompetent C150S protein, traffic differently in rods vs. cones , 14. As ke cells ). IP forrds-/-) and cone (rds-/-/nrl-/-) genetic backgrounds . We observe similar results with retinas from Y141C knockin mice (rdsY141C/Y141C). The Y141C mutation causes pattern dystrophy in patients [Fig 8B). These results suggest that though RDS/ROM-1 complexes assembly may be impaired to varying degrees by different RDS mutations, provided the abnormal protein complexes properly target to the OS, they retain the ability to bind Syn3B.We were also interested in the ability of Syn3B to interact RDS mutants which cause abnormal RDS/ROM-1 complex formation but not abnormal trafficking. The R172W mutation in RDS causes macular dystrophy  in patiepatients  and rod patients . Y141C ppatients . As in tHere we use multiple methods to demonstrate that Syn3B and SNAP-25 are novel interacting partners for RDS and ROM-1. Syn3B and SNAP-25 are known to play a role in rhodopsin trafficking to the OSs . Syn3B aSyn3B is hypothesized to serve the same function for rhodopsin , i.e. mein vivo, three formal possibilities exist. First, that VAMP2 is involved in RDS vesicle fusion, but does not bind to RDS or ROM-1 directly. This is possible since SNARE proteins do not necessarily bind to vesicular cargo; however the distribution of VAMP2 in the retina makes it unlikely. The second possibility is that another v-SNARE or VAMP is involved in RDS vesicle fusion, but that it also does not interact with RDS directly and thus did not appear in our LC-MS results. A third possibility is that Syn3B-mediated RDS vesicle fusion does not involve a v-SNARE. On the one hand, this seems unlikely; traditional SNARE-mediated vesicle fusion is well-characterized and requires both a t- and v-SNARE and RDS does not have a SNARE domain. However, RDS’ cytoplasmic C-terminal domain , a t-SNARE on the target membrane and a v-SNARE on the membrane of the arriving vesicle. We identified Syn3B as a candidate t-SNARE however; the only v-SNARE in our LC-MS analysis was VAMP2. Although VAMP2 is in the retina and interacts with Syn3B , 34, it equences ) is multequences , and criThere is precedence for syntaxins to bind to non-SNARE partners involved in vesicular transport, including vesicular cargoes such as vesicle coat proteins, tethering factors, and Rab GTPases thought to be involved in regulating the targeting and docking of vesicles , 44. In However, while it is possible that Syn3B-RDS interactions are a sign that Syn3B on the plasma membrane is involved in fusion of RDS-containing vesicles with that membrane, it is also possible that the interaction is a sign that the two proteins are actually trafficked together, i.e. that newly synthesized Syn3B is packaged into RDS-containing vesicles. Further testing of all these possibilities is required to understand the physiological roles of Syn3B/RDS/ROM-1 interactions and the role (if any) of RDS complex type in these processes.Though our main goal in this study was to identify and verify novel RDS/ROM-1 binding partners, our work investigating whether these newly identified binding partners interacted with conventionally or unconventionally trafficked RDS brought to light interesting results regarding the trafficking of different types of RDS complexes. Previous work has shown that OS-depleted retinal extracts contain primarily RDS/ROM-1 tetramers, while OS-enriched retinal extracts contain primarily covalently-linked higher order RDS complexes . This hards-/- [Widely varying blinding disease phenotypes are associated with point mutations in RDS, and these phenotypes have multiple different cellular/biochemical mechanisms , 47, manrds-/- , 41, andrds-/- , 14. FurS1 FigWestern blot incubated with the monoclonal Syn3B antibody shows a band of the expected size of Syn3B in mouse and bovine retinal extract, as well as in extracts from HEK293 cells transfected with a vector containing the cDNA sequence of Syn3B. There is no detectable Syn3B in untransfected HEK293 cells.(TIF)Click here for additional data file.S2 FigResultant WBs were probed for RDS (mAB 2B7) or ROM-1 (mAB 2H5) antibodies.(TIF)Click here for additional data file.


