Dengue virus infection has recently taken endemic proportion in India implicating all the four known dengue serotypes. There was a major dengue outbreak in northern India including Delhi in October- December, 2003 and again in 2004. We have carried out a detailed investigation of the 2004 outbreak by Serosurveillance, RT-PCR, nested PCR, virus isolation and genotyping. We also report the molecular epidemiological investigation of these outbreaks.The serological investigation of 162 suspected serum samples using an in-house dengue dipstick ELISA revealed 11%-IgM, 51%-IgG and 38%-both IgM and IgG antibody positivity. The RT-PCR analysis revealed presence of dengue RNA in 17 samples. Further subtyping and genotyping by nested PCR and nucleotide sequencing of C-prM gene junction revealed the association of subtype III of dengue virus type 3 in the outbreak.The sudden shifting and dominance of the dengue virus serotype-3 (subtype III) replacing the earlier circulating serotype-2 (subtype IV) is a point of major concern and may be attributed to increased incidence of DHF and DSS in India. The global incidences of the dengue infection has now increased enormously and an estimated 50–100 million cases of dengue infections are now reported annually from more than 100 tropical and sub tropical countries of the world [Flavivirus of family Flaviviridae. The genome of dengue virus consists of a single stranded, non segmented, positive sense ribonucleic acid (RNA) of approximately 10.7 kb in length [Aedes aegypti .Infection with any one of these serotypes generally leads to a mild, self limiting febrile illness ). However, in few cases DF also leads to severe life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Several hypotheses, like antibody dependent enhancement (ADE) in heterotypic secondary dengue infections, involvement of a virulent viral genotype, and host factors have been suggested to explain the mechanism of pathogenesis of DHF and DSS [Dengue virus infection is now recognized as one of the most important mosquito borne human infections of 21 and DSS .The number of DHF and DSS cases have increased enormously in the last two decades in India and DEN-2 has been implicated as the causative agent in most of these outbreaks ,5. It isIn the present study, we report the serological, virological and molecular investigation of the 2004 Dengue outbreak. We also report the molecular epidemiological investigation of the 2003 and 2004 Delhi outbreaks based on the nucleotide sequence analysis of C-prM gene junction.st to 3rd week of October. The clinical history revealed that all the patients had suffered from fever ranging from 38.5° to 40°C. Most of the prominent clinical symptoms include headache (75%), myalgia (66%), rash (48%), vomiting (42%), conjunctival hemorrhage (38%), epistaxis (17%) and melena (5%). The platelet count varies from 18000 – 2.8 lakhs . The epidemic affected males and females at a ratio of 2.6:1. Majority (52.5%) of the patients were found belong to the age group more than 25 years. The detail distribution of the disease in terms of the age and sex of the patients is listed in Table An outbreak of febrile illness was reported in Delhi, India, during September- October 2004. The trend of the epidemic indicated the maximum number of cases was reported from the 1The serological analysis revealed that a total of 141 samples (87%) are positive for the presence of dengue specific antibodies. Out of these antibody positive cases, 16 (11%) were found positive for IgM, 72 (51%) for IgG and 53 (38%) had both IgM and IgG antibodies.A total of 17 (10%) samples were found positive for the presence of dengue virus specific nucleic acid as demonstrated by the presence of dengue complex specific 511 bp amplicon in 2% agarose gel.Isolation of virus was attempted from all the RT-PCR positive samples in C6/36 cells. A total of four dengue viruses were isolated from these samples. The isolation was confirmed at each passage level by RT-PCR.The serotype of the isolated virus, as well as viruses directly from serum samples was determined by nested PCR using serotype specific primers. The result indicated that all the 17 samples were positive for DEN-3 specific RNA.The nucleotide sequence of the C-prM gene junction  of the nine representative dengue viruses and one NIV reference DEN-3 virus (isolated in Philippines in 1957) were determined in the present study. Detailed descriptions of these viruses were given in Table Two different dendrograms were drawn based on the pair-wise comparison of nucleotide sequence of partial prM sequence (nt. position 437–613) Fig . The denThe dendrogram based on the 435 nucleotide sequence of C-prM gene junction also clearly distinguished the two different genotypes (I and III). All the 2003 and 2004 Indian viruses except GWL-60 form a close branch along with SRIL-84; where as, GWL-60 forms a branch with SRIL-98 and GUET-98  revealed the involvement of DEN-3  and was ontinent -7. This ontinent . The appThe routine laboratory diagnosis of dengue virus infection is primarily achieved by the isolation of virus, detection of IgM/IgG antibodies by serodiagnosis and/or molecular detection by the demonstration of viral RNA by RT-PCR ,10. In tRT-PCR is one of the most important confirmatory test, employed to confirm dengue infection. However, it is only positive when sample is collected during the viraemic phase of the patient (preferably within first five days of onset of symptoms). In the present study, the identification of only 17 samples as dengue positive by RT-PCR, may be due to the fact that most of the patients were presented to the hospitals in the post-viremic phase. Further the lower rate of virus isolation may be attributed to the absence of live virus in the sample. This may be due to failure in maintenance of cold chain resulting in inactivation of virus. All the isolation was confirmed and identified by RT-PCR and nested PCR. Isolation and identification of virus from the clinical sample is considered the gold standard and gives a confirmatory diagnosis without any ambiguity ,12.Further, we have carried out the molecular epidemiology and genotyping study of the DEN-3 viruses, implicated as the causative agent of this outbreak. We have studied the sequence of these dengue viruses, directly from patient serum sample, as recently advocated by several researchers ,14. VariOn comparison of the sequence, it was found that all the Indian sequences were very closely related. It was found that the outbreak over fairly large areas of two provinces  were caused by the same type of DEN-3 virus. This indicates that the current virus is easily transmitted in human and mosquitoes and can adapt to a newer area efficiently. We have drawn two different dendrograms to study the evolutionary relationship of Indian DEN-3 isolates, due to absence of sequence of capsid gene of any of the DEN-3 viruses belonging to subtype II and IV. One phylogenetic tree was drawn based on the 177 nucleotide sequence of partial prM gene, classified all the 28 DEN-3 viruses, analysed in this study into respective subtypes, as designated in the classic paper of Lanciotti . All theThe dendrogram and sequence analysis clearly revealed that the subtype III of DEN-3 viruses are circulating through out the world, where as other subtypes are localized to a particular small geographical area. This indicates the higher potential of subtype III to spread, adapt and dominate in geographically diverse areas of the world. This subtype has also been implicated in major dengue/DHF epidemic from several parts of Asia, Africa and Americas; and has the potential to cause a trans-national dengue pandemic .Though all the four serotypes of dengue viruses were isolated from different parts of India, DEN-2 was considered as the predominant serotype circulating in northern India -7. DEN-3This study confirms that the major dengue outbreak in northern India in 2003 and 2004 was caused by dengue virus type-3 (subtype III). The reemergence of highly fatal subtype III of DEN-3 in a dominant form, replacing the earlier circulating subtype IV of DEN-2 in India is a matter of great concern. Detailed and continuous epidemiological surveillance is warranted to monitor the incursion and spread of dengue viruses, which will help to undertake effective control and management strategies at the earliest.An outbreak of febrile illness was reported in Delhi, India, during September-October, 2004. A total of 162 blood samples from clinically suspected dengue patients were collected from Delhi during this period. In addition, three viraemic blood samples collected from Delhi and Gwalior during October – December, 2003 [Staphylococcus aureus Cowan I, where as for detection of IgG antibodies, patient sera were used as such without any pre treatment. Goat anti-human IgM horshradish peroxidase (HRP) and goat anti-human IgG HRP conjugate  were used as secondary antibodies for the detection of IgM and IgG antibodies, respectively. The reaction was finally developed by dipping the projections in an insoluble substrate solution , 4-chloro-1-napthol and hydrogen peroxide). The results were visually recorded as filled brown dots, indicative of the presence of dengue specific antibodies.All serum samples were tested for the presence of dengue specific IgM and IgG antibodies using the dengue dipstick ELISA kit developed in our laboratory . Brieflyet al, 1992, with slight modifications [® ribonuclease inhibitor and Moloney murine leukemia virus reverse transcriptase (MMLV-RT)  with dengue virus complex specific antisense primer (D2) . The RT mix was incubated for 1 h at 37°C, before heating the reaction for 5 min at 99°C to inactivate the MMLV-RT enzyme. The amplification of cDNA was carried out in a total volume of 50 μl with PCR mix containing 10× PCR buffer, 25 mM MgCl2, NTPs, Taq-DNA polymerase , using dengue virus complex specific sense primer (D1),  in a thermal cycler . The thermal profile of the PCR reaction was- initial denaturation at 95°C for 2 min., followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 10 min. The PCR products were gel purified from 1.2% agarose gel using the QIAquick PCR purification kit .The identification of the virus isolates obtained from the clinical samples was carried out by RT-PCR followed by nested PCR by demonstrating the presence of virus specific RNA employing dengue group-specific as well as serotype-specific primers targeting C-prM gene junction following the protocol of Lanciotti ications ,23. Brie2 tissue culture flasks. The inoculum was incubated for 2 h before being replenished by 10 ml of fresh maintenance medium  with 10% foetal bovine serum . Suitable healthy cell controls were also kept along side. The cells were then incubated at 32°C and observed microscopically daily for the appearance of cytopathic effects (CPE), if any. The supernantants of infected cell culture were collected on the 6th – 7th post infection day (PID) and analysed for the presence of virus. Invariably, three subsequent serial blind passages were given in each case.Isolation of viruses from the acute phase viraemic samples was also attempted in the C6/36 cells, following the standard virus adsorption protocol . BrieflyDouble stranded sequencing of the C-prM gene junction was performed on an ABI 310 sequencer , employing Big dye terminator cycle sequencing ready reaction kit. Briefly, 2 μl (approximately 25 ng) of purified PCR product was mixed with 3.2 pmol of respective primer and a reaction mixture containing the four dye-labeled dideoxynucleotide terminators. Cycle sequencing was then performed as follows: 25 cycles at 96°C for 30 sec, 50°C for 1 min and 60°C for 4 min. The reaction mixture was purified by ethanol precipitation and the DNA was vacuum dried. The DNA pellet was resuspended in 10 μl of template suppression reagent (TSR) and preheated before loading on to the DNA sequencer.The nucleotide sequences were edited and analysed by using the EditSeq and MegAlign modules of the Lasergene-5 software package . Multiple sequence alignments was done employing CLUSTALW version 1.83 . The phyThe author(s) declare that they have no competing interests.PKD conceived the study, carried out the sequencing experiments and phylogenetic analysis and drafted the manuscript. MMP carried out the RT-PCR experiments, PS carried out the virus isolation experiments, AA carried out the clinical sample processing, immunoassays and sequence analysis study. CPS and KNT were responsible for collection and storage of clinical samples and meticulous collection of case history. AMJ and KS liaison between MCD and DRDE and coordinated this study. PVLR helped out to design and draft the manuscript and also revised it critically. All authors read and approved the final manuscript.


