Using phage display and IgG of a goat infected with Caprine Arthritis Encephalitis Virus (CAEV) we obtained families of 7 mer constrained peptides with consensus motifs LxSDPF/Y and SWN/KHWSY and mapped the epitopes mimicked by them at the Env 6-LISDPY-11 and 67-WNTYHW-72 sites of the mature gp135 amino acid sequence. The first epitope fell into the N-terminal immunogenic aa1-EDYTLISDPYGFS- aa14 site identified previously with a synthetic peptide approach; the second epitope has not been described previously. The first epitope is mostly conserved across CAEV isolates whereas the second newly described epitope is extremely conserved in Small Ruminant Lentiviruses env sequences. As being immunodominant, the epitopes are candidate targets for mimotope-mediated diagnosis and/or neutralization. Lentivirus genus of the Retroviridae family. Since the first comparison in 1995 of pol gene sequences of French MVV isolates with CAEV [small ruminant lentiviruses (SRLV) [Caprine arthritis-encephalitis virus, CAEV , and oviith CAEV  and subsith CAEV ), studies (SRLV) . Multipls (SRLV) ,9 in smas (SRLV) ,10. As ts (SRLV) ,8,11,12,s (SRLV) , immunols (SRLV) ). Testins (SRLV) -18. HereWe used the VR905, CAEV 75-G63 strain, cloned lot 2D, 91-12, ATCC (American Type Culture Collection). Cell cultures of Mycoplasma-free goat synovial membrane (GSM) cells that had been propagated in Dulbecco-modified Eagle's medium (DMEM) containing 10% fetal bovine serum, penicillin 100 U/mL, streptomycin 100 mg/mL, and 2 µM L-glutamine were used. Goat synovial membrane cell monolayers were used for virus multiplication and were monitored twice a week for evidence of cytopathic effects . The virus in cell supernatants was then titered through an endpoint dilution assay.®, Behring, IL, USA) [® ELISA Maedi-Visna/CAEV; One IDEXX Drive Westbrook, Maine, New England, USA).We infected a naïve 4-month old goat with the CAEV75-G63 strain. The goat was previously screened with a standard ELISA kit  ). The goIgG fraction purified from serum (see ) at 12-wThe deduced sequences of peptides from the 12-mer linear library did not show a detectable consensus motif Figure . ConversA homology search revealed two sites in the Env N-terminal region Figure  matching8 phages). Two libraries with different peptide lengths and conformation were used. As shown by our previous screenings, one of the libraries can mimic an epitope more successfully than the other depending on its structure [Previous work by Bertoni's ,17 and Vtructure ,21. Heretructure , a homoltructure ,26 was sThe coincidence of one of our mapped epitopes, aa6-LISDPY-aa11, with the previously identified immunogenic aa1- aa14 site  validateIn the experiments by Valas et al. , peptideIn conclusion, this first phage display study describes two discrete epitopes on CAEV gp135 N-terminal segment preceding the first variable V1 domain of the protein. One of them coincides with the previously reported N-terminal immunogenic site; the other was discovered for the first time. Apart from precise mapping of these epitope cores, the study highlights their critical (contact) amino acids and some secondary structure features. In forthcoming experiments the epitopes will be subjected to functional analysis to determine the potentials of their mimotopes for diagnosis of the infection and for virus neutralization .The authors declare that they have no competing interests.KG conceived the study, and participated in its design, coordination, contributed to the analysis of the results and preparation of initial and revised manuscript versions. AGS participated in designing of the experimental strategy, carried out the goat immunization, obtaining and characterization of sera; TG carried out the biopanning experiments, immunization of mice with mimotopes and immuno-testing the anti-mimotope sera; SAP - carried out the computer analysis of selected peptides and Env sequences, contributed to the preparation of the manuscript. All authors read and approved the final manuscript.Alignment of 16 env sequences showing the Ep1 (red) and Ep2 (yellow) sequence conservation.Click here for file


Drosophila, only a handful of viruses have been well-characterized. A viral metagenomic approach was used to explore viral diversity in 83 wild-caught Drosophila innubila, a mushroom feeding member of the quinaria group. A single fly that was injected with, and died from, Drosophila C Virus (DCV) was added to the sample as a control. Two-thirds of reads in the infected sample had DCV as the best BLAST hit, suggesting that the protocol developed is highly sensitive. In addition to the DCV hits, several sequences had Oryctes rhinoceros Nudivirus, a double-stranded DNA virus, as a best BLAST hit. The virus associated with these sequences was termed Drosophila innubila Nudivirus (DiNV). PCR screens of natural populations showed that DiNV was both common and widespread taxonomically and geographically. Electron microscopy confirms the presence of virions in fly fecal material similar in structure to other described Nudiviruses. In 2 species, D. innubila and D. falleni, the virus is associated with a severe (∼80–90%) loss of fecundity and significantly decreased lifespan.Little is known about the viruses infecting most species. Even in groups as well-studied as  The advent of high-throughput DNA sequencing technology has facilitated the discovery and identification of microbes from environmental samples. Though most of the focus has been on metagenomics of microbial communities, leading to the detection of a huge diversity of bacteria and their related bacteriophages Wolbachia, probably the most common vertically transmitted endosymbiont among insects Drosophila has not yet been explored. To investigate the relationship between insect hosts, endosymbiotic bacteria, and viruses, wild-caught Drosophila innubila females, about 1/3 of which are infected with WolbachiaThere is growing appreciation for the important role of interactions among symbionts in host ecology and evolution D. innubila is a member of the mushroom-feeding quinaria group of Drosophila. They inhabit woodlands and forests of the Sky Islands of Mexico, Arizona, and New Mexico. Adults feed, mate and oviposit on mushrooms and larvae burrow through mushroom tissue, feeding on it prior to pupation. Species in the quinaria group are hosts to endosymbionts such as Wolbachia and SpiroplasmaDrosophila species, very little is known about virus infection in natural populations.DrosophilaDrosophila Sigma Virus. Several studies have examined the frequency of Drosophila Sigma Virus infection in natural populations, indicating some degree of host specificity and infection frequencies ranging form absence to more than 70% Drosophila C Virus, which infects several species from across the genus, with little host specificity Of the roughly dozen different virus identified in Drosophila and other insects. This protocol revealed the presence of a new DNA virus that is both taxonomically and geographically widespread and is associated with significant mortality in at least two species of Drosophila. Wolbachia, however, appears to play no role in protection of D. innubila from the adverse effects of the virus.Reported here is the development and implementation of a new virus discovery protocol for et al.Wolbachia, and the rest of the carcass frozen at −80°C Wolbachia-infected and forty-one uninfected carcasses, spanning 2 collection years, were selected for viral screening. Flies that produced few or no offspring were overrepresented, in order to increase the chance of including virus-infected flies. One fly that was injected with, and later died from, Drosophila C Virus (DCV) was added to the Wolbachia-infected sample to assess the efficiency of enrichment for viruses.Flies for the metagenomic survey were collected in the Chiricahua Mountains, as described in Unckless A protocol was developed to remove as much host nucleic acid as possible, leaving capsid-protected viral nucleic acids intact before extraction, production of cDNA libraries, and sequencing . The proRNA was extracted using the E.Z.N.A. viral RNA extraction kit , which will also isolate DNA. Further sample preparation including library preparation , nebulization and emulsion was performed at the Engencore sequencing facility . The 2 samples were bar-coded and run on 1/4 of a chip using a Roche 454 machine with Titanium chemistry.Oryctes rhinoceros Nudivirus). All but the Viral RefSeq protein database were constructed de novo using NCBI's taxonomy browser. The Baculovirus, Nudivirus and Oryctes rhinoceros Nudivirus searches were added after identification of a putative Nudivirus in the initial searches (see below). Limiting the size of the database decreases the E-value of any particular match, increasing its significance, because the probability of a chance match decreases. For all searches, significant hits were characterized by parsing the BLAST output and accessing Genbank to identify genes and organisms for the hit. These scripts were written in PERL and utilized functions in BIOPERL Both raw reads and contigs  were analyzed. All searches were performed locally using stand-alone BLAST+ Drosophila were surveyed for infection with this virus. Flies were collected from Rochester, NY and the Southwest Research Station in 2009 and 2010. DNA from 7 Drosophila phalerata  individuals collected in Munich, Germany was kindly provided by Kelly A. Dyer. In addition, D. innubila were collected in 2010 at the Southwest Research Station and immediately dissected and extracted on site to minimize possible horizontal transmission of the virus among flies. DNA was extracted using the Puregene DNA purification kit . Flies were screened for the virus using standard insect COI primers (1490 and 2198) 5′–TGAAACCAGAATGACATATATAACGC and P47R: 5′–TCGGTTTCTCAATTAACTTGATAGC) for the P47 homolog found in the metagenomics search. For each species, the P47 locus from at least one individual was sequenced using BigDye Terminator v3.1  and deposited in GenBank (Accession numbers JN44311–JN44330). These sequences were used to build a phylogenetic tree using PhyML P47 ortholog from OrNV was used as an outgroup.After discovery of a