Estimates of healthcare costs associated with HIV infection would provide valuable insight for evaluating the cost-effectiveness of possible prevention interventions. We evaluate the additional lifetime healthcare cost incurred due to living with HIV.We used a stochastic computer simulation model to project the distribution of lifetime outcomes and costs of men-who-have-sex-with-men (MSM) infected with HIV in 2013 aged 30, over 10,000 simulations. We assumed a resource-rich setting with no loss to follow-up, and that standards and costs of healthcare management remain as now.Based on a median (interquartile range) life expectancy of 71.5 (45.0–81.5) years for MSM in such a setting, the estimated mean lifetime cost of treating one person was £360,800 . With 3.5% discounting, it was £185,200 . The largest proportion (68%) of these costs was attributed to antiretroviral drugs. If patented drugs are replaced by generic versions (at 20% cost of patented prices), estimated mean lifetime costs reduced to £179,000  and £101,200  discounted.If 3,000 MSM had been infected in 2013, then future lifetime costs relating to HIV care is likely to be in excess of £1 billion. It is imperative for investment into prevention programmes to be continued or scaled-up in settings with good access to HIV care services. Costs would be reduced considerably with use of generic antiretroviral drugs. Numerous studies have shown that effective antiretroviral therapy (ART) has reduced rates of HIV-related mortality, 2 and tWe have previously developed a stochastic computer simulation model of HIV progression and the effect of ART (HIV Synthesis) and used it to reconstruct the HIV-positive population in the UK and to predict future trends in key outcomes, 7. In aS1 File. In brief, it is an individual-based stochastic computer simulation model that generates simulated “data” on the progression of HIV infection and effect of ART on simulated individuals. For each simulated person, the model generates variables such as CD4 count, viral load, age, clinical events, use of specific antiretroviral drugs, resistance and adherence, all of which are updated in 3-month intervals. The model incorporates current estimated rates of virologic response to ART and subsequent long-term increases in CD4 counts. This version of the model does not include transmission so simulates HIV-positive individuals only. The model has been shown to provide a generally close fit to observed data relating to the natural progression and treatment outcomes[The HIV Synthesis progression model has been described previously, 6, 7 anIn the base-case analysis, we consider a scenario for a 30 year old MSM infected with drug-sensitive HIV in 2013. Outcomes were generated for 10,000 simulated people in this situation resulting in a distribution of possible outcomes for such a person. Each person was followed over 80 years to 2093) or until death and it was assumed that they were never lost from care at any stage in life. It was further assumed that current standards of healthcare management are maintained in future years and that associated costs remain as now. Age-specific mortality rates for England and Wales for 2011 were used to calculate non-HIV related death rates[3 or unti3 and 35% diagnosed late (CD4 count <350 cells/mm3 within 3 months of diagnosis) in 2011[3 (unless symptomatic). Individuals switch to further lines of therapy if virologic failure occurs but can also switch individual drugs for toxicity. Boosted protease inhibitor (bPI)-based regimens are modelled to be double-potency compared to non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens (i.e. acquisition of resistance mutations will impact suppression rates less on a bPI-based regimen). We assumed that 40% of MSM were smokers for life as observed[The rate of HIV diagnosis was chosen to reflect what has been observed recently in the UK for MSM in terms of CD4 count at diagnosis, i.e. median CD4 count of 422 cells/mm observed, 13 and  observed. It is a observed,16.3) to fit closely with the available data[3, on the basis that HIV infection raises the risk of common clinical conditions as mentioned previously (Table 1). The predicted average number of occurrences during a lifetime of each event listed above was multiplied by the unit cost  and then summed to give an estimate of the cost of treating one HIV-positive individual for a lifetime. All costs (2013 UK pounds) were obtained from published sources (Tables 2) and discounted by 3.5% per annum in accordance with NICE  recommendations. The presented lifetime costs also include costs of non-AIDS diseases in situations where the individual is seen in the same healthcare centre as for their HIV care.In this study we consider only costs associated with HIV-related healthcare. Costs for hospital or clinic services  per 3-month period were stratified by CD4 count category  would replace patented versions approximately three years after patent expiry. We