T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family  contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the  Toll-like receptors (TLRs) play a central role in innate immunity -3. TLRs Leucine-rich repeat (LRR)-containing domains are present in over 6000 proteins listed in PFAM, PRINTS, SMART, and InterPro data bases -8. All LThe indicated repeat number of LRRs and its "phasing"  in individual TLRs are different among the databases (or researchers) and species. This difference reflects the irregularity of LRR motifs in TLRs. Over one hundred complete TLRs are available. Several methods of protein secondary structure predictions such as Proteus and SSPro4.0 show a correspondence of about 75% -54. For 10-helix, polyproline II helix, or a tandem arrangement of β-turns , polygalacturonase-inhibiting protein (PGIP) [10GQ], nogo receptor/nogo-66 receptor (NgR) , glycoprotein Ibα (GPIbα) , decorin , biglycan [2FT3], Slit [1W8A], CD14 [1WWL], follicle-stimulating hormone receptor (FSHr) [1XWD], TLR3 , and human lingo-1 [2ID5].The structures of twenty-two different LRR proteins have been determined. They are ribonuclease inhibitor (RI) , GTPase-activating protein (RanGAP) , tropomodulin (Tmod) , S-phase kinase-associated protein 2 (Skp2) [1FQV], YopM [1G9U], four internalins, Inl-B [1D0B], Inl-H [1H6U], Inl-A  and Inl-C [1XEU], spliceosomal U2A' protein [1A9N], mRNA export factor (TAP) , rab geranylgeranyltransferase α-subunit (RabGGTα) , The LRRs alignments within the TLR family were made for TLR1 from four species ; TLR2 from 17 species , Q9DGB6 (TLR2.2)], bovine [Q95LA9], rat [Q6YGU2], dog [Q689D1], rabbit [AAM50059], goat [ABI31733], horse [AAR08196], hamster [Q9R1F8], Cynomolgus monkey [Q95M53], domestic water buffalo [Q2PZH4], Nilgai [Q2V897], Takifugu rubripes [Q5H725], zebrafish [Q6TS42], Japanese flounder [Q76CT8]); TLR3 from 9 species ]; TLR5 from 8 species ], rainbow trout [Q7ZT81]); TLR6 from 5 species ; TLR8 from 4 species ; TLR9 from 12 species ; TLR11 from mouse ; TLR12 from mouse [Q6QNU9]; TLR13 from mouse [Q6R5N8]; TLR14 from Takifugu rubripes [Q5H726] and zebrafish [XP_687315]; TLR15 from chicken [ABB71177], TLR21 from Takifugu rubripes [NP_001027751], TLR22 from Takifugu rubripes [Q5H723], TLR23 from Takifugu rubripes [AAW70378], and TLR from rainbow trout , Atlantic salmon [Q2A132], goldfish [Q801F9]), Japanese lamprey  and green puffer (Fragment) [Q4S0D3]).and Proteus [The protein secondary structure prediction by SSpro4.0 ,76,77and Proteus ,78 were  Proteus ,80. The  Proteus ,82. The  Proteus  was usedMultiple sequence alignments and sequence similarity searches were performed at Bioinformatic Center, Insitute for Chemical Research, Kyoto University . Dot-matToll-like receptor: FTLR. Toll IL-receptor: FTIR. LPS: Liopolysacchride. LRR: ECD: Ectodomain. Leucine rich repeat. HCS: Highly conserved segment of LRR. VS: Variable segment of LRR. SLRP: Small leucine-rich repeat proteoglycans: FSHr, Follicle-stimulating hormone receptor.NM (corresponding author) carried out the molecular genetic studies and wrote the manuscript. TT performed the dot plot analysis and contributed to the data analysis. EP performed the geometrical analysis of the known structure of TLR3. TM and MT participated in the sequence alignment. KY and YK conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


In 2005, ICTV , the official body of the Virology Division of the International Union of Microbiological Societies responsible for naming and classifying viruses, will publish its latest report, the state of the art in virus nomenclature and taxonomy. The book lists more than 6,000 viruses classified in 1,950 species and in more than 391 different higher taxa. However, GenBank contains a staggering additional 3,142 "species" unaccounted for by the ICTV report. This paper reviews the reasons for such a situation and suggests what might be done in the near future to remedy this problem, particularly in light of the potential for a ten-fold increase in virus sequencing in the coming years that would generate many unclassified viruses. A number of changes could be made both at ICTV and GenBank to better handle virus taxonomy and classification in the future. The VIIIth ICTV Report  lists moFor the past 20 years, GenBank has increased the number of sequences stored from one in 1982 to 42 million in 2002, and has increased the number of tools at the disposal of users to the point where today it is an absolutely necessary system for molecular biologists and virologists world-wide. In particular, GenBank has developed a complete database for taxonomy, including virus taxonomy, allowing virologists to search all viruses and to identify new sequences and new virus names. Were it not for the existence of this excellent system, we would not have known of the existence of these 3,142 unassigned "species" of viruses.ICTV also has been very active in the last 20 years, incrementally increasing the number of taxa and virus names from 369 in 1985 to 7,881 in 2004. Not only have the numbers increased exponentially (about 20-fold), but the complexity of virus nomenclature and taxonomy has become tremendously complicated and controversial, even puzzling for some. However, the overall stability of this virus classification, established in 1962 [The listing by GenBank, but not by ICTV, of 3,142 unassigned "species" clearly demonstrates a general problem of the application of the definition of virus species and that ICTV and GenBank must work in concert to cope with the day-to-day reality of virology and virus classification, and that collectively we need to improve the system in such a way that we will rapidly classify so-called "species" of viruses and change the current system, so that we do not find ourselves in a similar situation in the future.This paper will review the probable causes of the present situation and will identify actions that could remedy the situation both at GenBank and at ICTV. Finally a brief description of the Taxonomic Proposals Management System (TPMS), a new database to electronically handle all taxonomic proposals for ICTV, will be provided.Bluetongue virus in the genus Orbivirus, family Reoviridae; thus, 24 viruses assigned to a single species. Therefore sequences entered in GenBank must be associated with the name of a virus isolate. The term virus isolate has no specific definition and can be applied to any virus, so long as that virus has existed for some time. Several terms are used by virologists to define a virus entity below the species level and are somewhat variable and confusing, but for virus sequences the term "virus isolate" seems the most appropriate, practical and useful [A virus species, as with all other taxonomic levels, is a concept devised by humans to describe concrete  biological viral entities. The definition of a virus "species", established  and acced useful .The intent of GenBank is to collect sequences but the author, the person who enters the sequences in the database, is responsible for the accuracy of the sequences as well as for the validity of the associated information about the sequences, which includes the taxonomic information about the virus in question. The primary taxonomic information pertains to an isolate of a previously recognized virus or of a newly recognized virus. Consequently, if the author errs in naming the virus or in its classification, the entry will be incorrect and might remain incorrect for a long period of time. GenBank has personnel qualified to review all entries and they effectively and immediately correct a large number of obvious mistakes. However, with regard to "new species", GenBank personnel do not have the means to determine whether this new taxon is justified and acceptable or not. Obviously, this policy of GenBank cannot be changed from without, but it is certainly possible for GenBank to slightly modify the Entrez form in order to build in safety nets to avoid such situations in the future.The Entrez form of GenBank is a general form for all organisms, and one of the first questions asks the name of the organism. Whereas viruses are not organisms, this question immediately causes ambiguity because it is not clear whether the isolate name or the name of the species to which it belongs should be indicated, notwithstanding the fact that many investigators have not yet understood the difference between an isolate name, corresponding to a specific sequence of a virus, and the conceptual species name, to which a particular isolate has been assigned ,8   Fig. .Each SG comprises from three to 15 experts selected for their expertise in recognized and new viruses and new sequences of viruses in each family; obviously this is an increasingly difficult task, given the rapid increase in available data. These 82 SGs present taxonomic proposals from the level of species to the level of order, the proposals are reviewed by the Executive Committee (EC) of the ICTV and, 18 months later, through a posting on ICTVnet and two rounds of discussions, are then ratified (or not) and voted upon (or not) by the ICTV membership at large. This system, although well organized and in full compliance with the international code for virus taxonomy and nomenclature , is neitIt is not the responsibility of the authors to decide whether their new virus isolates belong to an existing or a new species, not the responsibility of GenBank to police authors and organize virus taxonomy and nomenclature, and not the responsibility of ICTV to deal with non-taxonomic issues, such as isolate names, but it is our communal responsibility to work together to make the system functional; and it certainly is feasible. Here we call for collaboration of all parties to establish a generally satisfactory system for virus taxonomy and nomenclature of virus isolates, sequence data, and all