putative DNA virus see , severalOryctes rhinoceros Nudivirus D. falleni and then PCR screened for the virus, using the methods described above. These samples were almost invariably positive for the virus, so fecal material was primarily used for imaging. To concentrate the virus on a microscope slide, a crowded vial of infected flies was inverted on a microscope slide and flies were allowed to defecate for 4 d. A small sample was scraped and PCR screened for the virus. An attempt was made to find virus particles in whole flies by dissecting out the digestive tract for imaging.Because transmission of the DNA virus may be fecal-oral, as hypothesized for the closely related The glass slide with deposited feces was fixed in 0.1 M sodium cacodylate buffered 2.5% glutaraldehyde for 24 hours and post-fixed in 1.0% buffered osmium tetroxide for 20 min. The slide was transitioned through a graded series of ethanol to 100% (×3) and infiltrated with Spurr epoxy resin overnight. The next day, size 3 BEEM capsules were filled with fresh resin and inverted and placed onto the glass slide over the fecal matter. The slide was placed into a 600°C oven and allowed to polymerize overnight. The polymerized BEEM capsules were removed from the glass slide using the “pop-off” technique Wolbachia. In September 2009, flies were captured near the Southwest Research Station as described above and transferred to sugar agar for transport to Rochester, NY, which took ∼6 d. In Rochester, flies were placed on mushroom food  for egg laying. Females were then moved to new vials every other day until they died. The experiment lasted for 55 days, at which time no flies were laying fertilized eggs. Females were screened for the DNA virus and their offspring were counted as they emerged. In July and August 2010, the same protocol was followed for D. falleni. In this case, flies were collected around Rochester, NY and were established in culture the day they were collected, but the experiment was terminated after 10 days. As described above, flies from the 2010 D. innubila collection were dissected on site and mature eggs (stage 10 or later) in both ovaries were counted to assess fitness costs associated with the virus. All flies were PCR screened for the DNA virus and, as a control, insect COI.Wild-caught females were used to assess the survival and fecundity of flies as a function of infection with virus and D. innubila females were homogenized individually in viral buffer (see above) and centrifuged at low speed for one minute to remove large fly debris. The supernatant was then spun through a 0.45 µm filter . 10 µl of the filtrate for each sample was used to PCR screen for the virus, using the DNA extraction and PCR protocols as described above. The filtrate from 4 flies identified by PCR as positive for DiNV infection were pooled as the virus-positive, and 4 flies screening negative for DiNV were pooled as the virus-negative control. Each sample was diluted 1∶10 before being used for injection. A separate control, sterile virus buffer was also employed. Six- to eight-day-old male and female D. innubila and D. falleni were injected with about 200 nL of one of the three treatments using a Narishige IM300 microinjector . Fly survival was monitored daily and flies were kept at low density  and transferred to new food every other day until most flies injected with the virus-positive filtrate were dead. Both the virus-positive and virus-negative samples were examined using electron microscopy to be sure a) only DiNV was present in the virus-positive samples and b) no bacteria were present in either sample.To directly assess the fitness consequences of viral infection, flies were injected with live virus and survival was monitored. Since cell culture for this virus has not yet been established, live virus was isolated as follows. Several wild D. innubila mothers found to be infected with the DNA virus, offspring were PCR screened to assess vertical transmission of the virus. Offspring were frozen 2–6 d after emergence, and DNA was later extracted and screened as described above. A total of 27 daughters and 2 sons of were screened from 10 virus-infected females. The low offspring production of virus-infected females limited the sample size and male-killing by Wolbachia resulted in the highly skewed sex-ratio.For a subset of wild-caught Drosophila are listed as threatened or endangered.All necessary permits were obtained for the described field studies. Since it involved protected lands, flies collected in the Chiricahua Mountains were collected with permission of the National Forest Service . Collections in the Chiricahua Mountains in 2010 were conducted on the grounds of the Southwest Research Station with permission from the station director, Dawn Wilson. Permits were not required for collections around Rochester, NY and all collections were performed on the property of the author and principal investigator, John Jaenike. Flies collected in Germany were processed and sent as DNA by a third party. No species of Wolbachia uninfected sample and 44,675 raw reads and 124 contigs (using 5939 raw reads) were recovered for the Wolbachia infected sample. Raw reads averaged 320.3 bp for the uninfected sample and 394.9 bp for the infected sample.A total of 1225 raw reads and 25 contigs (using 1154 raw reads) were recovered for the Wolbachia-infected sample was spiked with 1 fly that had been injected with Drosophila C virus (DCV). Two-thirds  of reads with significant hits in the infected group had DCV as the best hit, as opposed to less than 1% (9 reads) in the uninfected group, demonstrating both the sensitivity and specificity of the method. The All virus hits are summarized in Drosophila melanogasterDrosophila innubila.Three viruses in the list stand out. First was DCV, which was expected to be present in large quantities. The second most common virus hit was to Cricket paralysis virus (CrPV), which is closely related to DCV and therefore may represent poor quality reads that were actually DCV. However, CrPV has a broad host range and can infect Wolbachia-uninfected and Wolbachia–infected samples, respectively) had Oryctes rhinoceros Nudivirus (OrNV), a double-stranded DNA virus, as the best hit. Narrowing the search database to just viruses did not increase the number of sequences hitting OrNV, but searches restricted to Nudiviruses increased this to 31 and 7 . OrNV is a member of the small and unclassified Nudivirus group Drosophila species, including members of 3 subgenera, from both northeastern and southwestern United States, but is not found in a small sample of D. phalerata from Europe . A phylogenetic tree of P47 sequences from each species is presented in Drosophila simulans male and a Drosophila melanogaster or simulans female (both from the subgenus Sophophora) were identical, but were nested within sequences from other members of the subgenus Drosophila.DiNV virus is present in several m Europe . The preThe fecal material contained numerous virus particles morphologically similar to Baculoviruses and Nudiviruses . The capD. innubila collected in 2009, those infected with DiNV had significantly decreased lifespan . To assess survival after injection with the three treatments  a Cox proportional hazard model was used with treatment, species and sex as factors. Flies injected with the virus-negative filtrate experienced similar mortality to those injected with virus buffer (P = 0.44), suggesting that nothing in the virus-negative filtrate significantly affected fly mortality. This was further supported by the absence of any recognizable structures  in the electron micrographs of the virus-negative filtrate. Flies injected with the virus-positive filtrate experienced much higher mortality than those injected with the virus-negative filtrate . There was a moderately significant interaction (P = 0.06) between sex and treatment with males injected with the virus-positive filtrate surviving several days longer than females  than D. innubila regardless of treatment or sex.After injection, fly survival was monitored daily until most flies injected with the virus-positive filtrate had died .DiNV is associated with greatly reduced survival and offspring production among wild-caught individuals of D. falleni survived longer than D. innubila. The difference between species may be due to size differences in flies , or could reflect selection for increased virulence of the virus to its natural host, since the virus injected was from D. innubila. The difference between the sexes cannot be explained by size since males are smaller and survived longer. Interestingly, overall and in the 2010 D. innubila collection, males had higher rates of infection than females , it is probably safe to conclude that DiNV is not vertically transmitted and infection frequencies are high enough that infection via mites (which are found at relatively low frequency on D. innubila), the natural analog of microinjection, is unlikely. Therefore, DiNV appears to be a virus exhibiting high virulence without requiring a rather drastic injection to show such effects.Most well-studied viruses of Drosophila opens the way for study of host immune response to DNA virus infection in an easily cultured species. The system also lends itself to studies of host-pathogen coevolution between geographically isolated sister species and between semi-isolated Sky Island populations of D. innubila.The discovery of a DNA virus that naturally infects Material S1Sequences for short DiNV orthologs of other Nudiviruses. Note these are too short (<200bp) to be published in Genbank.(DOC)Click here for additional data file.Figure S1Schematic of viral nucleic acid enrichment protocol. Note there is no DNA digestion during the RNA extraction.(EPS)Click here for additional data file.Figure S2Distribution of BLAST hits corresponding to Drosophila C virus according to position in the genome.(PNG)Click here for additional data file.Figure S3Drosophila species collected near Portal, AZ (blue) and Rochester, NY (red). Branch labels are posterior probability/maximum likelihood bootstrap support.The phylogenetic relationships of DiNV isolated from (PDF)Click here for additional data file.


