We report the identification of a novel polyomavirus present in respiratory secretions from human patients with symptoms of acute respiratory tract infection. The virus was initially detected in a nasopharyngeal aspirate from a 3-year-old child from Australia diagnosed with pneumonia. A random library was generated from nucleic acids extracted from the nasopharyngeal aspirate and analyzed by high throughput DNA sequencing. Multiple DNA fragments were cloned that possessed limited homology to known polyomaviruses. We subsequently sequenced the entire virus genome of 5,229 bp, henceforth referred to as WU virus, and found it to have genomic features characteristic of the family Polyomaviridae. The genome was predicted to encode small T antigen, large T antigen, and three capsid proteins: VP1, VP2, and VP3. Phylogenetic analysis clearly revealed that the WU virus was divergent from all known polyomaviruses. Screening of 2,135 patients with acute respiratory tract infections in Brisbane, Queensland, Australia, and St. Louis, Missouri, United States, using WU virus–specific PCR primers resulted in the detection of 43 additional specimens that contained WU virus. The presence of multiple instances of the virus in two continents suggests that this virus is geographically widespread in the human population and raises the possibility that the WU virus may be a human pathogen. We have identified a novel virus, referred to as WU virus, in the family Polyomaviridae by screening of human respiratory secretions. Two human polyomaviruses, BK and JC, were identified in 1971 and infect the majority of humans around the world. These two viruses are closely related to each other and are both are pathogenic in immunocompromised individuals. Earlier this year, a third polyomavirus, KI, was described in human clinical specimens, although its pathogenicity and prevalence in humans has not yet been established. The discovery of WU virus brings the number of polyomaviruses detected in humans to four. WU differs from BK and JC significantly in its genome sequence and in its relative tissue tropism, suggesting that it is likely to have unique biological properties. This discovery raises many questions for further investigation, such as, Is WU virus a human pathogen? If so, what kind of disease does it cause? Where in the body does WU virus reside? At what age does infection typically occur? Perhaps most importantly, there are likely to be many more as of yet unidentified viruses infecting the human body. Viral infections of the respiratory tract are responsible for significant mortality and morbidity worldwide . DespiteViruses in the family Polyomaviridae possess double-stranded DNA genomes and infect a variety of avian, rodent, and primate species. To date, two polyomaviruses, BK virus and JC virus, have been unambiguously described as human pathogens. BK and JC viruses are ubiquitous worldwide, and in adult populations, seroprevalence rates approaching 75% and 100%, respectively, have been reported ,12. Inferas transforms primary human cells [Much of the interest in polyomaviruses and SV40 in particular derives from the transforming properties carried by the early transcriptional region of the viral genome that encodes for the small T antigen (STAg) and and large T antigen (LTAg). T antigen is capable of binding both p53 and Rb proteins and interfering with their tumor suppressor functions. The early region alone is sufficient to transform established primary rodent cell lines  and in can cells . This haan cells .We describe the identification and characterization of a novel polyomavirus initially detected by high throughput sequencing of respiratory secretions from a patient suffering acute respiratory disease of unknown etiology. The virus was detected in the respiratory secretions from an additional 43 patients in two continents, and the complete genomes of multiple isolates were sequenced.Haemophilus influenzae (three reads), Streptococcus pneumoniae, Corynebacterium pseudodiphthericum, and Leifsonia xyli (unpublished data). Upon further examination, the six viral reads were collapsed into three unique regions, each of which possessed only limited homology to known polyomavirus proteins  from a 3-year-old patient admitted to the pediatric ward of the Royal Children's Hospital in Brisbane with pneumonia was collected in October 2003. The patient had no other remarkable clinical traits other than the respiratory features of pneumonia. Testing of nucleic acid extracted from the NPA using a panel of 17 PCR assays for known respiratory viruses as described . One 384The complete genome of WU was sequenced to 3× coverage using cloned fragments of the viral genome generated by a series of PCR primers. Analysis of the DNA sequence revealed genomic features characteristic of polyomaviruses. First, the WU genome size of 5,229 base pairs (bp) was quite comparable to those of the primate polyomaviruses BK , JC , and SV40 . In addition, the overall GC content of the WU genome was 39%, which is quite similar to the GC content of BK (39%), JC (40%), and SV40 (40%). The genome organization included an early region coding on one strand for STAg and LTAg, and a late region coding on the opposite strand for the capsid proteins VP1, VP2, and VP3 . These t5CX7–8CXCX2CX21–22CSCX2CX3WF, that was present in both of the predicted isoforms of WU STAg. This motif, which is present in all STAgs, was perfectly conserved in WU virus with the exception of the initial cysteine residue.In the early region, an unspliced open reading frame of 194 amino acids was detected that possibly encodes for the STAg. As the paradigm in other polyomaviruses is that STAg is expressed from a spliced message, analysis of potential splice sites revealed the presence of a putative splice donor sequence just one nucleotide 5′ of the initially predicted stop codon. Splicing to a downstream putative splice acceptor site would excise an intron of 70 nucleotides and generate a slightly larger STAg of 217 amino acids . While tIn all polyomaviruses, the initial ∼80 amino acids of the N-terminus of the STAg and LTAg are identical; the LTAg is generated by alternative splicing of the early mRNA transcript. In WU virus, a conserved splice donor site was identified immediately after amino acid 84 of the early open reading frame. The position of the splice site is similar to that found in SV40, BK, and JC virus, which occur after amino acids 82, 81, and 81, respectively. Splicing to a conserved splice acceptor site would generate a predicted protein of 648 amino acids . The preBased on comparative sequence analysis of LTAgs, the polyomaviruses are classified into two subclasses: a primate-like group exemplified by SV40, and a mouse polyoma-like group exemplified by murine polyoma virus . Using tIn addition to encoding LTAg and STAg, murine and hamster polyomaviruses utilize alternative splicing to generate an intermediate-sized protein referred to as middle T antigen. The WU virus early region was scanned for splicing motifs similar to known murine and hamster polyomavirus splice donor and acceptor sequences, but no obvious combination of splice sites was detected that would yield a middle T antigen sequence in the size range of known middle T antigens. In addition, SV40, JC, BK, and baboon polyomavirus all encode a fourth late protein termed the agnoprotein. There was no open reading frame present in WU with any detectable homology to the known agnoproteins. Thus, our sequence analysis suggests that neither middle T antigen nor agnoprotein are encoded by WU virus, although it is possible that the sequences have diverged beyond our ability to recognize the appropriate splice sites or protein products.Multiple sequence alignments of the predicted STAg, LTAg, VP1, and VP2 open reading frames revealed that WU virus was clearly a novel virus that is most closely related to KI virus . NeighboPCR primers were designed to specifically amplify WU. The initial screen used primers targeting the VP2 region, which possessed less than 20% amino acid homology to JC and BK virus to minimize the possibility of cross reactivity with the known human polyomaviruses. Empirical testing of the primers on samples known to contain BK and JC confirmed that the primers did not cross react with either of these genomes (unpublished data). Positives in the initial screen for WU virus were sequenced and then further confirmed by a second PCR reaction using primers targeting the 3′ end of the WU virus LTAg coding sequence. All 43 positive samples in the initial screen were confirmed using the second pair of PCR primers. A subset of samples that tested negative in the initial screen was also tested with the second PCR primer pair, and none of those samples were positive.In order to assess the prevalence of WU polyomavirus, a cohort of 1,245 respiratory specimens collected in 2003 in Brisbane was examined. Thirty-seven out of the 1,245 (3.0%) samples tested were positive for the virus . In thisIn addition, we examined two cohorts of patients from St. Louis, Missouri, United States. In one set of upper respiratory specimens collected in 2006, five out of 410 were positive for WU virus in the PCR assay. In addition, 480 bronchoalveolar lavage samples from patients (mostly adults) with severe acute respiratory illness were tested, yielding one positive. Of the positive samples, all six were co-infected with other viruses . The ageTo assess the sequence variation within different isolates, we analyzed the 250-bp region encompassed by the initial screening primers for all 43 cases . SeveralBecause BK and JC virus are frequently excreted in urine, we examined urine samples from patient cohorts in both St. Louis and Brisbane for the presence of WU virus by PCR. In the St. Louis cohort, urine samples from 200 adult patients participating in a study of polyomavirus infections in kidney transplant recipients were tested . For mosWe used a high throughput sequencing strategy to search for novel agents that were present in respiratory tract infections of unknown etiology. The focus of this study was on individual clinical specimens that still lacked a diagnosis after analysis with an extensive panel of diagnostic assays for known respiratory viruses. In one such patient sample, novel sequences with limited homology to known polyomaviruses were detected. Complete genome sequencing and phylogenetic analysis revealed that the new virus clearly had the genomic organization typical of polyomaviruses but was divergent from all previously described polyomaviruses. In keeping with the two-letter virus names for human polyomaviruses, we have named this novel polyomavirus WU virus ,26. OverDetailed analysis of the viral DNA sequence