other virological data. Obtaining solutions will necessitate the collaboration of all those involved in producing, collecting and classifying viruses.Placing the 3,142 unassigned "species" (which likely are simply "viruses") registered at GenBank will require manual classification by the ICTV SG members. Most of these "species" are probably isolates of recognized species and are in the GenBank database by default as new "species" because, whether they have been named or not, they have not been entered in the GenBank forms. Some of these GenBank "species", however, may actually be new species that have not yet been submitted to ICTV and, if so, they should be proposed through the normal channels of the Taxonomic Proposals (TaxoProps) for evaluation and official ratification before they can be recognized as new species.As a remedy to the general problem of virus classification, we suggest a two-tier plan. Firstly, GenBank would work with ICTV to provide to the authors rolling menus, or automatic writing, offering a list of all the official ICTV names to select from. This would help avoid spelling errors or the creation of incorrect new names by authors. In the case of an unaccepted  virus name, the default should be "unassigned virus in the genus, or in the family, or in the order or in the virus kingdom". This assignation to an "unassigned category" would trigger an email to the relevant ICTV SG chair and to some EC members so that ICTV will be immediately aware of this new entry. This information also would be stored in a proposed new database called TaxoProp Management System (TPMS), with a pending status to be resolved promptly (see below). The second tier of the remedy would be to establish the TPMS database to handle all new TaxoProps containing information originating from an SG member, from any individual virologist or directly from GenBank.Resolving the mechanics of the system is only a partial solution and more details are needed in order for this to be functional in real time. ICTV has established a sophisticated and democratic system to receive, review, discuss, re-discuss and finally approve taxonomic proposals, whether they concern a new species or a new order . This syIn order to have taxonomic consistency, it is absolutely essential that we strengthen the relationships between ICTV and GenBank. This could be done with an electronic link between TPMS and GenBank, supplemented with improved and regular personal relations. For example, GenBank has appointed virus experts for a number of families of viruses. It would be more efficient to have GenBank somehow linked directly to the SG chairs of ICTV, so that GenBank would get taxonomic experts for advice in virus taxonomy. This, in turn, would provide a more active role for the SG members and would activate the overall system of virus taxonomy.Due to the rapid increase in the number of TaxoProps and the increasing difficulty in manually handling several thousand taxonomic names, it is planned by ICTV to create a database to handle all ICTV taxa, from inception to acceptance, and from species to order Fig. .The goal of the TPMS database is to electronically handle all the virus taxa at large (including virus isolates), i.e., those that have been inherited from previous reports, those that have been approved between 1999 and 2005, those in the process of being approved and those that will be approved in the future.At any time it will then be possible to:1- certify the approval status of any TaxoProp2- elaborate a complete list of approved taxa3- establish an electronic link with GenBank and all other virus databasesThe database will be accessible on line to all ICTV members and to the public at large. The database will comprise a collection of individual files, each one corresponding to a single taxon , and in addition will record isolates. Each TaxoProp will progress from the initial proposal to the approval of the taxon by the ICTV. The bulk of the database will be constituted by all the approved taxa and individual viruses and should be exportable to other databases for future use.de facto a ratified taxon of the ICTV.The TaxoProp approval system of ICTV is depicted in Figure TaxoProps can be filed by any virologist, but it is likely that most will be filed by SG members. There will be a set of TaxoProps to choose from on line according to the sort of taxon proposed: an isolate , a species, a type species, a genus, a subfamily, a family, an order, or any other taxon proposed by any virologist. Higher taxa cannot be proposed unless a lower level has been proposed and accepted. A genus must have a type species and at least one species or a list of species in the genus. Any proposed new taxa must have a name following the ICTV Code .For each genus, it will be possible to list the species demarcation criteria of that genus for the benefit of the author and readers of the species TaxoProps. These lists will be extracted from the VIIIth Report and from subsequent updates. It is envisioned that these lists of species demarcation criteria will become useful tools for ICTV to search for a better means of making the overall virus taxonomy more uniform in the future.Although isolates do not comprise formal taxa, recording and listing them are indispensable for the creation of species taxa and also for practical purposes. Virus species being defined from properties of virus isolates, ICTV records the existence of isolates in order to define species. There are no formal rules for naming isolates and, consequently, any name could potentially be accepted. However, it is common sense that these names follow previously established names and follow somewhat virus species names rules to avoid complete chaos ,9.TaxoProp species, after being proposed by the SGs, will automatically be reviewed to ensure that they follow the ICTV International Code and, if they do, will automatically be approved electronically. However, a vote by the ICTV membership at large will still be necessary for it to be considered as ratified by the ICTV; this also could be done electronically.All approved taxa, because they have been inherited, will be entered in the TPMS, so that the database will be updated automatically and in real time and can be linked to GenBank, ICTVdb (ICTV database) and other databases. Files for each taxon will archive all the comments and votes so that we can always return to this information if needed. However, public access to the TPMS for the ratified ICTV Master List will remain under control of the President of ICTV.Using TPMS, it will be possible to generate a complete list of approved taxa at any time, and this will be possible either alphabetically or taxonomically. Accepted taxa also will be presented according to the order of presentation of virus taxonomy: dsDNA, ssDNA, rtDNA, rtRNA, dsRNA, ssNRNA, ssPRNA, SAT (Satellites), VIR (Viroids), UN (unassigned). Within each category, the families will be classified according to the Order of Presentation of the Virus. Taxonomy Species will be assigned to a genus and classified alphabetically within the genus. The species taxa should comprise at least one isolate but include as many as described.Any virologist will have access to the TPMS database on line anytime so that he or she can verify names, either with an alphabetical pull down menu system or by use of a search engine.de facto become the definitive reference for virus taxonomy and nomenclature. Once approved by the ICTV President, typically after each ICTV EC meeting or after a certain period of time (such as six months), the TPMS database will be electronically linked with GenBank, ICTVdb, and other associated databases to ensure complete homogeneity of the virus taxonomy and nomenclature. This tool will be particularly important for the members of the International Virus Database Network, who recognize the critical need for such a system, and to homogenize and update in real time the virus taxonomy and nomenclature.TPMS will Because sequencing is much less expensive and increasingly done these days, it is assumed that investigators will generate many more sequences of known and unknown viruses in the very near future. The number of sequences is increasing exponentially and so will the number of virus names and virus taxa, as they are directly correlated with sequencing activities. It is clear that past management of virus taxonomy will become inadequate commensurate with this increase and that more rapid and better-adapted management is required. The electronic connection between databases storing sequences, storing biological data or handling taxonomic proposals is the only solution for the future. Human intervention is indispensable, but should be restricted to the decision-making process; all other interventions might best be done electronically. This will mean better collaboration between the people responsible for the different types of databases, the people involved in the ICTV organization, and individual investigators themselves. Software will have to be adapted or devised to better fit virus taxonomy and nomenclature, ICTV will have to better appreciate day-to-day realities such as dealing with virus isolates, and scientists will have to be better educated in virus taxonomy. Regarding the decision-making process for virus species and isolates, a much greater role for the ICTV SGs will be necessary because the ultimate expertise resides with them in determining which new viruses fit into existing taxa and which must be placed in defined new taxa. Consequently, it would be best for ICTV to work more closely with GenBank to establish the electronic TPMS database to work in real time and to cope with an acutely burgeoning number of taxa.The author(s) declare that they have no competing interests.The authors contributed equally to the writing of this paper.