Several previous global REDD+ cost studies have been conducted, demonstrating that payments for maintaining forest carbon stocks have significant potential to be a cost-effective mechanism for climate change mitigation. These studies have mostly followed highly aggregated top-down approaches without estimating the full range of REDD+ costs elements, thus underestimating the actual costs of REDD+. Based on three REDD+ pilot projects in Tanzania, representing an area of 327,825 ha, this study explicitly adopts a bottom-up approach to data assessment. By estimating opportunity, implementation, transaction and institutional costs of REDD+ we develop a practical and replicable methodological framework to consistently assess REDD+ cost elements.xxxx for deforestation and forest degradation drivers such as agriculture, fuel wood production, unsustainable timber extraction and pasture expansion. The mean opportunity costs for the three projects ranged between US$ 10.1 – 12.5 tCO2. Implementation costs comprised between 89% and 95% of total project costs (excluding opportunity costs) ranging between US$ 4.5 - 12.2 tCO2 for a period of 30 years. Transaction costs for measurement, reporting, verification (MRV), and other carbon market related compliance costs comprised a minor share, between US$ 0.21 - 1.46 tCO2. Similarly, the institutional costs comprised around 1% of total REDD+ costs in a range of US$ 0.06 – 0.11 tCO2.Based on historical land use change patterns, current region-specific economic conditions and carbon stocks, project-specific opportunity costs ranged between US$ -7.8 and 28.8 tCOThe use of bottom-up approaches to estimate REDD+ economics by considering regional variations in economic conditions and carbon stocks has been shown to be an appropriate approach to provide policy and decision-makers robust economic information on REDD+. The assessment of opportunity costs is a crucial first step to provide information on the economic baseline situation of deforestation and forest degradation agents and on the economic incentives required to halt unsustainable land use. Since performance based REDD+ carbon payments decrease over time (as deforestation rates drop and for each saved ha of forest payments occur once), investments in REDD+ implementation have a crucial role in triggering sustainable land use systems by investing in the underlying assets and the generation of sustainable revenue streams to compensate for opportunity costs of land use change. With a potential increase in the land value due to effective REDD+ investments, expenditures in an enabling institutional environment for REDD+ policies are crucial to avoid higher deforestation pressure on natural forests. In the process of developing national REDD+ frameworks, investors, donors and policymakers are highly interested in cost information in order to develop strategies, allocate budgets, and to assess the effectiveness of green house gases (GHG) emission reductions from avoiding deforestation and forest degradation, conservation, sustainable management of forests, and enhancement of forest carbon stocks in developing countries .Several REDD+ cost studies have been conducted -6 assert2 (median US$ 3.90 tCO2), indicating the high variability of opportunity costs within the country. Implementation costs for avoiding GHG emissions through investments in doubling agricultural output and in more efficient charcoal production exceeded opportunity costs, and ranged between US$ 1.63 – 17.05 tCO2 (median US$ 6.52 tCO2). It was concluded that the opportunity and implementation costs of avoiding GHG emission from REDD+ are likely to exceed estimates generated from previous global top-down models [In a previous study conducted in 2011, REDD+ opportunity and implementation costs for Tanzania were estimated using a bottom-up approach by taking into consideration regional variations at district-level . Opportun models . BuildinThe land use classification in the pilot project areas is presented in Table 2/ha, while in Kilosa and Lindi carbon stocks are almost twice as high at 145.3 tCO2/ha and 158.8 tCO2/ha respectively. With deforestation of one ha in the JGP, between 50 and 65 tCO2 are emitted, while in the TFCG Kilosa and Lindi projects the mitigation potential of one ha is significantly larger at 129 tCO2 and 105 tCO2, respectively.All three projects plan to account, monitor and receive payments only for avoided deforestation from aboveground and belowground biomass carbon pools. Table Following the methodological approach as described in the methods section, Table The NPV for natural forest in the JGP (U$ 924/ha) is significantly higher than in the TFCG / Mjumita Lindi and Kilosa projects (both US$ 95/ha). This difference is mainly due to the significantly larger use of non-timber-forest products and better marketing opportunities. In our modelling of unsustainable timber production for the JGP, which has led to deforestation and forest degradation over the past 10 years, we estimated that the forest is used for timber extraction over a period of seven years with an available extraction volume of 5.9 m³/year and a commercial recovery rate of 30%, sold at an average of US$ 112.5/m³ at the farm-gate. Subsequently the land is converted to agricultural land for shifting cultivation with four years of cultivation and 12 years of fallow period resulting in an NPV of US$ 1,687/ha. Unsustainable fuel wood collection, estimated assuming an annual average extraction rate of 3.6 tdm/ha over 30 years , results in a low NPV of US$ 533/ha mainly due to the low market prices (US$ 20/m³) for fuel wood in this region and the high level of household self-consumption, which is valued in the model. Agricultural land use in the JGP has a significantly greater NPV  than in Kilosa  and Lindi  projects, mainly due to higher productivity of the land and its proximity to the border with the Democratic Republic of Congo, which provides good market access. Regarding unsustainable charcoal land use in the Kilosa and Lindi projects, we assumed that one ha under deforestation and degradation pressure is cleared for charcoal production within one year extracting about 28 tdm/ha. Assuming a mean charcoal kiln efficiency of 19% ,17, this2 over 10 years , and the opportunity costs of land use change are negative (US$ -7.8 tCO2) for unsustainable fuel wood collection and US$ 7.3 tCO2 for avoiding the expansion of cattle grazing. The negative opportunity cost indicates that in the long-term, maintaining natural forest would result in a higher NPV than the NPV of unsustainable fuel wood collection and at the same time higher carbon stocks on a unit of land would be achieved in the long-term.Based on the aforementioned historical land use change patterns, carbon stock and economic data, the opportunity cost curve of avoiding the conversion of natural forest for the three pilot projects is presented in Figure 2  at costs of US$ 15.1 tCO2 for unsustainable timber extraction and US$ 28.8 tCO2 for avoiding conversion undershifting cultivation. The Kilosa and the Lindi projects have a large mitigation potential from avoiding the expansion of shifting cultivation and unsustainable charcoal production. In the Kilosa project opportunity costs for shifting cultivation amount to US$ 8.8 tCO2, while for unsustainable charcoal production opportunity costs are US$ 12.1 tCO2. In total, avoiding conversion of natural forest to these land uses has the potential to reduce emissions by 1.19 MtCO2 over 10 years . Similarly, in the Lindi project the avoidance of land use change to shifting cultivation and unsustainable charcoal production incurs opportunity costs of US$ 8.9 tCO2 and US$ 11.4 tCO2 respectively, and could reduce emissions by 1.76 MtCO2 over a period of 10 years .The two most expensive options are avoidance of unsustainable timber extraction and shifting cultivation. Together these two options have a total emission reduction potential of 488,791 tCO2, while the TFCG / Mjumita projects have lower opportunity costs at US$ 10.1 tCO2 and US$ 10.3 tCO2 for the Lindi and Kilosa sites, respectively.Taking the mean opportunity costs of each project, the JGP has opportunity costs of US$ 12.5 tCOFor the quantification of the implementation and transaction costs of the REDD+ pilot projects, we used available REDD+ project budgets. In our estimates we assume that no leakage effects will occur and that opportunity costs will be compensated by the project interventions or performance based payments. Implementation costs comprised between 89% and 95% of total project costs (excluding opportunity costs), ranging between US$ 7.9 ha/year (JGP), US$ 5.8 ha/year Lindi project and US$ 3.7 ha/year in the case of the Kilosa project Figure . Table 4Transaction costs for measurement, reporting and verification (MRV) and other carbon market related compliance costs ranged between US$ 0.9 ha/year for the JGP, US$ 0.3 ha/year for TFCG / Mjumita – Lindi and US$ 0.2 ha/year for the TFCG / Mjumita – Kilosa project. These implementation and transaction costs will be covered by the project development entities.In our study we assume that institutional costs are partly covered by the projects and partly by the government. One part of the institutional costs are comprised of a national average value of US$ 0.017 ha/yr, which is based on the budgets of the Forest Carbon Partnership Facility Readiness Preparation Proposal of Tanzania . These cThe other part of the institutional costs, to be covered by the pilot projects, are budget items related to institutional capacity building for district level governmental staff and knowledge dissemination of lessons learned which is an additional activity to build and strengthen REDD+ relevant institutions and capacity in Tanzania. The estimated institutional costs did not differ significantly among projects and amounted to US$ 0.07 ha/year for the JGP, US $0.07 ha/year for the TFCG / Mjumita - Lindi project and US $0.05 ha/year for TFCG Mjumita – Kilosa Figure .2/yr (1.65 MtCO2 over 30 years) while avoiding deforestation on about 20,000 ha, which is equivalent to 68% of the project’s reference emission levels. For the TFCG / Mjumita Kilosa project, emissions reductions will average 102,000 tCO2/yr (3.06 MtCO2 over 30 years), equivalent to the avoided conversion of 23,700 ha, and an 86% reduction compared to the reference emissions level. In the Lindi project, the total emissions reductions are projected to be about 120,000 tCO2/yr on average (3.6 MtCO2 over 30 years) which is equivalent to avoided deforestation and forest degradation on 34,340 ha and 68% of the reference emissions level. Assuming that the entire project implementation will be paid by carbon payments, in the JGP the total cost of avoiding the release of one tCO2 would be US$ 13.8, while the TFCG / Mjumita projects cost between US$ 4.8 (Lindi) and US$ 5.7 (Kilosa)  of a particular REDD+ activity location. A review of 29 empirical studies identified opportunity costs of REDD+ in the range of US$ 0.84 – 4.18 tCO2 with a mean of US$ 2.51 tCO2[2 with a mean of US$ 2.22 tCO2 for Africa. However, the studies quantified only opportunity costs and did not take into account other cost elements associated with implementation, measurement, reporting and verification, and institutional arrangements within REDD+ frameworks [2/ha) resulting in a global estimate of opportunity costs (US$/tCO2) [2 (midpoint: US$ 5.52 tCO2). The study considered only opportunity costs and administration costs of REDD+ and used a mean forest carbon density of 390 tCO2/ha. This approach ignored the significant variations in carbon densities between regions and led to highly aggregated estimates with a large uncertainty level. Global simulation models such as Global Timber Model (GTM), Dynamic Integrated Model of Forestry and alternative Land Use (DIMA) and Generalized Comprehensive Mitigation Assessment Process Model (GCOMAP), estimate REDD+ costs by simulating the development of the world economy taking into account the forestry, agricultural and fossil fuel using energy sectors [2). These models mainly also focus on opportunity costs without taking into account other cost elements of REDD+. A published study has estimated the costs of reducing global deforestation by 50% by 2030 to be in the range of US$ 9.27 – 20.57 tCO2. Reducing African forestry related GHG emissions by half was estimated to cost between US$ 5.20 - 12.3 tCO2[In general there are three different approaches to estimate cost elements of REDD+, i.e. local empirical models, global empirical approaches and global simulation models ,19. The S$/tCO2)  who esti sectors . The res12.3 tCO2.2). Comparing these estimates with data from Tanzania [2 with a median US$ 3.90 tCO2 for Tanzania.Opportunity costs are likely to vary significantly from place to place, depending on several factors such as agro-ecological conditions, climate, market access, scale of operation, inputs, technology and yields, so highly aggregated average values may be misleading ,22.This Tanzania  where op2/ha  were used. These are significantly higher than our reported project-specific carbon densities that range between 80.6 and 158.6 