and genomic organization confirmed the novelty of WU virus. At all loci, WU virus was most similar to KI virus, but the degree of divergence between WU and KI was greater than the divergence between SV40 and BK, indicating that WU and KI are clearly distinct viruses . Based oWe detected WU in 37 out of 1,245 (3.0%) patient specimens in Brisbane  and in six out of 890 (0.7%) patient specimens tested in St. Louis. As the positive specimens were all collected from 2003 through 2006, it appears that WU is currently circulating, and its presence in both North America and Australia suggests that the virus is geographically widespread in the human population. The age range of patients that tested positive for WU virus spanned from 4 months to 53 years. The majority (86%) of the cases were found in children 3 years of age and under. Of the four positive specimens from adult patients  causes interstitial pneumonia and significant mortality. MPPV also differs from other polyomaviruses in that besides the kidneys, it can also be detected in the lungs, liver, spleen, and blood of suckling mice . Thus, tOne striking observation from these studies is the relatively high frequency of co-infection detected in the respiratory secretions: 72% overall (100% in the St. Louis cohort and 68% in the Brisbane cohort). Although more extensive studies are necessary to confirm the generality of this observation, this raises several intriguing non-mutually exclusive possibilities to consider: 1) WU may be an opportunistic pathogen; 2) WU infection may predispose or facilitate secondary infection by other respiratory viruses; and 3) WU may be a part of the endogenous viral flora that is reactivated by inflammation or some other aspect of viral infection. Recent studies of the prevalence of the newly identified human bocavirus have also reported higher levels of co-infection than previously described for other viruses found in the respiratory tract, with co-infection rates as high as 50% reported ,29. In aExtremely high sequence divergence was observed in the capsid proteins VP1 and VP2 of WU virus and KI virus as compared to the other known polyomaviruses. This divergence may reflect a different “lifestyle” for the WU/KI branch as compared to known polyomaviruses. Our data demonstrating the presence of WU in respiratory secretions and its absence in urine samples suggest that the mode of transmission or the sites of persistence of WU may be distinct from the other human polyomaviruses. As such, the structure of the virion must be optimized to enable the virus to survive dramatically distinct physiological and environmental conditions. This may partially explain the observed sequence divergence in the capsid proteins.Another question raised by this study relates to the potential antigenic cross reactivity of the WU capsid proteins. In terms of establishing the seroprevalence of WU itself and determining whether seroconversion accompanies acute infection with WU, it will be essential to conduct these studies with consideration for potential cross reactivity to KI, BK, JC, and SV40 antibodies. In addition, it is tantalizing to speculate whether serum antibodies to WU have the potential to cross react to SV40-derived antigens, and if so, whether they may at least partially account for some of the studies that report the presence of SV40 antibodies in the human population that is too young to have suffered exposure from contaminated polio vaccination –32.In conclusion, we have identified and completely sequenced the genome of a novel polyomavirus. This virus appears to be geographically widespread in the human population as evidenced by the detection of 44 distinct cases in two continents. Based on preliminary analysis, WU and KI virus share some strikingly similar properties, including their complement of genes, phylogenetic relationship, and physical sites of detection in the human body. These data suggest that WU virus and KI virus define a novel branch within the Polyomaviridae family with unexplored biology and pathogenicity. Another implication of these results is that the diversity of viruses in this family may be far greater than currently realized. Further experimentation is now underway to determine the relative pathogenicity of WU virus in humans and to understand the molecular properties of the virus. Since the T antigen of WU is predicted to have transforming properties by analogy to other polyomavirus T antigens, one question currently under investigation is whether a subset of human tumors may be associated with WU.Brisbane cohort. A total of 1,245 specimens (predominantly NPAs) were collected between January 1, 2003, and December 22, 2003, from patients presenting to the Royal Children's Hospital in Brisbane, Queensland, Australia, with symptoms consistent with acute lower respiratory tract infection.St. Louis cohort #1. A total of 480 BAL specimens were tested. These included samples from a retrospective and a prospective collection. The retrospective specimens were from a sequential collection of BAL specimens submitted routinely to the Virology Laboratory at St. Louis Children's Hospital between December 2002 and August 2003 . For theSt. Louis cohort #2. This collection was made up of respiratory specimens, mostly nasopharyngeal swabs, submitted for routine virologic testing to the Virology Laboratory at St. Louis Children's Hospital between September 2005 and June 2006. The majority of these specimens were from children. Of the 410 specimens in this collection, 200 were selected because they had been found to be positive by fluorescent antibody staining or culture for influenzavirus A or B, respiratory syncytial virus, parainfluenza virus, rhinovirus, or adenovirus.n = 50), (ii) pediatric clinic (n = 52), (iii) antenatal clinic (n = 33), (iv) indigenous health clinic (n = 36), and (v) bone marrow transplant patients (n = 55).Brisbane cohort. Urine specimens (226) that were submitted during 2003 to the diagnostic laboratory for routine investigation were collected. These represented a diverse mixture of donors, including those from (i) sexual health clinic  according to the manufacturer's instructions. PCR assays for 17 known respiratory viruses were performed as described [Brisbane cohort. Nucleic acids were extracted from 0.2 ml of each specimen using the High Pure Viral Nucleic Acid kit . Specimens that were negative were also cultured using cell culture systems that could detect the same group of viruses plus rhinoviruses, cytomegalovirus, and herpes simplex virus. Total nucleic acid extracts were purified using a Qiagen M48 instrument (http://www.qiagen.com). Nucleic acid extracts were tested for a panel of respiratory viruses using the EraGen MultiCode-PLx respiratory virus panel , a multiplex PCR assay that detects the following viruses: influenza A and B, respiratory syncytial virus A and B, parainfluenza 1–4, human meatpneumovirus, adenovirus subgroups B, C, and E, rhinoviruses, and coronaviruses OC43, 229E, and NL63.St. Louis cohort. All respiratory specimens were tested originally by fluorescent antibody staining using a panel of monclonal antibodies directed against influenza A and B, respiratory syncytial, parainfluenza 1–3, and adenoviruses  for 60 min at 37 °C. Total nucleic acid was extracted using the Masterpure Complete DNA and RNA Purification Kit . Then, 100 ng of total nucleic acid was randomly amplified using the RdAB protocol exactly as described [http://www.usbweb.com) enabled second strand cDNA synthesis as well as random priming of DNA originally present in the total nucleic acid sample. Amplicons were then generated via 40 cycles of PCR using primer-B (5′ GTTTCCCAGTCACGATA) with a cycling profile of: 94 °C 30 s; 40 °C 30 s; 50 °C 30 s; 72 °C 60 s. The primer-B–amplified material was TOPO cloned into pCR4.0  and transformed into bacteria, and white colonies were picked into 384-well plates. DNA was purified by magnetic bead isolation and sequenced using standard Big Dye terminator (v3.1) sequencing chemistry. Reaction products were ethanol precipitated, resuspended in 25 ul of water, and loaded onto the ABI 3730xl sequencer.Samples were prepared in the following manner for high throughput sequencing analysis. A total of 200 ul of neat NPA sample was thawed and directly treated with DNase I  remaining reads were aligned to a bacterial database using BLASTn with an e−10 cutoff; and 3) remaining reads were aligned to the viral RefSeq protein database using BLASTx with an e−2 cutoff [Sequences were assessed for quality using Phred , and rea2 cutoff .The WU genome derived from the index case was sequenced to 3× coverage using six unique pairs of PCR primers for the amplification. Amplicons were cloned into pCR4.0 and sequenced using standard sequencing technology. All primers used for amplification and sequencing are listed in Protein sequences associated with the following reference virus genomes were obtained from GenBank: BK virus, JC virus, bovine polyomavirus, SV40, baboon polyomavirus (simian agent 12), finch polyomavirus, crow polyomavirus, goose hemorrhagic polyomavirus, African green monkey polyomavirus, budgerigar fledgling polyomavirus, murine pneumotropic virus, hamster polyomavirus, and murine polyomavirus (see Supporting Information for accession numbers). For WU virus, predicted open reading frames were used. For STAg, the predicted open reading frame of 194 amino acids was used for analysis. Multiple sequence alignment was performed using ClustalX (1.83). Neighbor-joining trees were generated using 1,000 bootstrap replicates.For all PCR assays, standard precautions to avoid end product contamination were taken, including the use of PCR hoods and maintaining separate areas for PCR set up and analysis. For initial screening of WU virus, PCR primers AG0044 5′ tgttacaaatagctgcaggtcaa and AG0045 5′ gctgcataatggggagtacc were used with Accuprime hot start Taq (Invitrogen) to amplify 1 ul of template using the following program: 40 cycles of 94 °C 30 s; 56 °C 30 s; 72 °C 60 s. For every 88 samples tested, seven no-template negative controls were interspersed between the actual samples. Products were visualized following electrophoresis on 1% agarose gels. The resulting 250-bp amplicon was sequenced directly in both directions using primer AG0044 and AG0045. These sequences have been deposited in GenBank (see Supporting Information for accession numbers). Secondary confirmation was performed using primers AG0048 5′ TGTTTTTCAAGTATGTTGCATCC and AG0049 5′ CACCCAAAAGACACTTAAAAGAAA that generate a 244-bp amplicon in the 3′ end of the LTAg coding region. The same cycling profile of 40 cycles of 94 °C 30 s; 56 °C 30 s; 72 °C 60 s was used. For detection of both BK and JC viruses, primers AG0068 5′ AGTCTTTAGGGTCTTCTACC and AG0069 5′ GGTGCCAACCTATGGAACAG were used with a profile of 40 cycles of 94 °C 30 s; 56 °C 30 s; 72 °C 60 s.Figure S1A) The initial six shotgun reads with homology to polyomaviruses. B) The three contigs derived from the six reads.