assumed that generics would be priced at an 80% reduction of paten3. Measured CD4 counts in the model are used only to determine the likelihood of ART initiation or regimen switch after virologic failure , and also whether PCP prophylaxis is used. In another strategy, we modelled all virologically suppressed individuals with no history of virologic failure to switch to ritonavir-boosted darunavir (DAR/r) mono-therapy. In this strategy, individuals start with standard triple-therapy (cART) regimens and if they fail the DAR/r-regimen after switching, they were modelled to switch back to an effective cART regimen. Finally, we also investigated the effect of 6-monthly and annual healthcare centre visits such that individuals attend clinics less and therefore have fewer CD4 count and viral load measures, but only if they have suppressed viral load, no history of virologic failure and with most recent CD4 count >350 cells/mm3,We also investigated the impact on lifetime costs of four alternative strategies to reduce HIV treatment and care costs. In one Univariable sensitivity analyses were performed to explore the effects of varying key assumptions on the overall lifetime costs and its various components. Assumptions which were varied include the age at infection, rate of diagnosis, rate of loss to care, ART initiation criteria, risk of non-AIDS deaths and population distribution of adherence. A multivariable sensitivity analysis was also conducted where the model was run 10,000 times, each time sampling values randomly from a distribution for different key parameters, to ascertain the variability and uncertainty associated with the estimates of lifetime costs.3 and 38% had CD4 count <350 cells/mm3 at diagnosis. Under the assumption that cART was initiated when the CD4 count dropped below 350 cells/mm3, the mean time spent from infection to treatment initiation was 5.2 years. Most (97%) people started first-line, 51% started second-line and 25% started third-line in their lifetime. Median CD4 count at time of starting ART was 301 cells/mm3. This was followed by a mean 19.4, 6.9 and 4.4 years spent on first-line, second-line, and third-line regimens and beyond respectively. At least one occurrence of treatment interruption occurred in 83% and of those who did interrupt, the mean total length of time spent off ART was six months. These outcomes led to a median (IQR) life expectancy of 71.5 (45.0–81.5) years.For the 10,000 simulated people, median  duration of time before diagnosis was 2.5 (1–4.8) years. Median (IQR) CD4 count at diagnosis was 422 (247–568) cells/mmTable 3). The majority, 68% , of projected lifetime healthcare cost was attributed to ART costs. Despite antiretroviral drugs used in second- and third-line regimens being more expensive in general, the average total undiscounted cost of first-line regimens at £158,900, was greater than subsequent lines  due to the longer time spent on first-line. Similarly, due to the longer time spent in the category ‘undiagnosed, or diagnosed and with higher CD4 counts’, compared to ‘diagnosed but with lower CD4 counts’, the costs incurred when in the former category are greater over a lifetime despite the lower costs incurred per person-year.The estimated mean lifetime cost was £360,800 and with discounting at 3.5% per annum, it was £185,200(Table 3). Therefore, by using generics with cost 20% that of patented drugs, the mean discounted lifetime costs were reduced by over £80,000 per person. In this scenario, costs of antiretrovirals contributed only 36% of the non-discounted projected lifetime costs, and use of healthcare centre services were now the largest cost burden. The average total undiscounted costs of first-line, second-line and third-line regimens were £45,500, £12,600 and £6,600 respectively.Under the assumption that patented antiretroviral drugs are replaced by generics three years after patent expiry, the estimated mean lifetime cost was £179,600 and £101,200 discounted(3 at most recent visit and with no history of virologic failure) reduced mean lifetime costs to £344,000 and £334,900 respectively. Healthcare centre visits costs were accordingly reduced by 20% and 37%. All alternative strategies did not change the projected life expectancy by more than 15 months except for the switch to DAR/r mono-therapy, which resulted in a median life expectancy of 74.3 years (probably due to the modelled high resistance barrier and high potency of bPIs).If CD4 count measures were taken yearly (instead of 3-monthly) in individuals with suppressed viral load and previously high CD4 counts, the estimated mean lifetime cost was £357,500 . CD4 count costs fell by £2,500 (a reduction of 57% compared to CD4 count costs in the base-case scenario). If virologically suppressed individuals with no history of virologic failure were switched from a cART regimen to DAR/r mono-therapy regimen, then the estimated mean lifetime cost was £330,600 . Total ART costs fell by £30,400 (a