tCO2/ha  over a period of 30 years. Comparing with other global REDD+ implementation cost studies, Nepstad et al. [2 in the Amazon region based on historical data. Grieg-Gran [2. However, these costs only considered the management of PES schemes and did not take into account additional investments in changing land use management. Thus, direct comparisons cannot be made and provide only a highly uncertain indication of actual REDD+ implementation costs. As an example from Tanzania, implementation of activities for agricultural intensification, improved charcoal production and improved energy efficiency were identified as the major strategies to avoid emissions from deforestation and forest degradation [2, with opportunity costs at US$ 1.90 – 13.40 tCO2. These estimates can be directly compared with data from the three REDD+ pilot projects described in this paper, that have implementation costs of US$ 4.5 - 12.2 tCO2 with mean project opportunity costs of US$ 10.1 – 12.5 tCO2. Our results indicate a close relationship between the level of opportunity cost and implementation cost that should be estimated in economic analyses of REDD+ to inform decision-makers of the potential cost-effectiveness and scale of REDD+ investments. Higher opportunity costs tend to have correspondingly higher implementation costs, which can be explained by the higher level of investment requirements to offset opportunity costs. Implementation of activities that focus on addressing the drivers of deforestation and forest degradation by increasing the profitability of one particular area of land, such as sustainable intensification of agriculture, may lead to the compensation of opportunity costs of land users with limited access to land from improved, more efficient and profitable production on a per ha basis. However, on a landscape level the opportunity costs may rise as the land profitability from alternative land uses increases, and this may even increase the pressure for deforestation [The implementation costs of the three pilot projects range between US$ 3.7 - 7.9 ha/year . Overall transaction costs are normally relatively scale invariant as project development entities normally have to invest into the establishment of a project-specific MRV system, undertake certification by internationally recognised standards and design and negotiate contracts [2 basis due to the distribution of costs over a larger amount of project area and carbon benefits [2 for large projects to US$ 4.05 for smaller projects with a weighted average of US$ 0.26 tCO2 for all projects [2 ranging between US$ 0.66 – 16.4 tCO2[2 ranging between 0.5% and 20% of projects’ total investments [In our estimates, REDD+ transaction costs for MRV and other carbon market related compliance costs ranged between US$ 0.2 and 0.9 ha/year which is equivalent to US$ 0.21 (Kilosa) - 1.46 tCOontracts . Therefo16.4 tCO2. The Worestments .2[These cost estimates are in-line with our project-specific estimates and it can be concluded that REDD+ transaction costs tend to increase with the reduction of the project area and carbon benefits due to economies of scale -34. Cons2.In previous studies, institutional costs were not explicitly estimated or described. Administrative and implementation costs are likely to comprise institutional costs. A strict differentiation between institutional costs and implementation costs is very difficult to achieve, since in some stages costs could be assigned either to implementation or institutional costs. Moreover, national expenditures for forest management may reflect the business as usual scenario and are not additional REDD+ investments, and thus cannot be reliably accounted for. Therefore, the biggest challenge to estimating institutional costs is the determination of incremental costs related to the design and implementation of a REDD+ scheme. Institutional costs of REDD+ are defined in the context of this study as costs incurred at the political-administrative level to develop, manage and enforce REDD+ policies and measures. These are typically costs incurred by the government to ensure a positive legal and regulatory environment, address governance issues and reduce unregulated / illegal forest use. Our institutional cost estimates on the national level of US$ 0.017 ha/year provides an initial benchmark on institutional REDD+ costs at national level. However, since Tanzania is still in the development of the REDD+ implementation framework it is likely that additional institutional costs will arise compared to those currently budgeted. Therefore our estimates tend to underestimate the actual institutional costs which will need to be further updated once the REDD+ implementation framework is operational.Global and national REDD+ cost assessments have mostly been conducted in moist tropics with high forest density and using highly aggregated data in REDD+ countries such as Brazil, Indonesia, Democratic Republic of Congo, Ghana, and Cameroon. These aggregated estimates do not reflect the economic conditions, regional variations and forest carbon densities of dry forest ecosystems such as are prevalent in Tanzania, and tend to underestimate the actual costs of REDD+ in these regions. The focus on opportunity costs of REDD+ is only one crucial part of REDD+ economics providing information on the economic baseline situation of deforestation and forest degradation agents and on the required economic incentives to halt unsustainable land use. However, since performance-based REDD+ payments are expected to decrease over time (as deforestation rates drop and because payments for each saved ha of forest occur only once), these agents will require new revenue streams that compensate their opportunity costs and help to maintain natural forests in the long-term. Therefore investments into implementation of REDD+ activities comprising about 89 - 95% of total costs (excluding opportunity costs), must result in a more sustainable land use systems that the returns exceed the opportunity costs. Thus investments in REDD+ implementation paid for by decreasing performance-based carbon payments have a crucial role in triggering sustainable land use systems by investing in the underlying assets of the project. Focusing only on carbon payments bears the risk of temporally shifting unsustainable land use without addressing the underlying causes of deforestation and forest degradation. With the increased profitability of land use due to investments in effective REDD+ implementation, investments in institutional costs must create an enabling environment for REDD+ and policies that do not impose more pressure on natural forest, e.g. through increased profits from agricultural intensification.Compared to top-down REDD+ cost assessments, bottom-up approaches to estimating REDD+ economics by considering regional variations in economic conditions is a more appropriate approach to provide policy and decision-makers robust economic information on which REDD+ decisions, policies, strategies and investments should be based on. Estimating the full range of REDD+ costs, including opportunity costs, implementation costs, transaction and institutional costs, must be complemented by the development and estimation of potential revenues to assess the potential effectiveness of REDD+ investments. This will provide data for the design of marginal abatement cost curves (MACC) as a robust economic decision-making tool which easily communicates information to multiple stakeholders on the mitigation potential and the incremental cost of different mitigation practices . Beyond th of October 2011 in Dar es Salaam (Tanzania).As part of the Tanzanian national REDD+ strategy, nine REDD+ pilot projects  have beeData collection was conducted through project visits and the collection of project related available information. Project information included socio-economic studies, analyses of the drivers of deforestation, documented project-specific carbon assessments, project funding proposals and existing project budgets and expenditure data. Missing information required for a complete REDD+ cost assessment was complemented by local expert estimates for the respective pilot project. Expert estimates made by the authors were verified by the local project staff members.Since cost definitions often vary among different REDD+ cost assessments, we defined the four REDD+ cost elements following Pagiola et al. and White et al. ,38.2. Opportunity costs are profits achieved by continuing “business as usual” and depend on the drivers of deforestation and forest degradation. Estimating these costs is also critical in order to understand the causes and the motivations of deforestation agents to deforest, and are a part of each REDD+ pilot activity nationally and sub-nationally.Opportunity costs are often the largest element of REDD+ costs. These equate to the costs of foregone benefits  incurred by retaining existing forest land that could have been otherwise used for the economic activities. REDD+ opportunity costs are the difference in net earnings from conserving or enhancing forests and the earnings from converting them to alternative land uses. These costs are compared with potential benefits of maintaining the forests and carbon, for instance, by conserving the forest or extracting non-timber forest products. Opportunity costs can be quantified in terms of monetary and physical units, such as US$ per tCOImplementation costs are defined as the costs and investments required to implement REDD+ activities and to avoid and minimize leakage effects. This includes, for instance the costs of guarding a forest to prevent illegal logging, costs of intensifying agriculture or pasture management, or improvement of energy efficiency so that people are not forced to collect fuel wood and produce charcoal for subsistence uses. Implementation costs also include capacity building of the project team to enable them to effectively manage and implement projects, costs of land use planning and other related activities required to achieve reduce emissions from deforestation and forest degradation.Transaction costs are expenditures that are required to realise a transaction involving a REDD+ payment  as well as external parties such as market regulators or payment scheme administrators . REDD+ tInstitutional costs are costs related to the implementation of conventions, norms and legal rules of a REDD+ framework so as to make REDD+ operational, including the costs of building institutions (and their capacity), strengthening land tenure, building an enabling policy environment. This includes expenditures associated with training, research, policy design, legal and regulatory processes, law enforcement as well as national stakeholder consultations and decision making costs. These costs may be very difficult to estimate but have to be taken into account ,22. For 2/ha). For each pilot project, we developed a standardized “REDD+ cost elements” analytical excel-based tool to separately estimate the opportunity costs, implementation, transaction and institutional costs. The tool is based on the methodological approach described in “Estimating the Opportunity Costs of REDD+ − A training manual” [Conceptually, this study estimates the REDD+ cost elements based on a project specific empirical approach using project-specific per-area estimates (US$/ha) and carbon density estimates  (3) and estimation of one ha economic models of the land uses (NPV in US$/ha) (4). Quantitative data from these steps was input into the Abacus software to generate opportunity cost curves [Estimation of opportunity costs was conducted in four iterative steps, including classification of current land use types in the project area (ha) (1), an analysis of historical spatial land use change patterns and trajectories (ha) (2), estimation of mean carbon stocks of the identified land use types  of a particular driver of deforestation and forest degradation, while Cnat is the mean carbon stock of of natural forest, expressed in tCO2/ha. The calculation of opportunity costs was conducted only for land that has been converted from natural forest to alternative land uses over the past ten years, as described in the results section (REDD+ opportunity costs curve). The mean opportunity costs of each project were calculated by weighting the relative contribution of each driver of deforestation and forest degradation to the total forest loss in the project area.Where OCIn the classification of land use types, each ha of the total project area must be assigned to a land use associated with a particular economic activity. In the three analyzed REDD+ pilot project, land uses such as natural forest, unsustainable charcoal production, unsustainable fuel wood collection, unsustainable timber extraction, agricultural (shifting cultivation) land uses and pastoral cattle grazing were identified. Unsustainable land uses are characterized by extraction rates that would not have been able to sustain the prevalent practices over 30 years, thus leading to deforestation and forest degradation.2/ha). The long-term mean carbon stock reflects the equilibrium carbon stock of a particular land use. If land use