(38 KB PDF)Click here for additional data file.Figure S2The consensus TAg binding motif is GAGGC. The known primate polyomaviruses SV40, JC, BK, and baboon polyomavirus all have four copies of the copies of the binding site oriented as shown above for SV40 (NC_001669). The first nucleotide of the third copy of the consensus TAg binding site is defined as nucleotide 1 for WU and SV40. Differences between SV40 and WU Virus are 1) one of the TAg binding sites in WU virus appears to be a non-canonical TAGGC; 2) the second and third consensus TAg binding sites in WU virus overlap; and 3) the nucletoide spacing between the TAg binding sites in WU virus varies from the prototype SV40 as shown. Shown in blue is the polyA/T tract that is commonly found to the late side of the origin in polyomaviruses(462 KB PDF)Click here for additional data file.Figure S3A consensus LTAg donor site was detected. Splicing to the consensus downstream acceptor would generate a LTAg of 648 amino acids. For STAg, an unspliced open reading frame of 194 amino acids was identified. A predicted slice donor site was also detected that would result in excision of a 70-nucleotide intron and production of a 217–amino acid open reading frame.(542 KB PDF)Click here for additional data file.Figure S4Multiple sequence alignment of WU virus LTAg with 13 other reference sequences reveal the presence of carboxyl terminus extensions in baboon polyoma, BK, JC, and SV40. WU virus does not appear to encode such a region.(5.4 MB PDF)Click here for additional data file.Figure S5Locations of original shotgun reads are depicted as indicated. Locations of all sequencing primers are mapped to the complete genome. Primers used for amplification are shown in red.(551 KB PDF)Click here for additional data file.Table S1(35 KB PDF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) protein sequences used in this paper are as follows:The GenBank (LTAg: African green monkey (NP_848008); baboon polyomavirus 1 (YP_406555); BK (YP_717940); bovine (NP_040788); budgerigar (NP_848014); crow (YP_529828); finch (YP_529834); goose (NP_849170); hamster (NP_056730); JC (NP_043512); KI Stockholm 60 (ABN09921); murine (NP_041264); murine pneumotropic (NP_041232); SV40 (NP_043127).STAg: African green monkey (NP_848009); baboon polyomavirus 1 (YP_406556); BK (YP_717941); bovine (NP_040789); budgerigar (NP_848015); crow (YP_529829); finch (YP_529835); goose (NP_849171); hamster (NP_056732); JC (NP_043513); KI Stockholm 60 (ABN09920); murine (NP_041266); murine pneumotropic (NP_041233); SV40 (NP_043128).VP1: African green monkey (NP_848007); baboon polyomavirus 1 (YP_406554); BK (YP_717939); bovine (NP_040787); budgerigar (NP_848013); crow (YP_529827); finch (YP_529833); goose (NP_849169); hamster (NP_056733); JC (NP_043511); KI Stockholm 60 (ABN09917); murine (NP_041267); murine pneumotropic (NP_041234); SV40 (NP_043126).VP2: African green monkey (NP_848005); baboon polyomavirus 1 (YP_406552); BK (YP_717937); bovine (NP_040785); budgerigar (NP_848011); crow (YP_529825); finch (YP_529831); goose (NP_849167); hamster (NP_056734); JC (NP_043509); KI Stockholm 60 (ABN09918); murine (NP_041268); murine pneumotropic (NP_041235); SV40 (NP_043124).WU complete genome sequences have been deposited under accession numbers EF444549–EF444554. VP2 partial sequences have been deposited under accession numbers EF444555–EF444593.


Multiple sclerosis (MS) is an incurable chronic disease that predominantly affects young adults. It has a high socio-economic impact which increases as disability progresses. An assessment of the real costs of MS may contribute to our knowledge of the disease and to treat it more efficiently. Our objective is to assess the direct and indirect costs of MS from a societal perspective, in patients monitored in our MS Unit  and grouped according to their disability (EDSS).We analysed data from 200 MS patients, who answered a questionnaire on resource consumption, employment and economical status. Mean age was 41.6 years, mean EDSS 2.7, 65.5% of patients were female, 79.5% had a relapsing-remitting course, and 67.5% of them were receiving immunomodulatory treatment (IT). Patients were grouped into five EDSS stages. Data from the questionnaires, hospital charts, Catalan Health Service tariffs, and figures from Catalan Institute of Statistics were used to calculate the direct and indirect costs. The cost-of-illness method, and the human capital approach for indirect costs, were applied. Sensitivity analyses were performed to strengthen results.The mean total annual cost of MS per patient results 24272 euros. This cost varied according to EDSS: 14327 euros (EDSS = 0), 18837 euros (EDSS = 1–3), 27870 euros (EDSS = 3.5–5.5), 41198 euros (EDSS = 6–7) and 52841 euros (EDSS>7.5). When the mean total annual costs was adjusted by the mean % of patients on IT in our Unit (31%) the result was 19589 euros. The key-drivers for direct costs were IT in low EDSS stages, and caregiver costs in high stages. Indirect costs were assessed in terms of the loss of productivity when patients stop working. Direct costs accounted for around 60% of total costs in all EDSS groups. IT accounts from 78% to 11% of direct costs, and decreased as disability progressed.The total mean social costs of MS in a cohort from Baix Llobregat  were estimated at 24272 euros per patient/year, and ranged between 14327 euros (EDSS = 0) and 52841 euros (EDSS = 7.5–9.5). Total costs, and particularly informal and direct costs, increased as the disability progressed. IT should be able to delay the progression of disability to be efficient and not only effective. Multiple sclerosis (MS) is a chronic, demyelinating inflammatory disease of the central nervous system (CNS). Its aetiology is still unknown. In Spain, the prevalence of MS is 53–58 per 100000 . It predThe prognosis for MS is unclear and is probably influenced by genetic, biochemical and clinical factors. Life expectancy seems to be relatively unaffected by MS, but morbidity is considerable: 50% of patients will need help to walk 10 years after diagnosis, according to natural history studies. HoweverMS represents the second most common cause of disability in young adults, after road accidents, and has a high socioeconomic impact.In the last decade immunomodulatory treatments (IT) have demonstrated their efficacy -8 in redIt is essential to assess the real cost of MS if we are to make a cost-effectiveness analysis whose results can be applied to daily clinical practice. Cost-utility analysis, a particular type of cost-effectiveness analysis in which outcomes are measured in terms of quality of life, may be particularly appropriate for a disease like MS, as the assessment of quality of life integrates physical and psychological components of wellbeing which are affected by the disease . The cosExpanded Disability Status Scale).Our study, the first of this type in our region , attempts to assess the overall cost to society of MS, calculated from a population of patients monitored in our MS Unit. The approach that we used could be extended to assess MS costs nationwide. Our intention was also to show how the costs of MS change as the disease progresses. Therefore, costs were calculated in different stages of disability as measured by the EDSS . We considered the clinical and demographic characteristics of the patients in the study to be similar to those of the general MS population seen in our clinics. No statistically significant differences were found between patients who answered the questionnaire and patients who did not : 23 patients; stage II : 107 patients; stage III : 42 patients; stage IV : 17 patients; and stage V : 11 patients. Patients' EDSS scores were obtained from their medical charts.The questionnaire was anonymous and voluntary. It was mailed to 454 patients with a statement of informed consent to be signed by patients and caregivers. A letter explaining the purpose of the study was also sent to every patient. We obtained 200 responses (44%). Data were collected on socio-demographic and clinical characteristics; work-status; resource consumption; and costs attributable to MS during the previous 15–365 days. The length of the recall period varied according to the item in question, to minimize any possible bias due to a patient's lack of memory. The recall periods for the different items were as follows:-previous year for hospitalisation, need for caregivers and sick leave, due to MS-previous 3 months for outpatient visits-previous 15 days for drug treatments, rehabilitation, visits to the General Practitioner, ambulance displacements, laboratory-imaging tests, and days of leisure time and/or daily activities lost due to MS.A recall period of 1–3 months has been shown to be generally reliable for retrospective data. For more important or less frequent events, such as hospitalisation, a 12-month recall period is considered appropriate -27,30,32Questions about home modifications referred to the full duration of the disease. The unit costs for adaptations were not available for our region. Therefore, we used the mean cost per patient per year of home adaptations made because of MS, which has previously been calculated in the European Community -27,29.Data obtained from questionnaires were verified and completed with data taken from inpatient records, medical files and the Medical Records Department, to improve reliability of the patients' responses. Data were then annualised when necessary. The study period was one year, from 1-8-2001 to 31-7-2002.The Ethical Committee of the University Hospital of Bellvitge and the Institute for Biomedical Investigation of Bellvitge , where the study was carried out, gave their approval for the whole project.Drugs and Chemists Pharmacy Database of the General Council of Official Colleges of Pharmacists, 2004); and investments required to overcome architectural barriers. Every unit cost was updated to the equivalent number of euros in 2004, as the resources consumed in 2001 and 2004 did not differ greatly.Unit costs were obtained from: Catalan Health Institute tariffs ; the Catalan Institute of Statistics ; market prices for pharmacological treatments  are included as direct costs -27,30.e.g, housewives), to avoid underestimating indirect costs in this group of patients. For the rest of the patients, we applied the salary cost per hour in each professional sector, as described in the "National list of Economic Activities in fourth trimester 2003 – CNAE 2003". Thus, the results obtained should be considered a minimum estimation of indirect costs.We used a Human Capital Approach to estimate the indirect costs. This approach involves analysing MS-attributable loss of productivity, in terms of morbidity and mortality. The number of days lost due to sick leave and changes in the work status of patients and caregivers because of MS are considered to be morbidity. Mortality, in terms of life expectancy, is not significantly affected by MS. Therefore it was considered irrelevant  in this study. A minimum hourly wage was applied to patients who did not carry out "productive activities" in the labour market , Barcelona, Spain.2, Student's t and U- Mann-Whitney tests were used.All statistical analysis were performed using SPSS software. X-We analysed data from 200 patients with definite MS. The average age was 41.6 (SD: 10.7), 65.5% of patients were female, and 78.5% had relapsing-remitting MS. The mean EDSS was 2.7 (SD: 2.2) and the median EDSS was 2. The mean number of years from MS onset was 12.3 (SD: 8.39)  independently of who pays for them. This is the most extensive approach, and enables data to be disaggregated for further analyses. This approach also enabled us to classify costs according to disability. In addition, we could obtain the costs derived from each disease-related item separately, which may be useful when establishing adequate therapeutic strategies and making cost-effectiveness analyses. An important disadvantage of this approach is the difficulty involved in selecting a representative sample. However, the alternative "top-down" approaches (when data about diagnoses and resources consumed because of MS are obtained from published sources), also have a significant disadvantage: available data are more limited and mostly retrospective. The sample we analysed in this study is representative of the MS population attended in our MS Unit. It included patients from the entire spectrum of the disease .The main purpose of the study was to obtain the costs of MS at different levels of disability. Therefore, we decided to group patients into clinically relevant stages of disability, in order to see whether the costs varied significantly among such stages . There were marked differences in costs among the five disability stages, particularly for direct costs Table . The difThe methodology used in our study has also been widely employed in the literature. Therefore, comparisons of our results with those obtained from similar studies carried out in other countries may be possible. However, direct comparisons with available data from other costs studies can not be made because of differences in healthcare systems, healthcare usage, absolute and relative costs.Major limitations of the study include: the retrospective collection of data, and the small number of patients involved (200). There was a low response rate to the questionnaire, which may have been due to the questionnaire's complexity. We did not carry out a second mailing, and did not personally insist on patients responding in order to guarantee their willingness. The fact that we did not find statistically significant differences between patients who responded to the questionnaire and those who did not minimizes the possibility of a selection bias.There was a higher proportion of patients on IT (67.5%) in our sample than in the general MS population. This was probably because treated patients tend to visit our MS Unit frequently . In addition, they may feel more involved with the MS Unit and therefore may be more willing to answer the questionnaire (47.5% of patients who did not answer the questionnaire were on IT). We made a rough estimation of costs for the general MS population by adjusting the costs  of IT toAnother limitation of the study was the lack of information on costs attributable to MS-associated neuropsychiatric complications and cognitive impairment. Such costs were not specifically measured in our patients, even though cognitive and neuropsychiatric factors have been shown to have a significant impact on MS patients .There are many controversial aspects in the methodology employed. Firstly, the "Human Capital Approach" has a significant limitation. It tends to underestimate the costs for people who are not in paid employment at the time of the study. Therefore, results obtained using this approach have to be considered a minimum estimation of costs. In spite of this, it is the most commonly used method in the literature. Secondly, if tariffs are used as unit costs when no other opportunity costs are available -as in our case- costs can be overestimated  or underWe consider that this study represents an exhaustive estimation of the real costs of MS. It shows that the direct costs are higher than indirect costs, and that the overall costs increase as the disability progresses. The main factors that contribute to direct costs differ depending on the EDSS stage. IT is the most important factor when the EDSS is below 7. Informal care is the most significant factor when the EDSS is above 7.5. IT accounts for 19% – 34% of total costs, depending on the percentage of patients treated .Our results are consistent with other costs studies reviewed in the literature -32, whicDespite the limitations described, cost-of-illness analyses like this study, which are based on real MS population data rather than on selected patients from trials, are important. They can be used to perform cost-effectiveness analyses which can demonstrate whether or not immunomodulatory agents are cost-saving in the long term, under real conditions of clinical practice.The present study reflects the significant social and healthcare impact of a chronic disease like MS. This impact increases with the progression of disability (EDSS).The total mean societal costs of MS estimated in our sample, a cohort of MS patients monitored in our MS Unit in Baix Llobregat , were 24272 euros per patient/year. Costs ranged from 14327 euros (EDSS = 0) to 52841 euros (EDSS = 7.5–9.5). The direct costs were higher than indirect costs in all EDSS stages. This was mostly due to IT use in lower EDSS stages, and to informal care in higher EDSS stages.Conclusions from this kind of analyses will be helpful for clinicians and for health care authorities, as cost-of-illness studies should be a first and indispensable step toward analysing the cost-effectiveness of IT. The cost-effectiveness of treatments is an increasingly important issue in policy decision-making which helps to optimise available resources.The author(s) declare that they have no competing interests.VC conceived of the study and participated in: its design, the data collection, the data analysis, and the draft of the manuscript. OC, LA, LR, EM, and LG participated in the data collection and contributed to the conception and design of the study. SMY and AMY participated in the design of the study and made substantial contributions to the data analysis and to the manuscript revision. TA coordinated the study, participated in its conception and its design, and reviewed the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In a recent study, the majority of three genomes of previously unknown positive-sense single-stranded (ss) RNA viruses were assembled from reverse-transcribed whole-genome shotgun libraries. The present contribution comparatively analyzes these genomes with respect to representative viruses from established viral taxa.RNA viruses have been isolated that infect marine organisms ranging from bacteria to whales, but little is known about the composition and population structure of the Picornavirales that fall into a well-supported clade of marine picorna-like viruses, the characterized members of which all infect marine protists. A temporal and geographic survey indicates that the JP genomes are persistent and widespread in British Columbia waters. The third genome, SOG, encodes a putative RNA-dependent RNA polymerase (RdRp) that is related to the RdRp of viruses in the family Tombusviridae, but the remaining SOG sequence has no significant similarity to any sequences in the NCBI database.Two of the genomes (JP-A and JP-B), appear to be polycistronic viruses in the proposed order Tombusviridae, SOG lacks a capsid and movement protein conserved within this family and SOG is thus likely more distantly related to the Tombusivridae than the RdRp phylogeney indicates.The complete genomes of these viruses permitted analyses that resulted in a more comprehensive comparison of these pathogens with established taxa. For example, in concordance with phylogenies based on the RdRp, our results support a close homology between JP-A and JP-B and RsRNAV. In contrast, although classification of the SOG genome based on the RdRp places SOG within the  RNA viruses of every classification have been isolated from the ocean; nevertheless, the marine RNA virus community remains largely uncharacterized. Although there are several examples of RNA viruses that infect marine animals  these orPicorna-like viruses are a "superfamily" of positive-sense single-stranded RNA (ssRNA) viruses that have similar genome features and several conserved protein domains . PreviouTombusviridae and the genus Umbravirus. Moreover, in both libraries, a high percentage of sequence fragments were part of only a few contiguous segments of sequence (contigs). Specifically, in the SOG sample 59% of the sequence fragments formed a single contig. Similarly, 66% of JP sequence fragments contributed to only four contigs that represented two viral genomes. Using a RT-PCR-based approach to increase the amount of sequence for each dominant contig resulted in the assembly of three complete viral genomes. This contribution analyzes these genomes from three previously unknown marine RNA viruses and investigates their similarities and differences with respect to representative genotypes from established viral taxa.In a recent study, reverse-transcribed whole-genome shotgun libraries were used to characterize two marine RNA virus communities . PositivThe two assembled genomes (JP-A and JP-B) from the Jericho Pier sampling site Figure  are singThe JP-A genome is positive-sense, 9212 nt in length with a 632 nt 5' untranslated region (UTR) followed by 2 predicted open reading frames (ORFs) of 5067 nt  and 3044 nt  separated by an intergenic region (IGR) of 149 nt Figure . ORF 2 iE value < 0.001) to nonstructural protein motifs of several viruses, including members of the families Dicistroviridae (Drosophila C virus), Marnaviridae (HaRNAV), Comoviridae (Cowpea mosaic virus) and the unassigned genus Iflavirus (Kakugo virus). The top matches for ORF 1 were to RsRNAV , a newly sequenced, unclassified positive-sense ssRNA