Hantavirus, family Bunyaviridae. The genomes of hantaviruses consist of three negative-stranded RNA segments  encoding the virus nucleocapsid (N), glycoprotein , and polymerase (L) proteins, respectively. The genus Hantavirus contains predominantly rodent-borne viruses, with the prominent exception of TPM virus which was isolated in India in 1964 from an insectivore, Suncus murinus, commonly referred to as the Asian house shrew or brown musk shrew. Analysis of the available TPM virus S (1530 nt) RNA genome segment sequence and the newly derived M (3621 nt) and L (6581 nt) segment sequences demonstrate that the entire TPM virus genome is very unique. Remarkably high sequence differences are seen at the nucleotide  and protein  levels relative to the rodent-borne hantaviruses, consistent with TPM virus having a unique host association.Thottapalayam (TPM) virus belongs to the genus  Hantavirus, family Bunyaviridae) are vectored by murid rodents of the Murinae, Arvicolinae, and Sigmodontinae subfamilies .TPM virus (strain VRC 66412) was grown in Vero E6 cells and harvested 12 days post-infection. Virus was inactivated in Tripure (Roche) and RNA isolated using the RNaid kit (Bio 101). The complete S segment sequence of TPM virus had been deposited in Genbank earlier by Song and colleagues  is predicted to encode a glycoprotein precursor of approximately 126 kDa. The overall structure appears similar to that of other hantaviruses, with approximate alignment of hydrophobic domains corresponding to signal peptide, and Gn and Gc transmembrane domains (data not shown). While most hantavirus glycoproteins have 5–7 predicted N-glycosylations sites, four potential N-glycosylation sites are conserved among all hantaviruses, three in Gn  and one in Gc (937 in PUUV) . The TPMInformation on characterization of L protein of hantaviruses is scanty. A previous study identified five conserved motifs  among all hantavirus RNA polymerases . As expeMurinae, Sigmontinae and Arvicolinae and the shrew-associated TPM virus , captured in Tamil Nadu, India [TPM virus was isolated from an Asian house shrew  captured in Guinea in western Africa [Finally, it was recently reported that a novel hantavirus was detected in the Therese shrew  declare that they have no competing interests.PDY participated in virus growth and RNA purification, design, optimization and execution of RT-PCR reactions, sequence analysis, phylogenetic analysis, and preparation of the manuscript. MJV participated in design of RT-PCR primers and experiments and preparation of the manuscript. STN conceived of the study, participated in the design and coordination of the experiments and preparation of the manuscript.


Little is known about the costs of community programmes to prevent cardiovascular diseases. The present study calculated the economic costs of all interventions within a Dutch community programme called Hartslag Limburg, in such a way as to facilitate generalisation to other countries. It also calculated the difference between the economic costs and the costs incurred by the coordinating institution.Hartslag Limburg was a large-scale community programme that consisted of many interventions to prevent cardiovascular diseases. The target population consisted of all inhabitants of the region (n = 180.000). Special attention was paid to reach persons with a low socio-economic status.Costs were calculated using the guidelines for economic evaluation in health care. An overview of the material and staffing input involved was drawn up for every single intervention, and volume components were attached to each intervention component. These data were gathered during to the implementation of the intervention. Finally, the input was valued, using Dutch price levels for 2004.The economic costs of the interventions that were implemented within the five-year community programme  were calculated to be about €900,000. €555,000 was spent on interventions to change people's exercise patterns, €250,000 on improving nutrition, €50,000 on smoking cessation, and €45,000 on lifestyle in general. The coordinating agency contributed about 10% to the costs of the interventions. Other institutions that were part of the programme's network and external subsidy providers contributed the other 90% of the costs.The current study calculated the costs of a community programme in a detailed and systematic way, allowing the costs to be easily adapted to other countries and regions. The study further showed that the difference between economic costs and the costs incurred by the coordinating agency can be very large. Cost sharing was facilitated by the unique approach used in the Hartslag Limburg programme. Many western countries have a very high prevalence of cardiovascular diseases (CVD) . In the Reducing risk factors for CVD might reduce the burden that they impose on public health and on the health care budget. There are many ways in which this can be achieved. A promising method to reduce the prevalence of CVD is the implementation of community programmes.While there is a considerable body of evidence on the effectiveness of community programmes to prevent CVD -6, littlIt is important that the costs are calculated according to guidelines because previous research has shown that considerable bias may occur in the costs when guidelines are ignored -19. OnlyThe value of presenting costs of community programmes in a detailed overview instead of presenting the costs at an aggregated level is twofold. First, providing insight into the costs of all individual interventions in a community programme facilitates budgeting for anyone who might want to implement such a programme. The combinations of interventions to change lifestyle that are used in community programmes are different for each programme and it is thus important to know which specific interventions are used, how they are implemented and what their individual costs are. The second advantage of presenting detailed costs instead of only final costs is that the latter may give rise to problems when generalising the costs to other regions and countries, because local factors are likely to affect the costs of a programme. Presenting detailed costs avoids these disadvantages of aggregated presentation.The present article reports on the costs of the community programme included in the Hartslag Limburg campaign, which aimed at preventing cardiovascular diseases.The aim of the study was to provide detailed information about costs of interventions within a community programme, calculated in accordance with guidelines for economic evaluation and in such a way as to facilitate generalisation to other countries.The intervention region consisted of the town of Maastricht  and four adjacent municipalities . The majority of the people thus lived in urban areas. 62% of the inhabitants of the intervention region had paid jobs, while 17% of the inhabitants were older than 65 years.In the intervention region, cardiovascular diseases accounted for 32% of all deaths. The prevalence of the acute heart infarction in the intervention region was 3.7%, which is almost 2% higher than the average prevalence in the Netherlands.As regards lifestyle, 32% of the inhabitants smoked and almost 50% of the inhabitants in the intervention region did not meet national recommendations for exercise (30 minutes a day for at least 5 days a week).The community programme in the Hartslag Limburg campaign aims at health promotion among all inhabitants of the intervention region, that is, the town of Maastricht and four adjacent municipalities . Special attention was paid to inhabitants of four low socio-economic status (LSES) neighbourhoods , in which 50% of the interventions took place. The study was approved by the Dutch Medical Ethics Committee TNO (reference CO/TW 2599/10049).The programme is based on a unique design. Before interventions were implemented, a network was formed consisting of large number of participating organisations. Amongst these organisations were the campaigns coordinating agency, which is the Regional Public Health Institute of Maastricht, as well as other health promotion agencies, the local hospital, general practitioners, welfare services and local authorities. This network can be described as the cornerstone of the campaign. Each of the agencies implemented a number of CVD prevention interventions, and also contributed financially. In this way, the network served as the starting point for the implementation of interventions. The network made it possible to implement a multiple intervention strategy and to reach inhabitants of the region in a variety of ways. Both would have been very difficult to achieve if a network had not been created first.The community-based health promotion within the campaign was implemented through nine local health committees: one in each of the five municipalities and one in each of the four LSES areas. Each committee consisted of employees of the Regional Public Health Institute and of welfare services, civil servants and individuals from the target population themselves. The committees approached individuals and groups within the target populations by means of health promotion projects and activities.The participation of community members in the local health committees was essential to facilitate the recruitment and participation of inhabitants. This was especially true for the LSES regions, because their residents are difficult to reach with general health promotion activities.The participation of civil servants was important as they could effect policy changes, and because health promotion activities are partially financed by city councils.The choices about which interventions to implement came about in a variety of ways. The most occurring were the following: (1) The coordinating agency or one of the other partners proposed an intervention, which was preferably evidence based. Together with one of the local health committees its value was discussed and the best way to implement the intervention was determined. When necessary partners or 'external' institutions were approached to collaborate in the implementation (2) The health committees explored the needs and wishes of the target-population. Based on this information, existing interventions were implemented or new ones were developed. When cooperation of institutions or organisations was needed, these were approached (3) Agencies outside the network contacted the coordinating agency of Hartslag Limburg with a suggested intervention. Together with the coordinating agency or a local health committee, the suggested intervention was evaluated on its merit for Hartslag Limburg and its attainability. In case of a positive attitude towards the intervention, implementation followed.The similarity between these ways is that they were based on the observed needs within the community and on the expertise of the various implementers about the methods to reach the inhabitants in the region.An adequate cost calculation requires three steps to be taken, namely identification, quantification and valuation .In the identification phase, decisions were made about the aspects of the complex Hartslag Limburg programme that were to be included in the cost analysis and those that were to be excluded.Starting from