is converted to another land use, we assume that carbon is emitted or sequestered immediately. In our study we considered forest carbon only from aboveground and belowground carbon pools, due to the fact that the projects are not planning to measure other carbon pools and receive payments related to these pools. Where natural forest land was comprised of more than one forest type with differing carbon stocks, we used area-weighted mean carbon stocks in which the carbon stocks of each forest type were multiplied by the area of the respective forest type and subsequently divided by the total natural forest area.Each identified land use type is assigned a long-term mean carbon stock value (in tCOThis step included a quantitative estimation of the project specific conversion of forest land to other identified land uses associated with a particular economic activity that has led to deforestation and forest degradation (in ha) Table . In our em are the total GHG emissions from conversion of natural forest to a particular land use  of net revenue streams associated with each land use as an economic indicator to estimate the profitability of land use. The NPV is the result of a Discounted Cash Flow analysis (DCF) of the costs and benefits for a certain land use over period of 30 years using a discount rate of 10%. Since our estimates are based on a period of 30 years and unsustainable timber extraction, charcoal production and fuel wood collection cannot be sustained over such a long time period, our model allowed a transition to another land use such as agriculture or pastoral use depending on local circumstances after the wood resource base would have been depleted, and this net revenues from this subsequent land use also contributed to the estimated NPV.For the estimation of implementation and transaction costs we used existing REDD+ project documents that included budgets and expenditures over a period of three to five years and that were extrapolated over the project lifetime of 30 years using expert judgement. For the identification of this comprehensive set of cost elements and for the verification of expert judgement, we worked in close cooperation with project development and implementation entities. The assignment of each budgeted or already spent money to the respective cost type was based on the REDD+ cost element definition as elaborated in the background section. Due to the lack of data and existence of solid revenue models, in the project scenarios we did not take into account potential non-REDD+ revenue streams. Therefore we assume that all costs are covered by potential carbon payments and estimate the required carbon price to cover these costs.For the determination of institutional costs we took into account only incremental REDD+ costs taken from the Tanzania’s REDD+ readiness and implementation plan prepared under the framework of the World Bank Forest Carbon Partnership Facility (FCPF) and UN-REDD programme budgets between 2010 and 2013, which amounted to US$ 3.47 million . SubsequWe did not add up the existing forestry related budget and expenditures of governmental institutions, as we consider these business-as-usual that would have occurred without a REDD+ framework anyway. Therefore our estimates include only REDD+ specific “additional” costs. In the framework of this study, “additional” costs are considered as expenditures being spent in the framework of national REDD+ Readiness and Implementation activities financed by the UN-REDD Programme, FCPF, Norway-Tanzania Climate Change Partnership and by the government of Tanzania .Furthermore for all projects, we assigned a small portion of the budget to local-level institutional capacity building and REDD+ knowledge dissemination, which we considered as an institutional cost according to the definition elaborated in the background section. These costs will be paid from project budgets. Finally we summed the national level and project level institutional costs.2 (US$/tCO2) we used the projected GHG emissions reductions from the avoidance of deforestation and forest degradation. Based on these estimates we divided the total implementation, transaction and institutional costs over a period of 30 years by the total projected GHG emissions reductions in the project scenario.For the estimates of REDD+ cost elements on a per ha basis (US$/ha) we divided the annual average implementation, transaction and institutional costs of the pilot projects by the total project area. For the estimates of the costs per avoided tCOREDD+ cost curves and estimates reflect only a static snapshot of one period of time and the current economic and political conditions prevalent in the project regions, and do not reflect potential future developments . ParticuAnother uncertainty of the REDD+ cost estimate lies in the future projection of project investments and estimates of the future GHG emissions reductions that will be actually achieved. It remains unclear whether the budgeted investments will actually result in the expected carbon savings, thus the effectiveness of REDD+ project investments still has to be proven by the successful implementation of the projects, supported by an enabling environment of the national REDD+ framework.The authors declare that they have no competing interests.EM designed the analytical REDD+ cost element tool, undertook the pilot project analysis, conducted interviews with local experts of the respective pilot project together with CH and drafted the manuscript. CH co-authored the manuscript. CH was mainly responsible for data collection, communication with the project development and implementation entities and quality control. TT and TB were responsible for review of the manuscript, provision of comments and quality control. All authors read and approved the final manuscript.EM works as a land-based climate finance project developer and analyst at UNIQUE forestry and land use. CH is Managing Partner of UNIQUE forestry and land use, heading the Forest Economics Division. TTe is Managing Director of UNIQUE forestry and land use and head of the Climate Division. TB is a freelance consultant regularly working for LTS International (UK). From 2003 to 2008 he worked as senior participatory forest management adviser for the Government of Tanzania within the Forest and Beekeeping Division.


We studied eight children who had measles at 6 to 10 months of age during the 1998 Argentine measles outbreak and in whom subacute sclerosing panencephalitis developed 4 years later. We report the genetic characterization of brain tissue–associated measles virus samples from three patients. Phylogenetic relationships clustered these viruses with the wild-type D6 genotype isolated during the 1998 outbreak. The children received measles vaccine; however, vaccinal strains were not found. Measles is often incorrectly regarded as a mild disease, and priority is not given to measles elimination programs in some countries  is not highly neurotropic, it can establish long-term persistent infection in brain cells. Three different neurologic complications result from interactions of measles virus with neural tissue. Acute postinfectious encephalitis, usually appearing 5–14 days after the rash, is thought to be a virus-induced autoimmune disease. Measles inclusion–body encephalitis, which occurs in immunocompromised patients after a latent period of 3–6 months, is believed to be a direct measles virus infection of neural tissue. The third form, subacute sclerosing panencephalitis (SSPE), manifests 2–10 years after primary measles infection as a progressive and fatal chronic neurodegenerative disease caused by persistent defective measles virus in neurons and oligodendrocytes. SSPE develops with high titers of anti-measles antibodies both in cerebrospinal fluid (CSF) and serum, which seem unable to eliminate measles virus from the brain  changes, raised CSF globulin levels without pleocytosis, raised CSF measles antibody titers, and typical histopathologic findings in brain biopsy materials (Antimeasles immunoglobulin (Ig) G antibodies in CSF and serum samples were assessed by using an automated qualitative enzyme-linked fluorescent immunoassay  and a quantitative indirect immunofluorescence test . In three patients, measles virus RNA was isolated from a punch of white matter obtained in the course of Ommaya reservoir implantation at SSPE onset. RNA was purified by the guanidinium-thiocyanate-phenol-chloroform method, and genetic characterization of measles virus was performed by sequencing the 450 nt from the carboxyl terminus of nucleoprotein (N) gene and a 377-bp fragment of the H gene  but also more replacement changes (18 vs. 12) and a higher κ ratio (2.1 vs. 1.2) as compared to H gene. However, we compared the ω ratio and found it to be higher for H gene (1.7 vs. 1). Fixed replacements were detected at position 357 in H protein and at position 467 in N protein. Variability was detected in both genes. Changes found in SSPE samples related to D6 genotype, and the consensus sequence from last Argentine outbreak are summarized in Distance methods were applied, and the matrix rendered similar results for both analyzed genes. The tree was built with measles virus genotype reference strains and Argentine strains previously characterized from 1991 and 1998 outbreaks. Phylogenetic relationships clustered the three SSPE strains with the wild-type D6 genotype. SSPE samples were strongly associated with wild-type D6 samples from the 1998 outbreak, supported by a bootstrap value of 100 out of 100 pseudoreplicates done. Divergence from genotype D6 (NJ-1 strain) was <2.2% in the N gene and <2.4% in the H gene. The unrooted tree for the carboxyl terminus of N gene was plotted .The measles vaccine was included in the regular immunization schedule in Argentina in 1978. Despite vaccination, several disease outbreaks have occurred  had a nonconservative V357S change at the same position (from hydrophobic to polar without charge)  and lower ω , and SSPE was not detected among patients >1 year of age at the time of acute infection; however, underdiagnosis is a permanent challenge. Time lag between acute and SSPE onset was approximately 4 years, according to the reported data, but the rate for developing SSPE was higher than previously described (Phylogenetic analysis of three SSPE cases from the last outbreak clustered with D6 genotype that circulated in Argentina in 1998. Although the original sequence of the wild-type virus that caused acute infection is unknown, we have a consensus sequence that summarizes the outbreak; therefore, we infer that changes may have occurred since then and contributed to the development of SSPE. Although all three patients had been immunized according to the schedule, vaccinal strains were not detected in brain tissue. These results agree with those recently reported for SSPE patients in the United Kingdom and Papua, New Guinea (


To the Editor: AbuBakar et al. described strain variation in Nipah virus during the 1998–1999 outbreak in Malaysia , which is consistent with the occurrence of human cases in Perak before the 1998–1999 outbreak. Evolution of the virus population in pigs, followed by the reintroduction in Perak of the original strain from bats and its subsequent southward movement in infected pigs, would explain observed strain differences. Models suggest that evolution of the virus within pig populations would result in lower death rates but prolonged illness. Although the pig-adapted virus strain may have circulated on both northern and southern farms, sampling biases in favor of the more virulent strain would be expected in areas of high death rates, which would explain the observed genetic relationships between sequenced isolates.We suggest that pigs be experimentally infected with the Perak strain of Nipah virus to determine whether differences exist in illness and death caused by this virus. Further sequencing of virus from archived pig samples will clarify with greater confidence whether multiple strains circulated in both regions. In response: Pulliam et al. (While the model is plausible, that NV infection was endemic among pigs before the 1998 outbreak is difficult to imagine in the absence of verifiable evidence. As with any virus that crosses species, NV would likely have caused severe infection, and what happened in 1998 is a classic example. Before NV could have evolved, become less virulent, and subsequently become endemic, it would have been first introduced to pigs. This initial introduction would have caused an outbreak, but no such outbreaks were reported before 1998. Furthermore, the life span of pigs reared in farms is relatively short before they are sent to the markets, which limits the time in which NV evolution could take place. Slaughtering these pigs would also have caused infection among abattoir workers and pork handlers. At present, the finding of 2 different NV strains from 2 different outbreak foci favors the suggestion that 2 possibly overlapping NV outbreaks occurred in Malaysia in 1998. Further investigation of NV archived materials would shed further light into the possible origin of NV in the 1998 Malaysia outbreaks.