virus that infects the widely distributed diatom Rhizosolenia setigera  and Drosophila C virus , a positive-sense ssRNA virus that infects fruit flies. Comparison of the protein sequence predicted to be encoded by ORF 2 of JP-A to known viral sequences shows that it has significant similarities to the structural proteins of viruses from the families Dicistroviridae (Drosophila C virus), Marnaviridae (HaRNAV), and the genus Iflavirus (Varroa destructor virus 1). The sequences that are most similar to ORF 2 of JP-A were the structural protein regions of RsRNAV , HaRNAV  and SssRNAV .Comparison to known viral sequences shows that the protein sequence predicted to be encoded by ORF 1 of JP-A contains conserved sequence motifs characteristic of a type III viral Helicase (aa residues 430 to 545), a 3C-like cysteine protease (aa residues 1077 to 1103) and a type I RdRp (aa residues 1350 to 1591)  searchesThe JP-B RNA genome is also likely from a positive-sense ssRNA virus. The 8839 nt genome consists of a 5' UTR of 774 nt followed by two predicted ORFs of 4842 nt  and 2589 nt  separated by an IGR of 298 nt (nt position 5617 to 5913) Figure . The 3' E value < 0.001) to nonstructural genes from positive-sense ssRNA viruses from a variety of families, including the Comoviridae (Peach rosette mosaic virus), Dicistroviridae (Taura syndrome virus), Marnaviridae (HaRNAV), Sequiviridae  and Picornaviridae . The top scoring sequences  were to a RdRp sequence from RsRNAV and a partial picorna-like virus RdRp from an unidentified virus  amplified from the same JP station during an earlier study  and HaRNAV  and SssRNAV [E value = 1 × 10-40. identities = 156/588 (26%)].The position of core sequence motifs conserved among positive-sense ssRNA viruses and BLAST searches of the NCBI database with the translated JP-B genome suggest that nonstructural proteins are encoded by ORF1, and the structural proteins are encoded by ORF2. We identified conserved sequence motifs in ORF 1 characteristic of a type III viral Helicase (aa residues 328 to 441), a 3C-like cysteine protease (aa residues 882 to 909) and a type I RdRp (aa residues 1143 to 1408)  searchesDicistroviridae. Several of these viruses contain internal ribosome entry sites (IRES) . This virus belongs to the genus Necrovirus in the family Tombusviridae that has a host range restricted to higher plants , a member of the unclassified genus Umbravirus whose known members infect only flowering plants [We identified the eight conserved motifs of the RdRp  in the Sr plants . SOG is g plants .Tombusviridae and genus Umbravirus, the remaining SOG sequence has no detectable similarity to any other known sequence. A Bayesian maximum likelihood tree based on alignments of the SOG RdRp with the available Umbravirus sequences and representative members of the Tombusviridae indicates that the SOG genome forms a well supported clade  with the single member of the genus Avenavirus, OCSV . The novelty of JP-A, JP-B and SOG, as revealed by sequence analyses and genome characterization, suggests that most of the diversity in the marine RNA virus community remains uncharacterized. Furthermore, these results raise the hypothesis that the genomes of these marine RNA viruses that we propose to infect single-celled eukaryotes may be more similar to the ancestral RNA viruses that gave rise to those that infect higher organisms.The shotgun libraries were constructed from seawater samples collected from two stations, JP (Jericho Pier), a site in English Bay adjacent to the city of Vancouver, British Columbia and SOG (Strait of Georgia), located in the central Strait of Georgia next to Powell River, B.C. Figure .The locations of the stations where one or both of the JP genomes were detected are shown in Figure Concentrated virus communities were produced as described by Suttle et al. . Twenty E values were < 0.001. The details for viruses used in phylogenetic analyses are listed in additional file A detailed description of the whole genome shotgun library construction protocol can be found in Culley et al. . BrieflyThe 5' and 3' ends of the environmental viral genomes were cloned using the 5' and 3' RACE systems (Invitrogen) according to manufacturer's instructions. The 3' RACE with the SOG genome required the addition of a poly (A) tract with poly (A) polymerase (Invitrogen) according to manufacturer directions before cDNA synthesis. cDNA was synthesized directly from extracted viral RNA from the appropriate library. Three clones of each 5' and 3' end were sequenced.Taq DNA polymerase (Invitrogen) in 1× Platinum Taq buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, and 0.2 μM of each primer  with primers JP-A 5 and 6 and JP-B 6 and 7  declare that they have no competing interests.AC contributed to the design of the study, performed the lab work, analyzed the data and drafted the manuscript. AL contributed to the design of the study, analyzed the data and helped prepare the manuscript. CS was involved in the conceptualization and design of the research and in manuscript preparation. AC, AL and CS have read and approved this manuscript.PCR primers used to complete the three genome sequences. The table provides detailed information about the primers used to complete the three viral genome sequences.Click here for fileVirus sequence details. Organized by taxonomic group, the table provides the full name, acronym and NCBI accession number for the viruses used in phylogenetic analyses.Click here for file


The cost-benefit of raccoon rabies control strategies such as oral rabies vaccination (ORV) are under evaluation. As an initial quantification of the potential cost savings for a control program, the collection of selected rabies cost data was pilot tested for five counties in New York State (NYS) in a three-year period.Rabies costs reported to NYS from the study counties were computerized and linked to a human rabies exposure database. Consolidated costs by county and year were averaged and compared.Reported rabies-associated costs for all rabies variants totalled $2.1 million, for human rabies postexposure prophylaxes (PEP) (90.9%), animal specimen preparation/shipment to laboratory (4.7%), and pet vaccination clinics (4.4%). The proportion that may be attributed to raccoon rabies control was 37% . Average costs associated with the raccoon variant varied across counties from $440 to $1,885 per PEP, $14 to $44 per specimen, and $0.33 to $15 per pet vaccinated.Rabies costs vary widely by county in New York State, and were associated with human population size and methods used by counties to estimate costs. Rabies cost variability must be considered in developing estimates of possible ORV-related cost savings. Costs of PEPs and specimen preparation/shipments, as well as the costs of pet vaccination provided by this study may be valuable for development of more realistic scenarios in economic modelling of ORV costs versus benefits. Rabies, caused by infection with a lyssavirus, has a worldwide distribution and has affected mankind since antiquity. Rabies is almost 100% fatal following serious neurological symptoms and great suffering . The recThe epidemiology of rabies in the U.S. has changed from a predominance in domestic animals to a predominance in wild animals . Bat rabIn the eleven eastern states from North Carolina to New York (NY), 82% of the counties reported the presence of rabid raccoons during 1977–1997 . AnotherA major cost associated with rabies is human postexposure prophylaxis (PEP), sometimes referred to as postexposure treatment. In the U.S., PEP consists of five doses of cell culture-derived rabies vaccine over the period of a month plus a single dose of rabies immune globulin . PEP is Primary prevention of rabies is based on animal vaccination , and manAlthough the use of ORV in Canada and France has been linked with reductions in rabid animals ,27, the NY is representative of many states with multiple rabies variants and a mixture of urban, suburban and rural areas with a large human population potentially at risk for exposure ,31. RaccBy law, NY places responsibility for rabies control and surveillance on the 57 local health departments (LHDs) outside New York City (NYC). NYSDOH operates a unique rabies reimbursement and reporting system for authorized PEPs , collection, preparation and submission of animal specimens to the State's Rabies Laboratory for testing, and five annual no-cost rabies vaccination clinics for dogs, cats and ferrets per county (recently reduced to three per year).LHDs submit quarterly vouchers detailing expenses for these categories of rabies prevention activity. PEP expenses are reimbursed if the case has been reported using the NYSDOH reporting system, and are limited to a maximum of $1,000 per patient. For animal specimens, reimbursement requires reporting species, laboratory ID number, date of shipment, and cost of preparation/shipping, and is limited depending on species. Vaccination clinic reimbursement is limited to $5000 per county per year .Previous studies of NY rabies PEP and rabies costs did not assess costs by variant ,17 or vaTo pilot test the computerization and analysis of rabies-control costs reported to NYSDOH, with emphasis on raccoon variant-associated costs to provide more specific cost estimates for ORV cost-benefit modelling, five counties were selected to represent diverse conditions of demographics, rabies epidemiology and reporting.This study includes reimbursement data from three consecutive fiscal years (April through March): 1999–2000, 2000–2001, and 2001–2002. Five counties were selected for the analysis, to represent diverse areas: the lower Hudson valley, upper Hudson valley, western NYS, central NYS, and northeastern NYS. One of the counties had an ORV program. Four counties have full service LHDs, including an environmental health program responsible for rabies exposure investigations. One of the LHDs is 'partial service' and relies on public health nurses for rabies follow-up. These counties represent 12.9% of the NY population, and 23.5% of the population outside NYC (Census 2000 data). With only one partial service county, which also was the only county with ORV, the association of cost data with being a partial service county and being exposed to ORV was not examined in this pilot study.Vouchers were reviewed to identify PEPs begun during the study fiscal years, and vaccination clinics and specimen shipments during this same time period. A master list of names was used to link computerized databases. After data matching, cleaning, and extraction, names were deleted retaining identification codes from original data sources. Documents used for specimen preparation/shipment cost calculations were private