this framework, the quantification step addressed the methods used to obtain an overview of the material and personnel resources necessary for the campaign interventions to be successfully implemented.Finally, the valuation phase applied the pricing methods.Since the present study focused on the costs of the actual interventions, costs related to creating and maintaining the network were excluded from the analysis. The reason for doing so was that the latter costs were dependent on local organisational aspects, which means that no context-independent estimate was possible. Furthermore, the network was not solely used for the CVD prevention programme, but might serve many purposes. Hence, we were unable to estimate which part of the network costs had to be attributed to the community program of Hartslag Limburg.We focused on calculating the costs of the interventions. Because of the large diversity of interventions, the aim was to calculate the costs of every single intervention separately. Within Hartslag Limburg, about 1000 interventions were implemented. About 800 of these were large-scale or frequently recurring interventions. In addition, there were about 200 small-scale, nonrecurring events. For practical reasons, only a random sample of the small-scale interventions was used to calculate costs according to the guidelines, and the resulting costs were then extrapolated to all other small-scale and non-recurring interventions. We think this is justified, because of the relatively small contribution of these smaller interventions to the total costs of the programme.development of new interventions were irrelevant because they do not have to be incurred again when the intervention is applied elsewhere, nor would future implementers have to pay indirectly for the development costs. Hence, they were excluded from the analysis. Because future programmes might involve developing new interventions, we provide an indication of the development costs within Hartslag Limburg, which are reported separately.Not all the costs related to the interventions were relevant to the present cost calculation. Costs incurred for the Finally, a cost calculation according to the guidelines implies that all inputs should be included to obtain a reliable overview of the actual costs of a programme. This also means that all input should be given a monetary value. For the present cost calculation this meant that (1) monetary input or input in kind that was received from other institutions  was included in the cost analysis, and (2) input from volunteers was also included. Although the guidelines recommend to include input from participants in the cost analysis as well, the current analysis did not include time input by participants, due to lack of data.In the quantification phase, an overview of all the material and staffing input involved (both in kind and in quantity) was drawn up for every single intervention. The overview was based on documentation, like budget statements, registrations of hours worked by staff, plans of action, evaluation reports and accounts. After an intervention had been implemented, we verified whether the actual input was in agreement with the anticipated input. This was done in regular meetings (once every two months) with health education specialists involved in implementing the interventions. This information was used for an accurate estimation of the resources that were necessary for the intervention to be successfully implemented.In the third and final step, the staff and material inputs were valued, using Dutch price levels for 2004. Interventions were mainly valued using prices.In order to obtain reliable estimates of the costs of staff input, and in accordance with guidelines for cost research -22, stafIn valuing staff input, we assumed an efficient use of input. This meant that adjustments had to be made for inefficient use of staff input by the programme due to start-up problems. When identifying staff input, we used the task level necessary to implement an intervention successfully to define the type of staff input and thus the price attached to it.The prices of the material input were derived from the Hartslag Limburg records.The study can be characterized as a descriptive analysis, in which all costs that were made during the community program of Hartslag Limburg were calculated on a detailed level.From 1999 until 2003, a total of 790 interventions were implemented. These included 21 different types of large-scale or frequently repeated interventions, accounting for 590 interventions over the five-year intervention period. Table The costs of the individual major interventions and the resulting total costs of the Hartslag Limburg community programme are shown in table As table There were also large differences between the costs of individual interventions. Some interventions were very cheap , whereas others involved very high costs, such as the interventions called 'Exercise TV', 'Tasty and Healthy' and 'Focus on Heart and Sports'. These three interventions accounted for 45% of the total costs.Of the total of €900,000 that was spent on the interventions, the coordinating agency, the Regional Public Health Institute, supplied €100,000, while the Netherlands Heart Foundation paid €185,000 and the municipal authorities contributed about €270,000. Other agencies, companies or organisations – including an well-fare agency – contributed the considerable amount of €345,000. This meant that only a fraction of the costs of the interventions was borne directly by the organising agency.As far as we are aware, this is the first study to present detailed costs of a large-scale community intervention programme, using the necessary material and staff input for every single intervention as a starting point and applying guidelines for economic evaluation.The costs of the total programme were about €900,000. The 800 large-scale and recurring interventions accounted for 95% of the costs, while the 200 small-scale and non-recurrent interventions accounted for the remaining 5%.The three most expensive interventions – 'Exercise TV', 'Tasty and Healthy' and 'Focus on Heart and Sports' – accounted for 45% of the costs. Because of the high costs incurred, it would be of great interest to assess the effectiveness of these interventions. However, the large number of interventions performed simultaneously within the Hartslag Limburg campaign makes it impossible to measure the effects of individual interventions.The present methodology has some major advantages over those used in previous studies in this field, in terms of validity, reliability, generalisability and health promotion practice.The accuracy with which the costs were calculated by using a bottom-up procedure based on necessary resources, instead of the specific resources used in Hartslag Limburg, improved the validity compared to previous studies. Previous attempts to calculate costs of community programmes were based on retrospectively analysing a programme's financial records. This can lead to considerable bias, because community programmes are often endowed with large contributions (monetary or in kind) by other agencies or organisations, which are often not incorporated in the coordinating agency's financial records and as a result are easily overlooked. Input from volunteers, which was included in the present study, is usually not shown in financial records either.The bottom-up procedure we used ensured that all relevant costs were included in the analysis, making the study more reliable than previous studies. Unlike previous studies, which used a retrospective design, the present cost calculation was made during the implementation of the Hartslag Limburg interventions, decreasing the degree of bias. Another aspect that contributed to the reproducibility of our results is that efforts were made to detach the costs from the local context, for example by using average salaries for particular task levels instead of using the actual salaries paid within the campaign we studied.Using average price levels instead of the actual price levels used within the Hartslag Limburg campaign makes the costs applicable to all parts of the Netherlands, and even to other countries with a price level comparable to that in the Netherlands.Generalisation to other countries is facilitated by the fact that we have provided a detailed overview of the necessary material and staff input. Generalisation is thus a matter of linking local prices to the necessary material and personnel resources (which are not expected to differ much between regions or even between developed countries).The insights our study has provided into the resources necessary to perform lifestyle interventions facilitate budgeting, allowing costs to be anticipated.The main shortcoming of our study is that the bottom-up procedure was not consistently used for the 200 small-scale and non-recurring interventions, but that their costs were estimated from the average costs of similar interventions. However, since these 200 small-scale interventions accounted for only 5% of the total costs, the bias resulting from this adjusted cost calculation method is not large.A practical disadvantage of the present study was that applying a bottom-up methodology to such a complex programme is very time-consuming.In spite of the fact that the present study paid more attention to the external validity than previous studies, it must be realized that the costs of community programs will never be completely independent of time and place.As was shown in the Results section, it is possible to share the costs of such interventions among various parties. If such a cost-sharing approach is to be used in future programmes, it is important to form a network of participating organisations. These partners should be well informed about the goals of the project and the advantages for their own organisation, in order to improve cooperation. Effective collaboration can be promoted by the use of written agreements, especially when private organisations are included. The involvement of the partners can be enhanced by informing them about the progress of the programme. Furthermore, the programme should be brought to the attention of the public, to increase the likelihood of attracting external funding. This public attention can for example be achieved by using the media. Within Hartslag Limburg, it was the task of the project leader to create, maintain and expand the network of partners. It is highly recommendable for future programmes to also appoint a person who can execute this task on a full-time basis. To give an indication of the staffing involved: the Hartslag Limburg coordinating agency invested an annual staff input of at least 1.5 FTEs .The main question that remains is whether spending money on programmes like Hartslag Limburg is useful. This question can only be answered by relating the costs of the programme to its effects. Schuit et al. (in press) have already analysed the overall effects of the Hartslag Limburg campaign, and their findings are promising: the community part of the campaign seems to have succeeded in reducing the age- and time-related increase in body weight and blood pressure among the community .Whether the effects of a community intervention