Triadica sebifera (L.) Small) invasions related to timber production in southern U.S. forestlands under different management strategies. Expected costs were confined to the value of timber production losses plus costs for search and control. We simulated management strategies including (1) no control (NC), and control beginning as soon as the percentage of invaded forest land exceeded (2) 60 (Low Control), (3) 25 (Medium Control), or (4) 0 (High Control) using a spatially-explicit, stochastic, bioeconomic model. With NC, simulated invasions spread northward and westward into Arkansas and along the Gulf of Mexico to occupy ≈1.2 million hectares within 20 years, with associated expected total costs increasing exponentially to ≈$300 million. With LC, MC, and HC, invaded areas reached ≈275, 34, and 2 thousand hectares after 20 years, respectively, with associated expected costs reaching ≈$400, $230, and $200 million. Complete eradication would not be cost-effective; the minimum expected total cost was achieved when control began as soon as the percentage of invaded land exceeded 5%. These results suggest the importance of early detection and control of Chinese tallow, and emphasize the importance of integrating spread dynamics and economics to manage invasive species.Economic costs associated with the invasion of nonnative species are of global concern. We estimated expected costs of Chinese tallow ( The ecological and economic costs of invasive species have been extensively discussed and the future impacts to global sustainability are a source of great concern Triadica sebifera (L.) Small, Euphorbiaceae, synonyms include Sapium sebiferum (L.) Roxb and Stillingia sebifera Willd.). Chinese tallow was first introduced into the United States in the late 1700's and repeated introductions have occurred throughout the southeastern US until the mid-1900's Among the most aggressive and costly invaders of southern U.S. forests is Chinese tallow tree  of Chinese tallow invasions on southern U.S. forestlands under different control strategies based on the percentage of land presently invaded by Chinese tallow tree. We consider the costs of timber loss as a result of Chinese tallow invasions and the costs associated with searching for and controlling Chinese tallow in invaded areas. This information on costs of different control efforts should help government agencies and private landowners decide whether and when to initiate control measures to maintain timber production.We modified the biological invasion model of Wang et al. To include the economic impacts associated with invasion in the model, we represented expected total costs as a function of three components: damage costs (timber losses in $), searching costs, and control costs as:We estimated damage costs as:We estimated control and search costs based on phone interviews conducted from July 15 to 21, 2009 with personnel from major invasive plant control companies in the region We estimated control costs based on the information from Superior Forestry Service, Inc. , which provided the most detailed information and was licensed to conduct invasion control services in several states . We assu© to Excel files and subsequently imported the Excel files into ArcView® to analyze spatial-temporal patterns of invasion.To evaluate the economic costs of controlling Chinese tallow, we ran 240, 20-year, Monte Carlo simulations from observed pattern of Chinese tallow invasion in the year 2003  under eaTo parameterize the Chinese tallow invasion model for eastern Texas, Louisiana, western Mississippi, and southern Arkansas, we obtained data on environmental and ecological characteristics of geo-referenced USDA Forest Service sample plots from the Forest Inventory and Analysis (FIA) dataset For cost estimation, we obtained data on timber productivity from the Forest Service Timber Product Output dataset Simulated Chinese tallow invasions, if not controlled, spread northward and westward into the forests of Arkansas and colonized lands along the Gulf of Mexico within 20 years, with about 14% of all forest cells invaded to some extent, and >62% of the invaded cells exhibiting >50% of the land occupied by Chinese tallow . AssuminWithout control, both the total area invaded by Chinese tallow and the expected total costs increased exponentially, reaching ≈1.2 million hectares  and ≈$30We have presented a dynamic bioeconomic approach for managing the impact of invasive species range expansion which combines predictions of the spatial-temporal advance of a biological invasion with estimations of the concomitant timber losses and expected total economic costs. Our approach integrates invasion ecology and natural resource economics within a spatially explicit, agent-based, simulation framework to compare the efficacy of alternative invasion control scenarios. In the following sections, we consider the theoretical and empirical basis for our approach, interpret our findings in terms of the bioeconomic implications for Chinese tallow management, and provide some suggestions for future study.Invasive species have had enormous negative environmental and economic impacts worldwide Ligustrum sinense Lour.), European privet (Ligustrum vulgare L.) and Japanese honeysuckle (Lonicera japonica Thunb) Ambrosia artemisiifolia L.) in Austria The dispersal of invasive species has been a subject of both theoretical and empirical study for decades It should be noted that had we assumed a different probability distribution to estimate dispersal distances, our projected dispersal velocities and the resulting economic costs would have been different. Had we chosen a fat-tailed distribution other than the lognormal , projected velocities and costs would have been similar since fat-tail distributions generate long dispersal distances. Had we chosen a thin-tailed distribution , projected velocities would have been slower and projected costs would have been smaller since thin-tail distributions generate short dispersal distances.The post-invasion growth of invasive species also has been a subject of much study, and population growth models have been firmly rooted in population dynamics theory , using both exponential and density-dependent growth models depending on purpose Identification of cost-efficient control strategies for invasive species ideally would be based on estimates of both the costs of control efforts and the economic losses due to damage to the resource Of course, had we assumed a different functional form of the relationship between area invaded and searching costs, we would have obtained different estimates of expected total costs. Had we assumed searching costs increase exponentially with decreasing invaded area Economic losses due to resource damage also are problematic to estimate because they include the impact of invasion on nonmarket as well as market values. A straightforward monetary measure of the impact of invasion on market values of many agricultural products is the average value of the product lost due to invasion damage Our aim in the present study is to provide spatially-explicit, temporally-dynamic, representations of the economic aspects of biological invasions. Most invasive plant management, including that of Chinese tallow, has emphasized controlling either highly-infested areas or areas in the early stages of recruitment and establishment Miconia calvescens) on the islands of Oahu and Molokai in Hawaii until 1400 and 2300 trees were found, respectively. If control was initiated when >5% of the area was invaded, overall total costs were markedly higher due to additional control costs and greater damage costs. Sharov and Liebhold Lymantria dispar) in the southern U.S. increased when the distance from the invasion front to the end of the uninfested area becomes <200 km.Our simulation results re-emphasized the importance of early detection and proactive control of Chinese tallow invasions in southern U.S. forestlands, a finding that echoes the almost universal conclusion of invasion studies focused not only on woody plants (e.g., Cacho et al. Currently, Chinese tallow occupies >5% of the area in ≈26% of the infested forestlands in our study area that it has invaded Providing useful predictions of the rate of spread of biological invasions and their associated economic costs remains a challenge which is of global concern We estimated damage costs based on data on timber productivity We estimated search and control costs based on recent information from invasion control companies The quantitative framework we have described in the present paper, unlike that of previous bioeconomic assessments of the spread of invasive species


To develop a more realistic assessment of costs, herein named “true” costs, the extra-cancer from medical radiation, environmental damage from imaging paraphernalia and radioactive wastes must be included as long-term costs from imaging examinations. It is urgent to define the “true” costs across imaging modalities as it interferes on physicians’ decision to request an exam and on research projects such as cost-effectiveness analysis. Cardiology is the specialty that most will benefit from the outcome as cardiovascular exams represent almost 30% of the total exams acquired annually worldwide. While innovations in medical imaging represent an exceptional success story, the escalating average costs of imaging is representing a major health economic and societal burden. The cost of diagnostic imaging in the United States (U.S.) is estimated in $100 billion per year . SeveralThe estimation of costs across imaging modalities is a crucial information, as it has impact not only in the clinical setting, i.e. physicians’ decision to request an exam; but also research results, i.e. cost-effectiveness analysis. Often, physicians lean toward the imaging modality with the lowest cost for any given comparable accuracy . As onlyFinal or total cost to produce an exam equals the sum of direct and indirect costs. Each institution has its own formula to allocate direct and indirect costs; so total cost variations between institutions is expected. In general, at the imaging department, the direct costs account for the higher percentage of total costs. Direct costs of producing an exam include, but are not limited to, labor, material, film, equipment; while indirect costs include transportation, internet, heating, sewer, lighting, etc.Herein, we suggest that three factors must be included in the total costs of producing an examination in order to correspond to what we denominate as “true” cost: environmental costs, extra-costs from  cancer caused by medical radiation, and radionuclide wasting costs. In addition, special attention must be given to the running costs (i.e. electricity) of each imaging modality as it affects greatly the environment.Figure The long-term costs of environment damage caused by imaging methods paraphernalia have been neglected. In the sustainability era where people are adapting their habits to a more “green” life with the expectation to preserve the ecosystem for future generations, it is more than fair that health care practitioners begin to consider the environment topic in their daily practice. Undoubtedly, the environment cost per imaging methods paraphernalia should be taken into account as part of the “true” costs as its impact not only the ecosystem but also the human health.The magnitude of the environmental impact, in an economic viewpoint, of certain products and materials during life time can be calculated through life cycle assessment (LCA). Damage