reduction of 12% compared to ART costs in the base-case scenario). Reducing the frequency of healthcare centre visits to 6-monthly and annually in stably suppressed people . If the assumed average age at infection is altered, it impacts both healthcare centre visit costs and ART costs due to the difference in number of years lived with HIV. If the rate of diagnosis is lowered such that MSM are only diagnosed when symptomatic or if AIDS develops, mean lifetime costs (and life expectancy) are reduced, mainly due to a 21% reduction in ART costs. Our results were not very sensitive to a slight change in the population distribution of adherence assumed in the model. However, if a much poorer distribution is assumed, this reduces ART costs by 38% as people with poor tendency to adhere are modelled to have a higher chance of treatment interruption and loss to care. The median lifetime cost from the multivariable sensitivity analysis was £328,300 and the 95% uncertainty bounds were . Further details are given in S1 File.We performed univariable sensitivity analysis to assess several key assumptions which were made in the model  of someone undiagnosed is the same as someone diagnosed with CD4 count >200 cells/mmWe assumed that the efficacy and tolerability of generics were the same as patented drugs, though we vary the assumption of adherence in sensitivity analyses. Some studies have shown that single-pill regimens improve adherence and reduce hospitalisation, 25. Nev3. Reducing healthcare centre visit frequencies to 6-monthly or annually saved on average £15,000 to £25,000 per person in a lifetime. Although as a proportion of the estimated lifetime costs these savings may seem small, in healthcare centres with large numbers of people in care, the total costs saved may be substantial.From a care provider prospective, it is plausible to reduce costs by way of reduced frequencies of CD4 count monitoring and clinic visits. However, our estimates of projected lifetime costs did not change substantially if CD4 count measures were taken yearly in individuals with suppressed viral load and CD4 count >350 cells/mmConversely, costs relating to quantity of antiretroviral drugs are not as easy to reduce. The strategy in which all virologically-suppressed individuals with no history of virological failure were switched to DAR/r did actually reduce lifetime drug costs by 12%. However, given that individuals in the MONET and MONOI trials, 27 who Further cost reductions may be possible if these alternative strategies are combined with other cost-saving approaches such as cheaper point-of-care assays and using large purpose-built treatment centres. Note that our estimates of lifetime costs do not include non-HIV related healthcare costs, such as costs relating to the treatment of cardiovascular diseases and cancers, nor do they include loss of income associated with illness.3 had a projected life expectancy of 24.2 years and projected lifetime cost of $618,900 in 2004 US dollars . In 2012, Sloan et al.[3. Although it is not suitable to directly compare given the differences in study setting, it is still interesting to note that our estimates are considerably below either of these , but all are consistent in that around 60 to 70% of costs are attributed to antiretroviral drug costs. Given that we have assumed an increased risk for non-AIDS deaths, when healthcare centre visit costs were also increased by 1.5-fold in diagnosed MSM with CD4 count <200 cells/mm3, the estimated lifetime healthcare costs rose to £404,500.There have only been a few studies which have investigated lifetime costs of HIV in the recent cART-era. In 2006, Schackman et al. estimatean et al. providedan et al. and projWalensky et al. have also recently assessed the cost-effectiveness of generics compared to patented drugs. They foOur analysis has a number of limitations. Firstly, due to modelling lifetime costs, we are inherently modelling over a long period of time. This means that the effect of discounting has a large impact. It also requires us to make an assumption that all-cause death rates remain at 2011 levels, rather than assuming they will continue on the current downward trajectory. It should also be noted that with an infectious disease such as HIV, evaluating the benefit of an intervention for reducing incidence of new infections must take into account reductions in future transmissions originating from the infected person. IdeallyIn view of the high lifetime care costs for HIV-positive individuals, there is large scope for preventative interventions to be cost-effective. Our results show that for settings with good access to cART and HIV care, it is imperative for investment into prevention programmes to be continued or scaled-up. Future reductions in drug prices by using generic antiretroviral drugs in place of patented drugs would reduce these costs considerably.S1 FileSupplementary Material. January 2015.(DOCX)Click here for additional data file.