like Hartslag Limburg will outweigh its costs will be addressed in a forthcoming cost-effectiveness study.The costs of the interventions of the community programme of the Hartslag Limburg campaign were calculated to be €900,000. Because the costs of all interventions were calculated separately, using a bottom-up procedure, our results should provide valuable information to health promotion specialists and policy makers. Furthermore, the methodology used improves the validity and reliability compared to previous studies in this field and it makes it easy to generalise the costs to other settings and countries.This study further shows that the costs do not have to be borne by one agency. Funding, subsidies and sponsoring can achieve that cost sharing. These can be regarded as valuable potential sources of funding. A network is required, however, to successfully implement a large-scale set of interventions like Hartslag Limburg.Finally, contrary to previous studies, the present study calculated the costs according to guidelines for economic evaluation. This means that costs can easily be related to the effects of the intervention to calculate a cost-effectiveness ratio, which can be used for decision-making at macro-level.The author(s) declare that they have no competing interests.ETR synthesised the analyses and coordinated the writing. WG conceptualised ideas, and helped to interpret findings. MS assisted in acquiring the data. ER coordinated the implementation of the interventions and contributed to data acquisition. AA conceived of the study, conceptualised ideas and supervised all of the study's aspects. All authors helped to interpret findings, reviewed drafts of the manuscript and gave final approval to the version to be published.The pre-publication history for this paper can be accessed here:Overview of the types and quantities of the resources necessary to implement the major interventions The file contains a table that list the resources that are necessary to implement the interventions that were carried out within the community programme of Hartslag Limburg.Click here for file


Rousettus aegyptiacus) in Gabon as shown by finding virus-specific RNA and IgG antibody in individual bats. These Marburg virus positive bats represent the first naturally infected non-primate animals identified. Furthermore, this is the first report of Marburg virus being present in this area of Africa, thus extending the known range of the virus. These data imply that more areas are at risk for MHF outbreaks than previously realized and correspond well with a recently published report in which three species of fruit bats were demonstrated to be likely reservoirs for Ebola virus.Marburg and Ebola viruses can cause large hemorrhagic fever (HF) outbreaks with high case fatality (80–90%) in human and great apes. Identification of the natural reservoir of these viruses is one of the most important topics in this field and a fundamental key to understanding their natural history. Despite the discovery of this virus family almost 40 years ago, the search for the natural reservoir of these lethal pathogens remains an enigma despite numerous ecological studies. Here, we report the discovery of Marburg virus in a common species of fruit bat ( Forty years after the discovery of Marburg virus as the cause of a hemorrhagic fever outbreak among laboratory workers in Germany, the natural reservoir for this highly pathogenic filovirus remains unknown Rousettus aegyptiacus) as evidenced by the presence of virus specific RNA and antibody.Here, we report testing of bats collected from Gabon and Republic of Congo and demonstrate Marburg virus infection in a common species of fruit bat  to inactivate virus. Each tissue was then diluted to 1X and homogenized for 2 minutes, at 1500 strokes/min using a ball-mill tissue grinder . Total RNA was extracted from ∼150 ul of the homogenate Epomops franqueti (N = 47) were used as the negative control group.IgG was detected from bat sera diluted 1∶100 using a previously described protocol modified for Marburg virus Bat derived sequence fragments (Genbank accession numbers EU068108-13) were concatenated then aligned with 18 MBG virus genomes  Maximum likelihood analyses (bootstrap 500 replicates) were completed . Genetic distances were calculated using the Wisconsin package of GCG version 10.3. .Bat species were morphometrically determined in the field using the species identification key developed by Bergman Rousettus aegyptiacus. Homogenized liver and spleen samples of 1138 bats were first analyzed using a Marburg virus-specific real-time RT-PCR assay to the VP40 gene recently used for human diagnostic testing during the 2005 Angola MHF outbreak Rousettus aegyptiacus, were positive at low levels  in the real-time assay. All four of these bats were trapped near caves in 2005 and early 2006 at two geographic locations in Gabon 250 km apart and approximately 700 km north of Uige, Angola. These four samples were then subjected to further analysis by conventional nested RT-PCR targeting the virus VP35 and NP genes  (Rousettus aegyptiacus while none of the other species tested (N = 196) had corrected OD values greater than this threshold value. Sera from three of the 29 bats had OD values greater than 0.13 when diluted 1∶400 while another five bats, including 1448 and 2296, met the same criteria at dilutions of 1∶1600  using a goat anti-bat conjugate made by immunization with IgG from multiple diverse bat species (both micro and mega-chiropterans). The results of this secondary testing were identical to the initial serological findings using protein A/G (data not shown). These data indicate a substantial fraction, almost 9%, of Rousettus aegyptiacus trapped at these locations may have low-level antibody to Marburg virus, while another 3% have more significant Marburg antibody titers. Among the R. aegyptiacus population tested for which age determinations could be made, evidence of Marburg infection in bats favored adults (24/138) over juveniles (4/86), 17.4% to 4.6% respectively . However, firm conclusions about the proportion of infected adult versus juvenile populations are difficult because the majority of ‘positive’ bats show only low titers of Marburg antibody while those bats with more conclusive evidence of Marburg infection, by being either PCR positive and/or having IgG titers greater than or equal to 1∶400 (N = 8), are more equivalently distributed (five adults and three juveniles). In addition, there could be residual maternal Marburg-specific antibody in the juvenile bat population. Fifteen of the bats, with OD values greater than 0.13, were males while among the three PCR-positive animals, two were adults  and one was a juvenile .After screening the collection of bats by real-time RT-PCR, we tested for Marburg virus specific antibody in sera (if available) from all bats trapped at the two locations (438 bats) from which PCR positive bats were found . Interesanqueti) . Moreovef 1∶1600 . UnfortuR. aegyptiacus, it is worth noting that all of the animals caught appeared clinically healthy and were strong enough to leave their roost to forage for food. Virus isolation attempts and antigen detection tests on the same liver/spleen organ extracts were negative, which along with the quantitative PCR data, indicate low levels of Marburg virus in these organs.The serological data, combined with the PCR data, are suggestive that these bats may represent a bon-a-fide reservoir species. However, we cannot rule out periodic contact by the bats with an as yet unnamed reservoir. The presence of both virus RNA and IgG antibodies in three (or possibly four) of the animals is consistent with extended viremias, but may well represent late acute phase infections. While it is difficult to determine from these data if Marburg virus causes significant morbidity in R. aegyptiacus representing a natural host for Marburg virus. Cave roosting is not generally observed with most fruit bats R. aegyptiacus is known to roost in caves, a behavior that correlates well with the epidemiologic linkage of greater than 80% of human cases in the Durba MHF outbreak to mining activity in a gold mine R. aegyptiacus encompasses the geographic origin of all known sources of Marburg virus outbreaks (Pteropodinae may serve as filovirus hosts and 3) the species of fruit bats that harbor Ebola and Marburg viruses are likely distinct, yet their home ranges may have large areas of overlap. Identification of the reservoir host should allow development of risk reduction measures to help mitigate the potential of future disease outbreaks.Although inconclusive, several other lines of evidence are consistent with utbreaks  [13] as


Children with congenital hearing impairment benefit from early detection and management of their hearing loss. These and related considerations led to the recommendation of universal newborn hearing screening. In 2001 the first phase of a national Newborn Hearing Screening Programme (NHSP) was implemented in England. Objective of this study was to assess costs and effectiveness for hospital and community-based newborn hearing screening systems in England based on data from this first phase with regard to the effects of alterations to parameter values.Design: Clinical effectiveness analysis using a Markov Model. Outcome measure: quality weighted detected child months (QCM).Both hospital and community programmes yielded 794 QCM at the age of 6 months with total costs of £3,690,000 per 100,000 screened children in hospital and £3,340,000 in community. Simulated costs would be lower in hospital in 48% of the trials. Any statistically significant difference between hospital and community in prevalence, test sensitivity, test specificity and costs would result in significant differences in cost-effectiveness between hospital and community.This modelling exercise informs decision makers by a quantitative projection of available data and the explicit and transparent statements about assumptions and the degree of uncertainty. Further evaluation of the cost-effectiveness should focus on the potential differences in test parameters and prevalence in these two settings. Between one and two children per 1000 live births have a moderate or greater bilateral permanent hearing loss . ChildreThese and related considerations led to a comprehensive review of the possible role of newborn hearing screening in the UK . The revThe first implementation phase of the Newborn Hearing Screening Programme (NHSP) includes sites where the screening is performed by Health Visitors at a home visit, usually at 10 days of age. This model is called 'community-based screening,' in contrast to the 'hospital based' model where babies are screened in maternity hospital by a new cadre of screeners prior to discharge with follow-up of missed cases in a variety of ways.a priori be expected are screen performance, maternal satisfaction or anxiety, and costs. The NSC and others have been clear that a national screen for newborn hearing is desirable, with national quality assurance processes and ongoing audit. The policy question is the extent to which a national screen could encompass the two different models of delivery, and if it could, on what basis areas might be permitted or encouraged