to mineral and fossil resources (millijoule surplus energy), damage to ecosystem quality (% plant species * Kilometers squared * year) and damage to human health [disability adjusted life years (DALY)] are the three final categories affected by several factors such as heavy metals, nuclides, hydrochlorofluorocarbon (HCFC), carbon dioxide (CO2), polycyclic aromatic hydrocarbons (PAH), fossil fuels, and land transformation. The same software used to calculate LCA also carry on life cycle costing (LCC) analysis. LCC is a tool that provides a cost estimation of the environmental damage costs and it is expressed through indirect costs.The environmental cost has impact solely in the indirect costs; however, some of the resources and materials included in the LCA and LCC analyses also affect direct costs Figure . TranslaRecently, Marwick and Buonocore  carried The inclusion of environmental costs in imaging cost assessment is a conceptual breakthrough: still, some limitations and appropriateness of the employed approach should be considered as a platform for future improvement and refinements of the model.The LCA results, and thus LCC, are greatly affected by energy consumption. According to COCIR -on the self-regulatory initiative echo-design of energy using products for medical imaging equipment on December 2011- the energy consumption from imaging methods corresponds up to 80% of environmental impact on LCA . In otheElectricity has impact in both direct and indirect costs; the latter through environment Figure . The dirThe U.S. is the leading country in CO2 emission per capita/year: 20 tons of CO2. The CO2 emission average worldwide is 3.1 tons, and in Italy is 8.1 tons. The cost of 1 ton of CO2 emission is $50. Ultrasound is the modality that emits lowest CO2 per exam  whereas, 3.0 T MRI emits the highest . The cost of CO2 per exam should be also account in the “true” costs.In the last 10 years, the medical imaging community has become increasingly aware of the need to include long-term cancer risk caused by ionizing radiation in the risk-benefit assessment of ionizing testing -10. ThisThe costs and savings of technological upgrading in terms of cancer prevention have been addressed by the food drug administration (FDA) some years ago. The group estimated that 723 lives per year spared radiation induced cancer mortality 30 years after the start of implementation of amendments. The average annual financial savings of $519 in the first 10 years of implementation greatly exceeds estimated average annual cost of $49 million to manufactures and to the FDA .3 in 1980, $14,286/m3 in 2005 and $35,714/m3 in 2010 [Aside electricity and extra-cancer, the cost of radioactive waste also should be accounted in the “true” costs. The costs of radioactive waste have been increasing steadily and it is estimate that in the past 30 years the cost had increased approximately 1000 times: $36/m in 2010 . The radThe assessment of economic, environmental, societal and biologic costs of medical imaging  is becoming increasingly important topic ,18. ThisThe authors declare that they have no competing interests.LB and EP designed research; LB conducted literature review, data analysis and manuscript drafting; BV contributed in depth to environmental data analysis and reviewed the manuscript; CGL contributed in depth to economic data analysis and reviewed the manuscript; EP had primary responsibility for final content. All authors read and approved the final manuscript.


To the Editor: Recently, the US government completed a targeted vaccination strategy limited to healthcare workers, first responders, and the military because of concern that variola virus, the etiologic agent of smallpox, might be used as a biowarfare agent  with major cutaneous reactions. Persons who participated in this study were selected from a randomized, double-blind, single-center study that compared the safety, tolerability, and immunogenicity of 3 smallpox vaccines . The 15-mL conical tubes were returned to the clinic in a cooler on cold packs the same day. Recovery of vaccinia virus was determined by infectivity assay. Samples were tested for infectious vaccinia virus by inoculation of fluid cultures of Vero cells grown in 12-well plates. A sample was defined as positive if cytopathic effects were observed  of dressing samples were positive for vaccinia   because we were concerned with transmission and infectivity. If we had chosen to measure noninfectious viral genomes, a positive outcome may have been likely. However, from a public health standpoint, such information would have been less valuable. Further studies will need to examine the viability of vaccinia virus in the environment to evaluate the possibility of contact transmission.


The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications. RNA interference (RNAi) is an intrinsic cellular mechanism for mediating gene expression, and has been established as a commonly used tool for gene down-regulation and loss-of-function studies in the life sciences et al. identified over 1,000 new genes required for cell division based on results from their high-throughput cell viability screening et al. systematically investigated and identified several novel components in the transferrin (TF) and epidermal growth factor (EGF)-related endocytic trafficking et al. demonstrated that esiRNAs are highly effective in RNAi-mediated gene silencing in embryonic stem cell (ESCs), while Tan et al. showed that esiRNAs were able to inhibit HBV replication more efficiently than synthesized siRNAs and tolerated limited target sequence variations without losing inhibitory capacity For example, Kittler The production of esiRNA, however, is a complex process involving multiple steps, including product purification, quantification and normalization. As RNase III enzymes do not completely digest long dsRNAs, the purification step is essential. In addition, normalization of esiRNA prior to transfection is a critical but tedious task. We previously demonstrated the manufacture of hundreds of esiRNAs using a polymer microbead-integrated chip The chip contained two components: a microwell array and magnetic microbeads coated with streptavidin . The utiFirst, a group of linearized DNA fragments, which could either be cloned cDNA fragments or chip-based, chemically-synthesized DNA oligomers, were dispensed into a microwell chip as the PCR template. One of the two PCR primers was biotinylated to facilitate the immobilization of the amplification products on beads after PCR and the subsequent easy separation from the supernatant containing dNTPs, DNA polymerase and buffer solution. To ensure that the templates were successfully immobilized, these beads were employed as templates for another PCR reaction. Amplification products were detectable in the supernatant when templates were generated from biotinylated primers, whereas no products were detected in the control reaction containing templates using non-biotinylated DNA primers .in vitro transcription buffer as well as T7 RNA polymerase, and large amounts of complimentary RNA strands were generated. Then, the DNA-immobilized microbeads were then removed, and a specific amount of magnetic microbeads tagged with a hybridization probe was added so that the beads would act as bait for the duplex via hybridization  CCTATAGTGAGTCGTATTACGAGGCCTTTCG TTGGGCTG -3′. Biotinylated PCR primer and reverse primer were as follows: Forward 5′-biotin-GCTCCGGAAAGCAACC CGAC-3′ and Reverse 5′- CAGCCCAACGAAAGGCCTCG-3′.For each target gene, we design and obtain a double-strand DNA template of 400–500 bp in length tagged with a pair of adapters, which were used for hybridization and Streptavidin (SA)-coated magnetic beads were purchased from Invitrogen . Biotinylated DNA templates were immobilized on these beads following the standard protocol provided by the manufacturer.The microwell chip was composed of two parts, a 96-well or 384-well plate, and a magnetic mask. The latter was assembled from a group of magnetic bars so that the bar came in close contact with the well when removing the magnetic beads.In vitro transcription (IVT) was carried out in reaction buffer containing T7 RNA polymerase (NEB). The magnetic beads containing immobilized DNA template were incubated with IVT buffer at 37°C for 4 h with shaking.Once transcription finished, the DNA template immobilized magnetic beads were removed, and tag-probe immobilized magnetic beads  resuspended in1 X SSC were added into the supernatant. The mixture was then heated to 95°C, slowly cooled down to 65°C, and incubated at 65°C for 1 h. After these performances, dsRNA duplex would anneal and hybridize onto beads via tag-probes. Washing with 0.5×SSC, at least 3 times, would remove the excessive transcription solution and DNA templates.The magnetic microbeads were easily removed after the transcription or the digestion step using a 96-magnetic needle plate, which was assembled with a group of electromagnetic steel needles.Finally, following the siRNaseIII (Takara) protocol, enzymatic digestion was performed at 30°C with shaking. After 1 h, enzymatic digestion was terminated by adding EDTA. The supernantant esiRNA products were stored at −80°C until the subsequently used for transfection. The digested products were transferred into another plate for transfection with the aid of the magnetic mask.esiRNAs were transfected into 293 T or Hela cells using Lipofectamine 2000 (Invitrogen) according to the manufacture’s instruction. Cells were collected at 48 h for the real-time PCR and western blot assay, or at 72 h for the cell survival and MTS assays.Cell lysis and protein extractions of 293 T or Hela cells were performed following the indicated procedures. Antibodies against TP53, PLAU, TGFBI, TGFBR2 and GAPDH were purchased from CST or Abcam, respectively. Sequences of qRT-PCR primers are listed in Real-time PCR and western blot assays were carried out following a standard protocol.The MTS Cell viability assay was performed following the instruction of CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay Kit .For transwell migration assays, Hela cells were seeded into the upper chamber of a Transwell insert  in 100 µl serum-free medium per well. Medium (600 µl) containing 10% serum was added in the lower chamber to function as a chemoattractant. Non-migratory cells were removed from the upper chamber by scraping the surface with a cotton bud. The cells remaining on the lower surface of the insert were fixed with 2% formaldehyde  and stained by DAPI . Self-assembled cell microarray screening assay was performed according to our previously described study Figure S1Schematic diagram of the process for manufacturing esiRNA using magnetic beads integrated on a chip. GSP represents gene specific primers.(TIF)Click here for additional data file.Figure S2Optimization of the concentrations of magnetic beads.A. Different amounts of magnetic beads were used during the immobilization step. The transcription products were normalized. B. Different amounts of Tag-probe immobilized beads were added before the hybridization step. The yield of esiRNA products was normalized.(TIF)Click here for additional data file.Table S1Migration assay using the esiRNA tyrosine kinase library. The esiRNAs were manufactured using the magnetic bead-integrated chip approach. Samples 1, 2 and 3 represent three different esiRNAs all targeting one gene, respectively.(DOC)Click here for additional data file.Table S2Sequences of qRT-PCR primers used in the experiments.(DOC)Click here for additional data file.


CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies.Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene ( The abbreviation B12 covers all forms of cobalamins  and not only cyanocobalamin, which is the vitamin B1212 serves as coenzyme for 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) and methylmalonyl coenzyme A mutase (MUT). Deficiency of cobalamin leads to reduced activity of both of these enzymes resulting in an increase of methylmalonic acid (MMA) and total homocysteine (tHcy). Therefore, measurements of these metabolites allow the assessment of cellular cobalamin availability and are the tests of choice to detect early or mild cobalamin deficiency in humans Cobalamin is a member of the B-group, water soluble vitamins. Cobalamin is also known as vitamin BMTR is involved in the synthesis of DNA. Therefore, a lack of cobalamin affects rapidly dividing cells and leads to changes in the hematopoietic system such as megaloblastic anemia. Chronic deficiency of cobalamin also leads to neurological symptoms and irreversible damage of the brain and nervous system The uptake of dietary cobalamin in mammals is a complex process requiring several endogenous proteins. In the small intestine the dietary cobalamin (“extrinsic factor”) is bound to a secreted protein, called gastric intrinsic factor (GIF). The uptake of the cobalamin-GIF complex from the intestinal lumen into the body is mediated by a specific membrane protein complex termed cubam receptor AMN or CUBN genes lead to Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption Mutations in either the AMN gene cause IGS in Australian Shepherd Dogs and Giant Schnauzers, respectively Primary cobalamin absorption disorders have also been reported in several dog breeds including Australian Shepherd Dogs We previously described clinical and laboratory findings in young Border Collies with IGS and established breed-specific reference parameters for serum cobalamin, urine methymalonic acid, and plasma homocysteine concentrations in Border Collies We previously described the clinical phenotype of IGS in Border Collies in detail −5), we retained 112,323 markers for the final genome-wide allelic association study. Three best-associated SNPs in the GWAS had identical raw p-values of 4.6×10−6 , were non-informative (MAF <0.05), or showed a strong deviation from Hardy-Weinberg equilibrium in the controls  for the causative mutation and flanking chromosomal segments. We analyzed the cases for extended regions of homozygosity with simultaneous allele sharing. Only one genome region, which coincided with the associated interval on CFA 2, fulfilled our search criteria. Here, all 7 affected dogs were homozygous and shared identical alleles over 229 SNP markers corresponding to a ∼3.5 Mb interval. We concluded that the causative mutation should be located in the 3.53 Mb critical interval between the closest heterozygous markers on either side of the homozygous segment . The CUBN:c.8392delC variant is predicted to result in a frameshift and early premature termination codon in the open reading frame encoding cubilin  MRC1, CUBN is thus an excellent functional candidate gene for the hereditary cobalamin malabsorption disorder observed in Border Collies. We therefore conclude that the CUBN:c.8392delC variant is the most likely causative defect for the phenotype, which we propose to call IGS in analogy to the human disease. According to our knowledge, these are the first dogs with a characterized molecular defect in the CUBN gene. Together with AMN mutant Australian Shepherd Dogs and Giant Schnauzers CUBN mutant Border Collies now complete the repertoire of molecular characterized dog models for IGS in humans.The CUBN:c.8392delC variant in a cohort of nearly 200 randomly selected Border Collies is relatively low at 6.2%.The carrier frequency of dogs being heterozygous for the MRC1 gene. The MRC1 variant is located 849 kb away from the CUBN frameshift variant on dog chromosome 2. Both variants appear to segregate only in Border Collies and have thus most likely arisen within the last 200 years. Unfortunately, the MRC1 and CUBN variants were in perfect linkage disequilibrium in all available dogs of our study, so that we could not disentangle the functional effects of each variant separately. Nonetheless, the seven IGS affected Border Collies did not show any obvious differences in their clinical phenotype with respect to other dogs with primary cobalamin absorption disorders. In particular they showed no signs of immunodeficiency or exaggerated inflammatory reactions (data not shown). It therefore appears that a spontaneous inactivation of the MRC1 gene in dogs does not result in any obvious clinical phenotype, similar to what has been observed in Mrc1 knock out mice This study highlights the power of complete genome re-sequencing. Using this approach we were able to quickly identify the most likely causative variant for IGS in Border Collies. This variant could also have been obtained by a conventional candidate gene approach. In the case of IGS however, the two known candidate genes would have comprised a total of 79 exons, and the individual design of PCR primers and traditional workflow of PCR-amplifying each exon followed by Sanger sequencing would have required about the same amount of time and money in our laboratory as the whole genome re-sequencing experiment. In contrast to data from a conventional and more limited candidate gene approach, our analysis has also revealed the unexpected finding of a nonsense mutation in the In conclusion the identification of a candidate causative mutation for IGS in Border Collies provides the first dog model for IGS with a molecular characterized CUBN defect. Our findings will allow the development of a genetic test and eradication of IGS from the privately owned Border Collie breeding population.All animal experiments were performed according to the local regulations. The dogs in this study were examined with the consent of their owners. The study was approved by the “Cantonal Committee For Animal Experiments” .We used 7 Border Collie cases, which could be unambiguously phenotyped based on small growth, poor BCS, undetectable serum cobalamin concentrations, methylmalonic aciduria, homocysteinemia (4/7) and complete clinical response to exclusive parenteral cobalamin supplementation. These were all affected dogs that we could obtain for the study and thus represent a convenience sample. Two of the cases were full-siblings. The 7 control Border Collies for the GWAS were judged to be healthy based on unremarkable history and physical examination, as well as normal normal results of CBC, serum biochemistry, urinalysis, serum cobalamin, urinary methylmalonic acid, and plasma homocysteine concentration. We classified additional dogs as controls based on owner-reported unremarkable histories. The complete cohort for this study consisted of 200 Border Collies and 357 dogs of diverse other breeds . We collWe isolated genomic DNA samples from EDTA blood with the Nucleon Bacc2 kit . Genotyping was done on illumina canine_HD chips containing 173,662 SNP markers at the NCCR Genomics Platform of the University of Geneva. Genotypes were stored in a BC/Gene database version 3.5 (BC/Platforms).−5). We performed an allelic association study using the –assoc command of PLINK. We also used PLINK to search for extended intervals of homozygosity with shared alleles as described previously We used PLINK v1.07 2 tests of the allele frequency in cases vs the allele frequency in controls for each marker. After the filtering procedures described above, we had 112,323 markers left for the final analysis. In order to correct for the multiple testing situation, we determined an empirical significance threshold by performing 100,000 permutations of the dataset with arbitrarily assigned phenotypes.Raw p-values in the GWAS are based on χCUBN gene corresponds to the accessions NM_001003148.1 (mRNA) and NP_001003148.1 (protein).We used the dog CanFam 3 genome assembly derived from a Boxer as reference genome sequence. All numbering within the canine http://sourceforge.net/projects/picard/). We used the Genome Analysis Tool Kit . We obtained a total of 125,457,731 paired-end reads or roughly 10× coverage. We mapped the reads to the dog reference genome with the Burrows-Wheeler Aligner (BWA) version 0.5.9-r16 We used Sanger sequencing to confirm the illumina sequencing results and to perform targeted genotyping for selected variants. For these experiments we amplified PCR products using AmpliTaqGold360Mastermix (Applied Biosystems). PCR products were directly sequenced on an ABI 3730 capillary sequencer (Applied Biosystems) after treatment with exonuclease I and shrimp alkaline phosphatase. We analyzed the sequence data with Sequencher 4.9 (GeneCodes).Table S1Genes in the 3.53 Mb critical interval on CFA 2.(XLSX)Click here for additional data file.Table S2Non-synonymous and associated variants in the 3.53 Mb critical interval on CFA 2.(XLSX)Click here for additional data file.Table S3List of control dogs and breeds that were used for the association study.(XLSX)Click here for additional data file.


The complete genome sequences of a cytopathic/noncytopathic pair of border disease viruses (BDV) that were isolated from a sheep with mucosal disease-like lesions were determined. The cytopathic virus possessed an insert of 288 nucleotides that was derived from the Jiv cellular transcript and had 37 nucleotide differences from the noncytopathic virus. Pestivirus genus of the Flaviviridae, is the causative agent of border disease (BD) in sheep and goats. BD is characterized by abortions, stillbirths, congenital deformities, and weak lambs. Like bovine viral diarrhea virus (BVDV), BDV can be classified as noncytopathic (ncp) or cytopathic (cp) based on its growth characteristics in vitro. For BVDV, the cp form arises from the ncp virus by insertions of cellular sequences or rearrangement and duplication of viral sequences (–Border disease virus (BDV), a member of the equences –5. When The BDV strains Coos Bay 5 ncp (CB5nc) and cp (CB5c) were isolated and biologically cloned in the late 1980s from a sheep with mucosal disease-like lesions. Viral RNA was extracted following treatment of the virus stocks with a nuclease cocktail . Both viThe libraries yielded 13,755  and 41,679  sequences for CB5nc and CB5c, respectively. The genomes of CB5nc and CB5c were 12,300 and 12,588 bases. The single open reading frame (ORF) of CB5nc was 11,694 bases  and the ORF of CB5c was 11,982 bases . CB5c had a 288-base insertion derived from the Jiv cellular transcript . ExcludiKJ463422 and KJ463423, respectively.The genomic sequences of the Coos Bay noncytopathic and cytopathic viruses have been deposited in GenBank with the accessions numbers