Highly pathogenic avian influenza (HPAI) subtype H7N8 was detected in commercial turkeys in January 2016. Control zone surveillance discovered a progenitor low pathogenic avian influenza (LPAI) virus in surrounding turkey flocks. Data analysis supports a single LPAI virus introduction followed by spontaneous mutation to HPAI on a single premises. In January 2016, the Indiana State Board of Animal Health (BOAH) veterinarians were notified of increased mortality at a commercial turkey operation . The H7N8 HPAI virus contained a 9-nucleotide insertion at the hemagglutinin gene cleavage site translating to basic amino acids (PKKRKTR*G).The H7N8 virus is also highly similar across six of eight genes (except nucleoprotein and matrix) to a recent H7N8 LPAI virus isolated from a hunter-harvested male lesser scaup (after-hatch year) in Kentucky during November 2015 as part of the national wild bird surveillance effort. This indicates that reassortment likely occurred prior to virus introduction in turkeys in Indiana. However, mutation to HPAI likely occurred during replication of the virus in poultry.http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.04_AI.pdf). Mechanisms for acquiring multibasic amino acids include (a) insertion of basic amino acids from codons duplicated at the cleavage site during transcription , LPAI A/turkey/Indiana/16-001573-2/2016(H7N8), and LPAI A/turkey/Indiana/16-001574-7/2016(H7N8) are deposited in GenBank under accession numbers


Infection by hepatitis C virus (HCV) leads to one of two outcomes; either the infection resolves within approximately 6 months or the virus can persist indefinitely. Host genetics are known to affect the likelihood of clearance or persistence. By contrast, the importance of the virus genotype in determining infection outcome is unknown, as quantifying this effect traditionally requires well-characterized transmission networks, which are rare. Extending phylogenetic approaches previously developed to estimate the virus control over set-point viral load in HIV-1 infections, we simulate inheritance of a binary trait along a phylogenetic tree, use this data to quantify how infection outcomes cluster and ascertain the effect of virus genotype on these. We apply our method to the Hepatitis C Incidence and Transmission Study in prisons (HITS-p) data set from Australia, as this cohort prospectively identified incident cases including viraemic subjects who ultimately clear the virus, thus providing us with a unique collection of sequences from clearing infections. We detect significant correlations between infection outcome and virus distance in the phylogeny for viruses of Genotype 1, with estimates lying at around 67%. No statistically significant estimates were obtained for viruses of Genotype 3a. Flaviviridae, genus Hepacivirus) is estimated to infect around 170 million people worldwide, and is a major cause of chronic liver disease  and virSimmonds .IL28B gene that correlate with an increased probability of HCV clearance have been detected  region of the HCV genome harbours a hypervariable region, which is a target for neutralizing antibody response, which in turn has been associated with driving viral evolution  or it develops into a ‘chronic’ infection that generally persists for life unless cured by antiviral therapy. Spontaneous clearance can occur after 6 months, albeit with a very low probability  and several closely related subtypes  that differ approximately by 30% in nucleotide and amino acid sequences , New South Wales Department of Corrective Services (reference number 05/0884) and the University of New South Wales (reference number 05094), all located in Sydney, Australia. The project was approved by the relevant institutional review boards. All subjects provided written informed consent.Hepatitis C virus genome data used in this study were obtained as part of the Australian ‘Hepatitis C Incidence and Transmission Study in prisons (HITS-p) cohort . The second-round cycling conditions included 95°C for 5 min and 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 1 min. The amplified products were identified by agarose gel electrophoresis. PCR products were purified and sequenced directly on an ABI 3730 DNA Analyzer  using dye-terminator chemistry.The region encoding the last 171 bp of core, E1 and HVR1  was amplified by nested RT-PCR as previously described  and 0.4Sequences of the E1-HVR1 region of the HCV genome were available for 92 subjects infected with genotype 1a, 1b or 3a viruses. Of these, 19 were from subjects who ultimately naturally cleared the HCV infection . For the study, we only used the earliest-obtained HCV sequence for each patient and discarded subsequent sequences. Preliminary analysis showed that within-host evolution contributed minimally to the phylogenetic relatedness between subjects, as all the longitudinal samples present in the cohort clustered with the subject itself (result not shown). Due to some incomplete sequencing, subsequent phylogenies were built using a 657 bp length of sequence, covering the core and E1 regions. Although Gray et al.  found thP = 0.0619, Fisher's exact test).We performed an initial analysis on the complete data set. However, to prevent confounding of any detected genetic signal with the HCV genotype, we subsequently extracted the genotypes from the two major observed subtypes and performed analysis on these separate data sets, which we denoted as ‘Genotype 1’ (containing viruses of subtype 1a and 1b) and ‘Genotype 3’ , we did not find a positive correlation between the sampling date and root-to-tip divergence for either subtype. Both estimates were not significantly different from zero . Therefore, it appeared that no molecular clock signal is present in the data. We therefore proceeded using a strict clock model as the simplest possible scenario and checked afterwards that the estimated substitution rates were in line with previous findings for HCV. The outputs seemed to make sense, as substitution rate estimates that we found lied around 10−3, as found by Gray et al.  that were chosen from a uniform distribution between 0 and 1. A value of c = 0 means that there is no trait correlation between related strains, so daughter infections are assigned a trait value at random. On the other hand, a value of c = 1 means that the ‘offspring’ of an infection caused by a specific strain is certain to retain the same outcome as its ‘parent’ infection. For each of these 20 chosen values of c, we started by either assigning the trait of interest (such as HCV infections that clear rapidly) to the root with probability p, where p is the observed frequency of the trait in the actual data set or the alternative trait (such as chronic HCV infection) with probability 1–p. From this tip, we went through each node of the tree in turn. At each new node, we assigned a trait to this node. This trait is either that of its ancestor with control probability c. With probability 1 – c, the outcome is unrelated to its ancestor, so instead the trait was chosen from a Bernoulli distribution with mean p  and subsequently assigned to the node. For each branch, we repeated this process for generations, which were chosen from the following distribution:A schematic of these simulations is shown in Fig. L/Tg)g = 1 + Poisson  and the log-likelihood for the estimates. Note that in order for the analysis to proceed, there needed to be heterogeneity in trait outcomes. That is, there has to be at least one tip of each category present at the end of the simulation, to cause transitions between the two traits. If all the simulated tips were of one type only, we artificially introduced an outcome of the opposite type at a random tip on the phylogeny.After we simulated infection outcomes, we calculated the associated clustering statistics for a simulated outcome. To achieve this, we used the ‘ace’ function, provided as part of the ‘ape’ package  for R . The total number of clearers appeared to fluctuate around 7 for most correlation values, but if the correlation value used as an input approached 1, then this total number could reach high values , or more likely to go to zero. This behaviour is due to the final trait allocations being more likely to reflect the initial state assigned to the tree root , which executes the maximum-likelihood analysis of Pagel . A lower value (0.59) was observed for the birth–death model, which was also marginally significant , and subtype 3a Fig. . ResultsThe high level of virus control found for randomized tips likely reflects that, although the log-likelihood of the switching rates decreases with increased virus control, the likelihood surface can be flat for a broad range of control values. This effect can reduce the power of the method to detect low levels of signal  are much more likely to achieve clearance  are more likely to clear the virus  as the trait of interest instead of the chronic/clearer outcomes. No significant correlations were obtained, although p values tended to be close to significance for the IL28B-917 SNP . Results were quantitatively similar if using a birth–death phylogeny  to recalculate estimated transitions rates of the infection outcome, given that they could be affected by a secondary trait . We did not investigate the effect of IL28B-860 SNP as a co-factor as we did not find nearly significant evidence of this trait clustering on any of the phylogenies. This second analysis essentially removed the variance in infection outcomes solely caused by host differences and focused on the virus control on the residual variance. We then used these new rates to calculate estimates of virus control from the simulated data set of infection outcomes.As there could have been potential confounding effect from the host in determining potential influence of the virus control on infection outcome, we repeated the first analysis while accounting for the host's P = 0.015) and high virus control value of 0.87 was obtained if infection outcome covaried with the IL28B-917 SNP. The control value obtained using the birth–death phylogeny was slightly lower (0.84) but remained strongly significant , as opposed to two rates . We found that the four-rate model provided a better fit, as determined by a lower BIC value, irrespective of whether we used a coalescent or a birth–death phylogeny (data not shown). This result reflects the large phylogenetic distance between Genotype 1 and 3 clades . However, for Genotype 3, a close but nonsignificant value of 0.573 (P = 0.099) was found. Phylogenetic signal for the birth–death phylogeny remained significant for Genotype 1 .It was then determined whether there was any potential clustering of host genotypes along these subclades  free from selection. This analysis produced essentially the same estimates of phylogenetic signal as found using a phylogeny built using the whole genetic region . However, despite a higher control value being obtained for Genotype 3 than before (0.64), it was nonsignificant following the randomization test. The estimated control value using the birth–death phylogeny was higher for Genotype 1 (0.69) and remained significant. Although a similarly high value was found for the Genotype 3 birth–death phylogeny (0.66), it was not significant due to the high estimates obtained from randomized tips , implying that the level of virus control could have been