to select one or the other model. As the first step, the NHSP Steering Group wished to verify that the data on screen performance, maternal satisfaction/anxiety, and costs did not argue strongly against the community-based model.The evaluation of the first phase of the NHSP, commissioned by the Department of Health in accordance with National Screening Committee (NSC) policy, included a comparison of hospital-based and community-based screening as a main comparison of interest. The measures on which differences between the two models might The aim of the modelling presented here was to determine the costs and effectiveness for hospital and community-based systems of Neonatal hearing screening in England and Wales with a special regard to the effects of reasonable alterations to parameter values.We modelled the cost-effectiveness of the two screening systems, hospital- and community-based screening using some already-available costs data and screen performance data from the first phase implementation, data from the published literature on newborn hearing screening, and further data collection on costs from the first phase of the NHSP. We used a modified version of a decision-analytic model which has been developed for a German Health Technology Assessment funded by the German Federal Ministry of Health .We estimated absolute and incremental costs and effectiveness of two newborn hearing screening settings. The recommendations of the Panel on Cost-Effectiveness in Health and Medicine were followed . Target see Figure A state-transition (Markov) model  was deve- Unknown status- Healthy (hearing) confirmed by diagnostic test or screening – true negative- Healthy (hearing) not confirmed by diagnostic test- Hearing impaired confirmed by diagnostic test or screening – true positive- Thought to be healthy (hearing) but hearing impaired – false negative- Thought to be hearing impaired but healthy (hearing) – false positive- Not followed up/not compliantThe model starts with a cohort of newborns being of unknown status and applies transition probabilities recursively to simulate how children progress through different states. In each cycle (lasting one month) children can undergo several possible transitions which accrue costs and utility weights. Ultimately all children from the initial cohort are diagnosed as healthy or as impaired or, if they are healthy, some remain 'undiagnosed' .A predefined and externally reviewed literature search on newborn hearing screening on all relevant electronic databases has been performed. Search strategy and methods have been reported in detail elsewhere . DetectePrevalence of congenital hearing disorders was derived from comprehensive literature searches. The probability of hearing children presenting with falsely suspected hearing disorder was estimated by a panel of experts. The probability of being detected at a certain age without screening was estimated from a survey of activity in an area of Germany in 1998 and 1999 . PositivThe slope of the weighting function was estimated by experts making the following assumptions: each month detected before the age of 6 months is weighted with 1, on the general assumption that children detected (and treated) within the first 6 months of life can develop typical speech and language abilities. If not detected within the first 12 months, profoundly and severely impaired children will end up with a weight of 0.85, and moderately impaired children with a weight of 0.90. Presuming that 50% of the children with permanent congenital hearing disorders are moderately impaired gives a weight of 0.875 for every month which is detected after the first birthday. The weights between 6 and 12 months were calculated by linear extrapolation.Screening and diagnostic procedures are presented under the assumption of conditional independence, i.e. test parameters are independent of the prevalence of the condition and test results of diagnostic testing are independent of test results of screening procedures. This is plausible because screening and diagnostic testing are based on different testing principles.Screen performance data and costs for screening and diagnosis have been derived from empirical data from the NHSP first wave sites.st May 2002 – Avon, Barnsley, Bradford, Buckinghamshire, Dewsbury, Manchester, North Staffordshire, Northumberland, and Oxford – were included in the study. Four community-based areas and seven hospital-based areas were able to provide data. Table All community-based areas – East Sussex, Shropshire, Wiltshire (Bath) and Wiltshire (Swindon) – and all hospital-based areas that had started NHSP before 1The following data have been provided by the 11 sites and included into the cost/effectiveness calculation: Screen performance data , staff grade and full-time equivalent , quantity, make and model of screening equipment, quantity, make and model of computers and printers, quantity and make of consumables, travel costs (exclusive travel associated with training) and any additional costs . Additional information was obtained from National Health Service salary scales, the National Health Service Rehabilitation Services Catalogue (screening equipment and consumables), the Medical Research Council Institute of Hearing Research for calibration costs, IT costs and training costs. Additionally, training costs during screening and IT training have been obtained. Audiology services reported costs for 10 consecutive referrals and all true cases for audiology follow-up costs.Incidence and prevalence of congenital permanent bilateral hearing loss were assumed equal.To calculate salaries, midpoint was taken.Within the community model, health visitors' time for NHSP was estimated at 1%. This estimate was based on a Health Visitor screening on average 1.3 children per week and spending ca 20 minutes on the screen, which is based on data from the sites and Netten et al .National insurance and superannuation was taken as 13%.Non-staff related costs refer to the overheads, building capital and equipment costs associated with running audiology services. Most NHSP services use a number of different facilities to deliver the different components of the programme and do not have these figures readily available. Hence, to determine these costs, the following steps have been taken: Allowances for indirect overheads  have been taken as fixed cost of £2216, and building capital (the costs assigned to treatment and non-treatment space) relative to the level of pay scale based on Netten et al [Direct overheads i.e. the costs associated with lighting, heating and cleaning were assumed to be 11% of the sum of staff costs, indirect overheads and building capital. This was based on previous studies carried out in hospital settings where the direct overheads were found to account for 4% to 18% (midpoint 11%) of total costs ,18. As tWhen equipment is totalled over 10 years, a 5% annuity for each year of life has been allowed for. VAT at 17.5% has been included.The sites provided information of the quantity of consumables they used in November 2002 and prices obtained from NHS Purchasing and Supply Agency. The figure was multiplied by 12 for the whole year cost. VAT at 17.5% has been included.Calibration costs were based on the manufacturers' specifications.Only staff travel costs directly associated with the screen were included. Data was obtained from the proformas filled in by the Team Leaders.Initial training cost calculation was based on cost of attending, cost of conducting the training, venue costs. Cost of attending and conducting the training consist of travel and accommodation costs and cost of time spent by participants and deliverers. Cost of time spent was calculated as number of days attending/delivering training divided by number of workdays per year multiplied by annual salary. Data was obtained from the proformas filled in after each training event. Refresher training cost calculation was based on an assumption that refresher training would be 0.5 day a year per screener.We assumed the typical audiological assessment which confirms false positive status consisted of an ABR (Automated Evoked Brainstem Response). We assumed a cost of £160 per referral.Costs to the families associated with NHSP screening  were not included.Costs and their standard deviations were calculated separately for both settings and weighted with the number of children screened per site.Future costs were discounted at a rate of 6% per year, future effects at a rate of 1.5% per year. Yearly discount rates have been converted to monthly discount rates.One-way and multiple sensitivity analyses were performed on all relevant parameters.Multivariate simulations were used for probabilistic modelling (Monte Carlo). The simulation associates with each of the model variables a probability density function which represents our uncertainty about a fixed but unknown value. The ranges for test parameter estimates derived empirically and from the literature assumed beta distribution based on available ranges of estimates, and ranges for empirical cost data assumed gamma distribution. The model was evaluated for 1,000 trials.As the number of sites was small the parameter estimates were estimated in a context of uncertainty. We wanted to evaluate the impact of extreme parameter changes on outcome and decision between alternative settings. As described by Felli and Hazen , Monte C- to show the probability that one setting  is more cost-effective than the other under the assumption that the two sites differ in one parameter, and- to indicate which difference in a certain parameter between sites might result in substantial differences in costs.This was achieved by the following procedure: The simulation was run twice with all parameters except one held fixed, the first time with the extreme high estimate of the parameter, the second time with the extreme low estimate of the parameter. This resulted in "high" and "low" estimates for costs and QCM for each setting. Differences of costs and QCM were then calculated using the "high" estimate for hospital and the "low" estimate for community and vice versa. This was done for each of the parameters mentioned. If QCM between hospital and community did not vary, only cost differences were calculated. If both costs and QCM varied the resulting distributions in mean differences of costs and QCM were combined using the Net Benefit Approach . The IncData 3.5 (TreeAge Inc.) was used to construct and run the Markov model and Excel (Microsoft Corp.) was used to validate the model and to perform the Monte Carlo simulations.We modelled costs and effectiveness of universal newborn hearing screening in two different settings. As test parameters were held to be constant across hospital and community sites there was no difference in effectiveness, only in costs. Both hospital and community settings yielded 134 true positive cases  and 794 quality weighed detected child months (QCM) at the age of 6 months with total costs of £3,690,000 per 100,000 screened children in hospital and £3,340,000 in community. Table Higher prevalence in hospital resulted in higher costs figure  and highWe applied a decision-analytic Markov model to empiric data of first stage implementation areas of NHSP in England