carrier bills (UPS/Airborne Express), veterinarian bills/vouchers and information on personnel salaries.Variables included expenses and dates for PEP treatments, specimen preparation/shipment, and vaccination clinics; amount paid by third party insurers for PEPs; amount of donations collected for vaccination clinics; amount reimbursed to LHDs by the State; and species involved in incidents or submitted for testing. When the amount of donations collected for vaccination clinics exceeded costs, the county cost was considered zero and the donation excess was not accounted in the total clinic cost.LHD telephone interviews were used to determine how the LHDs calculated the specimen shipment costs and the information sources used for the calculations; and to understand the meaning of low values reported in the cost of PEPs and specimen shipments.To determine which control costs might be reduced or eliminated due to a successful ORV program, animal species involved in human rabies exposures were grouped into bats and non-bats . Most of the rodents were grouped together with the exception of squirrels and woodchucks because there were rabies cases in these species in recent years in the U.S. . All terThe NYSDOH Wadsworth Center's Rabies Laboratory tests the variant for all rabid animals other than raccoons, and for a periodic sample of raccoons. A 20 year assessment identified variants other than raccoon (bat variants) for only 14 (0.1%) of 14,582 rabid terrestrial animals . Thus, fIn addition to a calculation of overall costs, statistics were calculated by county and fiscal year. Correlation coefficients were calculated for PEP and specimen costs with county population. A Pareto analysis was done for PEP and specimen costs by species.To determine whether PEP costs due to terrestrial rabies are the same for patients exposed in the county of treatment and those exposed outside the treatment county, the average PEP cost was calculated and compared for the two groups. The null hypothesis was that the average cost of PEP was the same for both groups. This hypothesis was tested using Students' t-test for comparison of means. For this analysis we assumed the exposures are independent events.Data management and statistical analyses were conducted using MS Access, MS Excel, and SAS. For graphics S-Plus, MS Excel and MS PowerPoint were used.For the study counties, the total cost of prevention activities for both terrestrial and bat rabies during fiscal years 1999–2001 was $2,143,905. Of this total, $1,948,008 (90.9%) represented PEP costs, $100,278 (4.7%) represented specimen preparation/shipment costs, and $95,618 (4.4%) represented pet vaccination clinic costs. Table The total expenses that may be attributed to terrestrial rabies in this study were $784,529 (36.6%) for the three-year period, if including all vaccination clinic costs. Without clinic costs, the total was $688,911 (32.1%) . Terrestrial rabies-associated PEP costs were $618,274 (32%) (Table 2%. TerreCats were associated with 43% 281) of the terrestrial rabies PEPs, dogs with 27% (179), and raccoons with 16% (102)  of the patients receiving PEP due to terrestrial rabies exposures were exposed in a county other than the one providing the treatment and twenty-nine (4.4%) were exposed outside the State or country. The average PEP cost for patients exposed in the county providing the treatment was $959.25, compared to an average cost for patients exposed elsewhere of $793.19 . The association of this factor (treatment within or outside exposure county) with completion of treatment was not assessed.The average per capita cost for terrestrial rabies PEPs during the three-year study period was $0.25, with a range of $0.08 to $0.34. The county population size was highly correlated with the number of PEPs (r = 0.86) and PEP costs (r = 0.75), but not with PEP average cost (r = -0.21). A cost < $300 was reported for 132 terrestrial rabies-associated PEPs. LHDs reported these represented patients not receiving a full five-dose vaccine series and immune globulin, PEP for lower weight children (because HRIG costs are based on body weight), or incomplete submission of bills. The reasons provided for not receiving a full treatment series included repeat PEPs that only require two doses of vaccine, stopping the PEP when the animal was found not to be rabid, and stopping the PEP after it was started by an emergency room, private doctor or health center out of county after determining it was not needed. Having the exposure and PEP in different counties was also reported as a cause for incomplete submission of bills to the LHD.During the study period, 4,393 specimens were submitted for rabies testing from the study counties at a cost of $95,618. About 46%  of the specimens were terrestrial animals, but these specimens accounted for 70% of the submission costs  ; dogs 19% 392); raccoons 15% (305), and skunks 11% (218) . The county population size was highly correlated with the number of specimens (r = 0.80), specimen preparation/shipment costs (r = 0.81), and the average specimen cost (r = 0.76).LHD interviews indicated that veterinarians are contracted for specimen preparation in three counties. One county uses its own personnel. In two counties, veterinarians are paid according to the species, whereas in a third county veterinarians are paid a fixed fee per specimen. However, the reimbursement vouchers for that county did not itemize the veterinarian fee and the vouchers reported varying costs. In one county, veterinarians ship the specimens and report the cost to the LHD. Two counties report no cost for some shipments, usually for bats. Although shipment costs vary based on specimen weight, one county calculates a fixed-cost per specimen for reimbursement purposes. The formula is based on the average time to prepare a specimen, staff salary for that time, and average shipping costs.There were 26,185 animals vaccinated in study county-sponsored clinics. The total cost was $95,618, of which $34,362 (36%) was reimbursed by NYSDOH . County population size was not correlated with the number of animals vaccinated (r = -0.02), but was inversely correlated with clinic total costs (r = -0.57) and partially correlated with the average cost per animal vaccinated (r = 0.32).The cost summary presented in this pilot study is an initial quantification of rabies reimbursement data in NYS for PEPs, specimen preparation/shipment, and pet vaccination for five counties over a three-year time period. This data may assist in determining the parameters for the potential savings to be achieved if ORV or some other prevention/control program is used to reduce or eliminate the raccoon rabies virus variant in an eastern U.S. state. This study found that terrestrial rabies accounts for more than a third (37%) of the reimbursed rabies prevention and control costs. The study found that in NYS, the State bears the largest proportion of the reported costs. About half of all expenses were covered by the State in the five counties studied. The LHDs covered 7% of the total expenses. While the amount spent by LHDs is a small proportion of the total costs of terrestrial rabies control reported in this study, for some counties this may represent an important impact to their budget.The per-capita cost of terrestrial rabies including PEP, specimen cost and pet vaccination clinics costs was estimated to be $0.32. However, use of this average in estimates of potential savings from ORV may considerably over- or under-estimate the potential savings as indicated by the county variation in average costs from $0.10–$0.77 per person. Although counties with larger population sizes had greater PEP and specimen costs, the per capita cost was highest in the smallest county, perhaps reflecting economies of scale in larger counties able to prorate their costs over a larger number of exposures. The fact that total terrestrial variant-reimbursed costs are increasing despite a relatively stable enzootic state of rabies in NYS, and active prevention and control programs, should be considered when estimating the future savings potentially associated with use of control programs.PEP expenses account for about 90% of the overall costs reported in this study. Thus, PEP is a large component of possible cost savings to be produced by public health intervention to control raccoon rabies. If ORV is used effectively to eliminate the raccoon variant, raccoon attacks are likely to be minimized and there will be no spillover of rabies to other species from this variant, thus reducing the total number of rabid animals and human exposures. In this study about 16% of the PEPs attributed to raccoon variant rabies were related directly to raccoon incidents, and 4% were due to skunk incidents. With raccoons and skunks unlikely to attack people if not rabid, it can be assumed that raccoon rabies elimination would eliminate most of the PEPs related to these species. Exposure to these two species represented 131 incidents for the 5 counties in the 3-year study period, at an estimated PEP cost of $125,271 representing 16% of the total terrestrial-associated costs.Humans have more frequent contact with domesticated species than with wildlife, and bites to humans from these species frequently occur for reasons unrelated to rabies. In this study bites from dogs and cats accounted for 70% of the total terrestrial-associated PEP costs. Although elimination of the raccoon variant with ORV would reduce the number of rabid pets and other domestic animals and thus exposures to rabies, bites from pets will continue. Thus elimination of raccoon variant would result in a reduction in PEP costs associated with dog and cat incidents, but the reduction may not be substantial because most dog and cat bites are not due to the animal being rabid. PEP costs associated with pet exposures will likely not decline significantly until there is sufficient assurance that the raccoon variant has truly been eliminated and the probability of a pet being rabid is sufficiently low that PEP is not warranted even when the pet's rabies status cannot be verified. Changes in policies about providing PEP when a pet's rabies status cannot be determined would require a sufficient period without raccoon variant and without it encroaching from neighboring areas. These changes also assume no other terrestrial variants with frequent spillover to pets. If the raccoon variant had been eliminated from these five counties during the three-year study period and there was sufficient confidence that terrestrial animal-related exposures did not require PEP consideration, the maximum potential savings would have been 657 PEPs avoided at an estimated cost savings of $618,274.With control of raccoon