inflated when we pooled all samples due to the extra phylogenetic structure caused. We further investigated this result using power simulations, to verify that our estimates for Genotype 1 are accurate.Overall, this analysis suggests that the phylogenetic signal obtained for the complete data set was strongly driven by viruses of Genotype 1. After correcting for In the previous analysis, one of the more glaring outcomes is that even randomized tips gave rather high estimates of virus control, lying at around 40%. The randomization test determined that our data from Genotype 1 showed evidence of phylogenetic signal compared with randomized tips, but the question remained as to whether the magnitude of the estimates of virus control we obtained are meaningful.c), with each outcome produced along a separate tree from the posterior distribution. We then used our method to estimate the level of virus control from these simulated data sets as before, except that we tested for significance using 100 randomized tips as these tests were computationally intensive.To determine the accuracy and the potential power of our method, we simulated 100 different tip outcomes along the Genotype 1 phylogeny, for a known virus control value . In addition, the estimated level of virus control from the significant estimates increases with the input value (the slope of the linear fit is 0.35). Although error estimates are quite large for inputted c = 0.55, we see that the estimated values are fairly accurate in estimating the actual input by comparing mean points to the y = x line. However, if the input value is very large, then the control value is underestimated .Figure Overall, these power simulations showed that given a result is significant, the obtained estimate of virus control is likely to lie close to the actual level of phylogenetic signal that is present. However, this method also shows that our method can be prone to noisy estimates, which reduced the power of the analysis. This result makes clear the need for future work to increase the precision of phylogenetic inference . In addWhile the clustering of infection outcomes suggests that there is an effect of virus genetics on infection outcome for Genotype 1, caution should be taken in interpreting the magnitude of these estimates as phylogenetic signal could also arise due to the action of an unknown co-variate that has an effect on virus persistence, such as age or gender. Additionally, it could be argued that infection outcomes cluster due the effect of a different disease that is co-transmitted with HCV. With the HITS-p cohort, the overwhelming majority of transmissions are caused via contaminated injecting or tattooing equipment  and also shared the same transmission history. In this regard, no known first-degree relatives were enrolled in the cohort, so this type of sampling bias should not exist.An important discussion point is why we detected relatively high and near-significant rates of correlation amongst the host IL28B genotypes that increase clearance rate. Another explanation is that the host IL28B genotype selects for specific virus mutations from amongst the quasispecies shortly after the infection has occurred, explaining their genetic similarity. This would be a case of parallel evolution . However, as phylogenetic signal was nearly significant for only one host SNP (IL28B–917) for the complete data set only, and the same degree of signal was also present on a third-site phylogeny (where most selective effects of the virus should be absent), then these findings do not strongly suggest that there is major selection acting by the host. Importantly, this significant interaction we detected between the virus phylogeny and the host genetics should not be mixed with the known correlation between host genetics and infection outcome (Thomas et al. IL28B polymorphism and infection outcome. This has been done for HIV (Bhattacharya et al. A more exciting explanation is that there was an interaction between the lineage of the virus and the host genetics. This effect could have arisen if the host acts as a virus filter, so only some HCV viruses can infect hosts that have It is becoming increasingly recognized that the variance in viral infection outcomes is not just due to host genetics; differences in the virus also plays an important role (Alizon et al. These findings also shed some interesting light on the process of virus evolution. Superficially, it seems baffling as to why HCV infections that clear immediately after the acute stage should be maintained by the virus genotype, as their presence appears to reduce opportunities to spread the pathogen. It could be that ‘clearer’ HCV genotypes are weakly deleterious, as reflected in the low frequency of clearers that are observed. These weakly deleterious genotypes could be maintained between hosts at low frequencies via recurrent mutation Wright , which cOur method is able to elucidate correlations between virus genetics and categorical infection outcomes that may not be detectable using standard clustering statistics (Parker et al. In summary, by creating a method to simulate inheritance of a binary trait along a set of phylogenetic trees, we have found that the HCV phylogeny controls 67% of the phylogenetic signal of acute HCV infection for Genotype 1. This outcome could have arisen due to an inherited or transmitted trait within the HCV genome. Overall, this finding not only suggests that the virus genotype can affect infection outcome, but this is also dependent on the specific subtype of HCV, and host genetics. These findings should motivate further research into host–parasite interactions that affect virus evolution.


The aim of the study was to provide detailed herd level cost information about an outbreak of interdigital phlegmon (IP), which has been an emerging problem with enlarged loose house barns in Finland in recent years. During enlargement, the farmer’s financial situation is sensitive because of the large investments to the farm business and unexpected costs can risk the farm’s survival.The University of Helsinki research herd and three commercial herds having outbreaks of IP in 2012 or 2013 were visited to collect detailed information about the costs and economic impact of the outbreaks. The majority of the costs came from the discarded milk due to the antibiotic treatments. In Finland IP is usually treated with parental benzylpenicillin for 5 days which result in discarded milk for a total of 11 days. Third generation cephalosporins, widely used in other countries, have no milk withdrawal time. However, the use of these antibiotics is not recommended in Finland since these antimicrobials are critically important for human health. Herd-level costs varied between 4560 and 28,386 € depending on the herd size, the frequency of the infected cows, the antibiotics used and other costs involved. The average cost per infected cow was 489 €.The outbreaks of IP cause severe economic losses to dairy farms and the costs are lower if cows are treated with antibiotics with no withdrawal time. However, other costs, such as involuntary culling, reduced production and fertility also produce substantial costs to the farms. Early detection of sick animals, rapid treatment and control measures to limit the outbreak of IP can lower the costs. Because of the high costs farms should concentrate on preventing the disease. Fusobacterium necrophorum (which is believed to be the primary pathogen), Dichelobacter nodus, Porphyromonas levii, P. asaccharolytica, Prevotella intermedia, and P. melaninogenica [F. necrophorum is commonly found in the soil and can be isolated on the feet, rumen and feces of healthy cows [Interdigital phlegmon (IP) or foot rot is a necrotizing soft tissue inflammation in the interdigital region of the foot –3. IP isnogenica . Howeverthy cows . In receIn Finland IP is usually treated with parenteral benzylpenicillin for 5 days (20 mg/kg) with a milk withdrawal time of 6 days, which results in discarded milk of a total of 11 days. Additionally, cows are usually treated with non-steroidal anti-inflammatory drugs and in some cases foot baths for the healthy cows are recommended for controlling the infection in the herd. If IP is diagnosed and treated early the antibiotic treatment is usually successful and most cases respond rapidly . AccordiThe aim of this study was to provide detailed cost information about an outbreak of IP at the herd level. Such information is not possible to obtain from the standard Finland health or economic recording and no previous studies have been undertaken to compile the economic costs associated with an IP outbreak.my) in the previous test day recording before the clinical case of IP was diagnosed, the wait time (wt) due to antibiotic use and the milk price (mp) at that time.vf) and medicines (m).ts) on the clinical case and the hourly wage claim (wc) of the dairy farmer in 2013 in Finland (14.90 €) [rt).tc), the price of the culled cows (pc) which varied between 500 and 1250 € depending on the parity, milk yield and fertility status and the price of the lost calves (plc) which was 100 €/calf.The costs presented in this study do not include taxes (VAT 0 %). In the University research farm the accurate daily discarded milk yields were available. In the other farms losses due to the discarded milk were calculated based on the number of the clinical cases of IP, the milk yield (14.90 €) . The estOther costs, such as the increased use of copper sulfate in the foot baths, were also included. IP can also lower the milk production, prolong the calving interval and predispose cows to other diseases . A decreUniversity of Helsinki research herd (A) and three commercial loose-housed herds (B–D) having outbreaks of IP in 2012 or 2013 were visited to collect detailed information about the costs and economic impact of the outbreak. These farms had not had IP outbreaks before and the previous status of other infectious claw disorders was comparable to other farms in Finland. The commercial farms had already participated in the “Infectious hoof diseases in dairy cows in new loose housing system” project funded by the Ministry of Agriculture and Forestry. The researcher visited the farms at the beginning of the outbreaks and diagnosed IP based on clinical signs  and bacteriology. Later IP cases at the farm were diagnosed by the local veterinarian. Farm location, number of cows, number of cows treated and antimicrobials used are described in Table The costs varied between the farms Table , herd-leIP epidemics cause economic losses to dairy industry and the majority of the costs come from the discarded milk due to the treatments with antibiotics. Also, other costs involved  are substantial. However, rapid treatment and control measures to limit the outbreak of IP will greatly reduce the total herd cost. Based on this study the average cost per infected cow can be assumed to be 600–700 € when the cow is treated with benzylpenicillin. Because of high costs of IP it would be more efficient on the economic point of view to focus on the prevention of the disease.