variant rabies, there should be fewer wildlife specimens to submit. This study found that 70% of the specimens submitted for testing were for terrestrial species, primarily dogs, cats, raccoons and skunks. If all raccoon and skunk submissions were eliminated by eliminating raccoon variant rabies, the cost savings for the study period would have been 26% of the total cost for terrestrial rabies specimen preparation/shipment, representing a savings of 523 specimens that would not have been prepared and shipped. This would yield an estimated cost savings of $24,228, accounting for 3% of the total terrestrial-associated costs.Dog and cat specimens are submitted when the animal is showing signs of rabies because neurologic animals must be euthanized and tested after a bite to a human. Even with elimination of the raccoon variant, there will be occasional bites from neurologic pets due to bat rabies or other diseases. However, specimen shipments are likely to be considerably reduced. In NYS, avoiding 4,393 specimen shipments for all species at a cost savings of $95,618 is an upper limit of the potential savings in five counties over a three-year period.NYS law requires rabies vaccination of pet dogs, cats and ferrets. This requirement would not be modified in a scenario where ORV controlled or eliminated raccoon rabies because bat variants remain. The possibility of translocation or reintroduction of rabies in wildlife also remains, therefore pets will continue being vaccinated.An elimination of terrestrial rabies could lead to a reconsideration of the need for State resources to pay for pet vaccination clinics. Thus, State-associated costs of $34,362 could be considered a possible savings to be achieved with ORV. However, if pet vaccination continues as recommended by national guidelines and required by state laws, total vaccination costs may potentially increase if the costs associated with individual veterinarian vaccinations are higher than mass clinic costs. The current study indicates a wide disparity in the per-animal cost of rabies vaccination, ranging from $0.33 for a large county that offers mass vaccination clinics to $15.00 for a large county that reimburses a flat fee to veterinarians for vaccinating pets at their regular practice.An initial pilot study of the reimbursements provided for rabies costs in five NYS counties over a three-year period indicates a wide variability in costs by county. Counties with larger populations experienced larger PEP and specimen shipment/preparation costs. The study indicated that there are wide variations in the ways that LHDs estimate rabies-associated costs. Such potential variation must be considered in developing estimates of possible ORV-related cost savings. Within the limitations of LHD estimations of cost data, this study offers specific cost data for two critical factors, PEPs and specimen preparation/shipments, as well as the costs of pet vaccination. Actual data such as that obtained in this pilot study are critical for basing the parameters chosen for economic modelling of potential ORV benefit on realistic scenarios.Interviews with counties indicated that cost data is not universally complete or accurate if based on estimates. Year-specific costs may be affected by lags in reporting PEP costs associated with insurance paperwork.Overall averages for specimen preparation/shipment were artificially lowered by the inclusion of a county with the NYSDOH Rabies Laboratory which can hand deliver specimens and thus incurs almost no specimen shipment costs. The unexpected lower average cost for large animal specimen preparation/shipment may also lead to an underestimate of the average cost for all specimens. In one county, species vaccinated was not reported, and in another county, the reported cost per animal was below the actual cost of vaccine and other supplies.The five upstate New York counties may not be representative of other areas, although they represent almost a quarter of the State's non-NYC population. With a larger study, factors such as population density, health care access, LHD capabilities, and animal rabies incidence could be assessed for their impact on rabies-associated costs. The influence of ORV itself could not be estimated with only one ORV county in the study.Assuming that these costs associated with raccoon rabies would all be saved if ORV eliminated the raccoon variant is only applicable to areas with no other terrestrial variants. With bats having an insignificant impact on rabies in most other species (except humans), eliminating the raccoon variant could indeed result in elimination of most of the costs associated with the current raccoon-variant outbreak. However, PEP costs are clearly the largest component of the costs documented in this study. Until health care and public health professionals are sufficiently confident of the low risk of rabies from terrestrial animal exposures in areas where terrestrial variants have been eliminated, PEPs will continue to occur in relation to these exposures. With the evidence of raccoon variant in skunks tracking similarly to the raccoon variant in raccoons , estimatThe most significant limitation on potential PEP savings from terrestrial variant control is the fact that in NYS only 31% of the PEPs were due to terrestrial animal exposures, representing only 29% of the total PEP costs. There are also many other rabies-associated costs that require better data in order to appropriately model potential ORV-associated savings, including costs associated with animal confinements, quarantines, and euthanasias, personal costs to those receiving PEP including time lost, pain and suffering, and costs to governmental agencies for managing the rabies cases and the consequent consulting, investigation, and prevention/control measures.The current study has demonstrated the feasibility of obtaining actual cost data for several important rabies-associated cost variables, and the variability in such costs across time and geographic area. Additional studies are required to include a larger area and additional important cost variables. In the interim, the data from this pilot study may be helpful in refining the parameters used in any additional ORV cost-benefit analyses for the elimination or reduction of raccoon-variant rabies, including modelling studies of animal to human transmission risks. In these studies, it will be critical to include varying levels of bait density and geographic size of baited areas, in addition to estimates of vaccine efficacy, as components of the cost-benefit assessments.The author(s) declare that they have no competing interests.SR carried out the computerization of the cost data, performed statistical analysis and drafted the manuscript. BC participated in the analysis design and result interpretation. ME conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Most of us don't have much trouble recognizing what we see. Whether it is a face in a crowd, a bird in a tree, or papers on a desk, our brains expertly distinguish the target from the clutter. It is a simple skill most of us take for granted, but object recognition is not hard-wired. As we navigate our environment, the brain's visual centers continually reorganize themselves, classify novel features, and learn to pick out important objects from the background. Just how the human brain does this is not well understood, but new research by Zoe Kourtzi and colleagues may have uncovered some important clues.To investigate how the human brain learns to separate targets  from noise Kourtzi et al. showed subjects pictures of novel shapes embedded in a cluttered background and asked the subjects to determine whether or not the shapes were symmetrical. The researchers recorded the subjects' responses while using functional magnetic resonance imaging (fMRI) to measure neuronal activity in brain regions associated with visual processing. Each subject was tested using two sets of novel shapes: high-salience shapes (shapes easily distinguished from the background), and low-salience shapes (shapes camouflaged by the background). After the initial testing, the subjects were trained to recognize a subset of the new shapes from each group, and then re-tested.Visual input is thought to go through a hierarchy of processing centers that transform retinal images into complex objects and scenes. Kourtzi et al. recorded responses from both early  and late  stages of visual analysis in 26 subjects. The authors found that subjects demonstrated an increased number of correct responses for shapes they encountered during the training sessions, regardless of the type of background the shapes were presented on. By contrast, the fMRI responses differed dramatically, depending on whether the surroundings made the shapes easy or difficult to detect. Low-salience shapes triggered an increased fMRI response across all brain regions following training; high-salience shapes precipitated a decrease in fMRI response in the regions of the lateral occipital cortex, but produced no change in any of the early visual areas . These results demonstrate that the ability to learn to detect novel shapes is independent of the degree of difficulty, but suggest that the brain employs different mechanisms of perceptual learning depending on whether the objects stand out from their surroundings, or are obscured by them. Learning to detect highly camouflaged shapes results in increased brain activity levels that are presumed to reflect an increase in signal processing at the level of both the early visual areas and higher levels of cortical analysis. On the other hand, the reduction of neural activity that occurs during learning of more distinctive shapes likely reflects efficient neural coding of the critical features for their recognition at later stages of visual analysis.According to Kourtzi and her colleagues, their results provide evidence that the visual brain is capable of tailoring the mechanism of perception to best suit the task. When the signal is weak—as in the case of viewing camouflaged targets—learning amplifies neural responses to the target shapes and drowns out the noise. But when the signal is strong—as in the case of viewing easily distinguishable, highly salient targets—neural activity in the visual cortex is reduced, possibly because training engages smaller populations of neurons that respond much more selectively to distinctive features of the stimulus.In other words, all visual stimuli are not treated equally, and with just cause: the brain's unique ability to treat ambiguous signals differently than robust ones likely allows it to optimize neural coding, and in doing so, learn to increase detection of a broad spectrum of visual signals.


Natural products have numerous medicinal applications and play important roles in the biology of the organisms that accumulate them. Few methods are currently available for identifying proteins that bind to small molecules, therefore the discovery of cellular targets for natural products with pharmacological activity continues to pose a significant challenge in drug validation. Similarly, the identification of enzymes that participate in the biosynthesis or modification of natural products remains a formidable bottleneck for metabolic engineering. Flavonoids are one large group of natural products with a diverse number of functions in plants and in human health. The coupling of flavonoids to small ceramic and glass beads provides a first step in the development of high-throughput, solid-support base approaches to screen complex libraries to identify proteins that bind natural products.The utilization of small glass and ceramic beads as solid supports for the coupling of small molecules was explored. Initial characterization of the beads indicated uniform and high capacity loading of amino groups. Once the beads were deemed adequate for the linking of small molecules by the coupling of NHS-fluorescein followed by microscopy, chemical hydrolysis and fluorometry, the flavonoid naringenin was modified with 1,4-dibromobutane, followed by the attachment of aminopropyltriethoxysilane. After NMR structural confirmation, the resulting 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin was attached to the ceramic beads.Our results demonstrate that ceramic and glass beads provide convenient solid supports for the efficient and facile coupling of small molecules. We succeeded in generating naringenin-coupled ceramic and glass beads. We also developed a convenient series of steps that can be applied for the solid-support coupling of other related flavonoids. The availability of solid-support coupled naringenin opens up new opportunities for the identification of flavonoid-binding proteins. Natural products are small molecules synthesized by bacteria, algae, fungi and plants, as part of their biotic and abiotic interactions with the environment. They provide a valuable source of pharmaceuticals, with 45% of the most popular drugs derived from natural products . Howeverin vivo animal and plant cellular targets for flavonoids are largely unknown.Flavonoids, derived from phenylpropanoids, are widely distributed throughout the plant kingdom and are abundantly present in many fruits and leaves . They arFew methods are currently available for identifying proteins that bind to small molecules of plant origin, for example flavonoids. The development of high-throughput methods for the identification of flavonoid-binding proteins could significantly advance our understanding of the mechanisms by which flavonoids modulate plant hormone transport , contribHere, we describe the efficient coupling of the flavanone naringenin, a central intermediate in the flavonoid biosynthetic pathway , to smal3 and a diameter of approximately 2 μm, about 5 times smaller than the glass beads . Thus, not only were the beads efficiently loaded with amino groups, but this loading was also uniformly distributed among the beads. Together, these results indicate that both the ceramic and the glass beads met the desired requirements for serving as solid supports for the coupling of flavonoids, and the amino-modified beads have further potential for solid phase synthesis.In order to determine the effective loading of amino groups on the beads, both naïve (unmodified) and amino-modified glass and ceramic beads were incubated with NHS-fluorescein in the dark at 4°C for 24 hrs. After completion of the reaction, the beads were washed with water and dried. Naïve glass or ceramic beads displayed no fluorescence, nor did they fluoresce after incubation with NHS-fluorescein, while amino-modified glass and ceramic beads fluoresced after reaction with fluorescein Fig. . To quane.g., proteins) that were intended to be probed.Initially, it was envisioned that the amino-loaded ceramic beads could be used for the direct coupling of naringenin, in the presence of a convenient linker such as dimethyl suberimidate (DMS). This approach, however, resulted to be impractical because technical limitations prevented the quantification of the amount of naringenin coupled to the beads, and it was feared that non-reacted amino groups on the beads or imidoester groups on the DMS linker would react with components of the biological system (1) with little amount of 7,4'-di(4-bromo-n-butoxy) naringenin as a by-product [2). The compound was purified by chromatography on silica gel and confirmed to be 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin by NMR. Once the identity of compound (2)was confirmed, its coupling to sodium hydroxide-treated ceramic beads in 95% methanol was performed overnight. Separation of the beads from non-conjugated compound (2)was accomplished by filtration and washing.Therefore, a different strategy was utilized, where the linker was first linked to the organosilane group on one end and naringenin on the other Fig. , followe-product . After s2)Fig.  was coup2)Fig. . The flu2)Fig. .2)was first calculated at 314 nm, corresponding to one of the absorption peaks of this compound that remained in solution after a coupling reaction was carried with a slight molar excess of 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin (assuming a bead capacity of 0.34 μmol/g). The loading of compound (2)was established to be 0.465 μmol per gram of beads, corresponding to approximately 5.5 × 106 molecules of 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin per ceramic bead. Using a similar approach, the coupling of 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin to the glass beads was established to be 0.00016 μmol/g, corresponding to 5.3 × 104 molecules per glass bead.To further quantify the amount of 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin coupled to the beads, the molar extinction coefficient (ε) of compound . Fluorescence was measured in a FLEX Station™ Instrument  with the SOFTmax@PRO program.Scanning electron micrographs were obtained on a Philips XL Series Scanning Electron Microscope (SEM). Fluorescence micrographs were obtained on a Nikon Eclipse E600  microscope equipped with a OSRAM HBO Mercury Short Arc Lamp (λ1H and 13C NMR spectra were recorded on a Bruker NMR spectrometer (DRX-250 and DRX-500) in acetone-d6 (Aldrich). UV absorption spectra were performed on a Cary 50 Bio UV-Visible spectrophotometer. Data was analyzed by the Varian Cary WinUV Scan application.Column chromatography was performed on silica gel  using the indicated solvents. TLC analyses were carried out using silica gel plates (Aldrich).Five grams of ceramic beads (3M™ Zeeospheres™ Ceramic Microspheres) or glass beads (Aldrich) were shaken in 20 mL of 10% sodium hydroxide solution overnight, and subsequently washed with water (40 mL), 1% hydrochloric acid (40 mL), water (40 mL) and methanol (40 mL), three times each. Then, the ceramic beads were shaken in 30 mL of 3% aminopropyltriethoxysilane solution made in 95% methanol at 4°C overnight, followed by washing with methanol (40 mL) and water (40 mL), three times each. After air-drying, the amino-modified ceramic beads were stored at 4°C.Amino-modified ceramic beads (0.3 g) were added to 1 mL of 50 mM sodium bicarbonate buffer (pH ~8.5). In a dark room, 0.2 mg of NHS-fluorescein  were dissolved in 200 μL of DMSO, and then the NHS-fluorescein solution was added to the sodium bicarbonate suspension of beads and mixed well. The suspension was protected from light and shaken at 4°C for 24 hrs. After the liquid was removed by centrifugation and washed extensively with water, the fluorescein-bound ceramic beads were dried and stored at -20°C for future use. To compare with the control, bare (not amino group loaded) ceramic beads were treated under the same conditions. To test the fluorescence of bare and modified beads, all beads were separately shaken in a mixture of 200 μL of DMSO and 1 mL of 50 mM sodium bicarbonate buffer and dried.Fifty mg of fluorescein-loaded ceramic beads were shaken in 1 mL of 1 N hydrochloric acid solution at 4°C for 4 days in the dark. After filtration through a 0.45 μm cellulose filter and washed with water, the filtrate was neutralized with 50 mM sodium bicarbonate buffer. To compare with the control, all bare beads incubated with or without fluorescein were treated under the same conditions. Fluorescence was measured on 200 μL of serial cleavage solution for each kind of beads in a 96 well cell microplate from both the top and bottom faces. A pure NHS-fluorescein solution was prepared as a calibration standard to calculate the absolute amount of fluorescein released form the beads.1H NMR  δ 1.86–1.97 , 2.00–2.09 , 2.74 , 3.19 , 3.57 , 4.11 , 5.46 , 6.03 , 6.04 , 6.89 , 7.38 , 8.48 , 12.11 ; 13C NMR  δ 28.34, 30.18, 34.27, 43.46, 68.38, 79.98, 94.94, 95.88, 103.72, 116.17, 128.99, 130.67, 158.68, 164.15, 164.95, 168.09, 197.53.Naringenin  was dissolved in acetone (5 mL), then potassium carbonate  was added and heated to reflux for 30 minutes. To this suspension, 1,4-dibromobutane  was added and stirred at reflux for 8 hours. After cooling to room temperature, the mixture was concentrated on a rotary evaporator to become a thick slurry. The slurry was re-dissolved in ethyl acetate (30 mL), washed with distilled water (three times with 10 mL) and brine (10 mL), then evaporated. Purification was performed by silica-gel column chromatography (3:1 hexanes/ethyl acetate) to give pure 7-(4-bromo-n-butoxy)naringenin as a white solid : 1H NMR  δ 0.72 , 1.16 , 1.42 , 1.79 , 1.89 , 2.9 , 3.20 , 3.48 , 3.54 , 3.80 , 4.00 , 5.12 , 5.77 , 5.88 , 6.88 , 7.36 , 8.58 . This is an easy, but not efficient procedure. We successfully completed this reaction independently twice, however, we failed in other opportunities to obtain the desired product for unexplained reasons.7-(4-bromo-n-butoxy)naingenin  and pyridine  were dissolved in dry DCM (1 mL), 3-aminopropyltriethoxysilane  was then added and shaken for 48 hrs. After removal of DCM, the residue was purified by silica-gel column chromatography (2:1 hexanes/ethyl acetate) to give pure 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin as a pale yellow solid : Ceramic beads (30 mg) were first treated with 10% sodium hydroxide solution and washed with water, hydrochloric acid and methanol as above, then shaken with 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin (20 mg) in 95% methanol (1 mL) for 24 hrs. After filtration, the naringenin-coupled ceramic beads were washed three times each with methanol, water and methanol, then vacuum dried. The obtained beads were treated with NHS-fluorescein and detected by fluorescence microscope, as described above.2) was dissolved in 95% methanol (concentration from 128 μM to 12.8 μM) and measured as a calibration. Based on Beer's law, the molar extinction coefficient (ε) was determined to be 17244 cm-1M-1.7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin  was shaken with ceramic beads (500 mg) in 3 mL of 95% methanol for 24 hrs. After filtration, 1 mL of the filtrate was measured by absorbance at 314 nm. 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin (compound YL carried out all the chemical experiments described, NGI helped with the fluorescence microscopy and fluorometry. EG conceived the project and participated in the design and coordination of the study.