to evaluate cost and effectiveness of different settings for newborn hearing screening. Base case assumptions with constant test parameters but cost difference between hospital and community settings yielded a cost difference of £25 per quality weighted detected child month (QCM): To detect one hearing impaired child one month earlier produced costs of £268 in hospital settings and of £243 in community settings. This cost difference, however, was not statistically significant. Probabilistic multivariate Monte Carlo simulation revealed that in nearly half of 1000 simulated trials community settings would yield higher costs than hospital settings. The cost-effectiveness of the two newborn screening models – hospital-based and community-based – did not differ significantly, assuming comparable screen performance for the two newborn screening models. Projected magnitude of costs per detected child was comparable to the costs found by other UNHS models , provingUp to now preliminary data were too sparse to detect any differences of important input parameters – like screen performance and program costs – between settings. Extremes analyses showed that any statistically significant difference in prevalence, sensitivity, specificity and costs would result in significant differences in cost-effectiveness between settings. Any further evaluation of cost-effectiveness between different programme alternatives should evaluate in the first place if there is substantial difference in terms of these parameters.Our study has several limitations. Even though QCM was weighted, it is a surrogate parameter for the actual burden of disease for the child. To date, however, there is no study yielding empirical data on a more general effectiveness measure such as quality adjusted life years. There are drawbacks of this study concerning uncertainty on model parameters. There is still only limited evidence for further benefits of early diagnosis and intervention. Weighting assumptions in the presented model are therefore only estimates of the potential impact of late diagnosis and the actual child's burden of disease. To date there is no further studies should be conducted on this issue. Other parameter uncertainties should be ruled out as soon as long term data from the NHSP implementation are available.Probabilistic analysis of the incremental cost-effectiveness ratio can be used to give ceiling values. Policy makers can then decide on a fixed incremental effectiveness they would like to obtain by introducing a screening program and the model will show how probable this outcome will be under the assumption of parameter uncertainty.As a part of ongoing and future research the NHSP Evaluation aims to identify factors predicting high cost-effectiveness for either model and to compare maternal satisfaction/anxiety in hospital-based and community-based setting.The value of this modelling exercise lies in the provision of information to decision makers by a quantitative projection of available data and the explicit and transparent statements about assumptions and the degree of uncertainty. This has been achieved at an early stage of implementation. The evaluation of the NHS Newborn Hearing Screening Programme will serve as a valuable tool and example to justify and improve large scale screening programmes.The author(s) declare that they have no competing interests.EG carried out the statistical analyses and drafted the manuscript. KU designed and coordinated the study and participated in the writing process. FH, RST and JW participated in the statistical analyses and in the writing process. LD participated in the design of the study. JB conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


With the recent advances in high-throughput genotyping technologies that allow for large-scale association mapping of human complex traits, promising statistical designs and methods have been emerging. Efficient simulation software are key elements for the evaluation of the properties of new statistical tests. SLINK is a flexible simulation tool that has been widely used to generate the segregation and recombination processes of markers linked to, and possibly associated with, a trait locus, conditional on trait values in arbitrary pedigrees. In practice, its most serious limitation is the small number of loci that can be simulated, since the complexity of the algorithm scales exponentially with this number.I describe the implementation of a two-step algorithm to be used in conjunction with SLINK to enable the simulation of a large number of marker loci linked to a trait locus and conditional on trait values in families, with the possibility for the loci to be in linkage disequilibrium. SLINK is used in the first step to simulate genotypes at the trait locus conditional on the observed trait values, and also to generate an indicator of the descent path of the simulated alleles. In the second step, marker alleles or haplotypes are generated in the founders, conditional on the trait locus genotypes simulated in the first step. Then the recombination process between the marker loci takes place conditionally on the descent path and on the trait locus genotypes. This two-step implementation is often computationally faster than other software that are designed to generate marker data linked to, and possibly associated with, a trait locus.Because the proposed method uses SLINK to simulate the segregation process, it benefits from its flexibility: the trait may be qualitative with the possibility of defining different liability classes  or it may be quantitative and normally distributed. In particular, this implementation is the only one available that can generate a large number of marker loci conditional on the set of observed quantitative trait values in pedigrees. In recent literature, algorithms and software were developed to simulate the segregation process of marker loci that are in linkage disequilibrium (LD), some allowing for the possibility for the markers to also be associated with a trait -3. TheseMost recently, the software SimPed  has beenThe SIMLA software ,2 has reAn advantage of SIMLA over other gene dropping simulation tools (such as SimPed or SimM ) is thatIn contrast to these gene dropping simulation tools, SLINK ,11  and at the same genetic position. At the same time, a perfectly informative marker  is generated at the same genetic position as the trait locus; this marker serves to describe the segregation path of the chromosomes in the pedigree, and is known hereafter as the descent marker. The descent marker allele frequencies are irrelevant, and the marker is generated in LE with the trait locus. In the output of FastSLINK, the alleles of the genotypes are ordered in such a way that the phases of the chromosomes (or haplotypes) are fully known; thus, the descent marker provides complete information about the identity by descent state between any two trait locus alleles in the pedigree.Given a pedigree, a set of observed trait values, and a genetic model that translates genotypes into trait values, the following implementation relies on FastSLINK to simulate the trait locus genotypes conditional on the observed trait values. Since FastSLINK can generate LD between a trait locus and a marker locus, it is possible to obtain in the output the simulated genotypes at the trait locus  simply by simulating a marker locus that is in perfect LD with the trait locus  are simulated for all individuals in the pedigree. First, chromosomes in the founders are generated according to user-specified alleles and haplotype frequencies (when LD is desired) and conditional on the trait locus allele already found on the chromosome . Then segregation of the chromosomes at the position of the trait locus follows the path of the descent marker, while recombination occurs on both sides of the trait locus, according to a user-specified genetic map , without interference.Figure Even though there is no serious limit to the size of the pedigrees that FastSLINK can handle, there is a limit on the number of alleles a marker can have: the implementation of FastSLINK uses a bitshift operator to shift a long integer by a number of bits that corresponds to an allele number. Thus, for most computers the total number of alleles a marker may have cannot exceed 32, which restricts the above implementation to pedigrees having at most 16 founders, which is still four times as much as the number of founders SIMLA handles.The second step of the procedure described above has been implemented in a C++ program named SUP .F/2 sib-pairs, where F is the number of founders in the pedigree. All individuals in the last generation were assumed to be affected; the affection status of all other individuals was assumned to be unknown, and thus free to vary.The performance of the combination of FastSLINK and SUP to simulate genetic data linked to, or linked to and in LD with, a susceptibility locus was compared with that of SIMLA v3.1 and ALLEGRO v1.2c , which aOne susceptibility locus or two interacting ones were simulated for a dichotomous trait. In the case of one susceptibility locus, the high-risk allele frequency was taken to be 15%, and penetrances were chosen under a multiplicative model such that the relative risk for the heterozygote at the trait locus is 1.5 (2.25 for the high-risk homozygous). In the case of two susceptibility loci, a two-locus interaction threshold effect was simulated (see e.g. ): carrieIn all cases, two chromosomes each having 6 or 6000 markers were simulated. If only one susceptibility locus was simulated, then the second chromosome was generated under a null model. When simulating under LE, each marker had four equifrequent alleles. When simulating under LD, only 6 bi-allelic markers were simulated on each chromosome (the default limit of SIMLA) forming the 12 haplotypes found in Table While FastSLINK can only generate a single trait locus in a single run, it can be used to simulate multi-locus susceptibility between unlinked loci in multiple runs: in the case of two loci, given a set of two-locus penetrances, the marginal penetrances can be calculated for one of the loci and, given the possible genotypes at that locus, conditional penetrances can be calculated for the second trait locus  than to create a single replicate of a set of 1000 families . SIMLA always generates genotypes at two trait loci, even if only one is desired (in which case the user must select a null genetic model for the second trait locus). It is thus not surprising to see similar times for the one or two trait locus scenarios. If the two trait loci lay on the same chromosome, then SIMLA's times would be slightly longer [single replicate of only 3 markers  in family 2F, while this time increased to more than 85 minutes for 4 markers. It was not possible to simulate more loci with FastSLINK due to memory constraints.To get an idea of the increase in time it takes FastSLINK in its standalone version to simulate an increasing number of marker loci, note that it took 8 seconds for FastSLINK to generate a SUP's execution time scales linearly with the number of markers that are in LE, and linearly with the number of haplotypes with non-zero frequency. For illustration purposes, it was able to handle 100000 markers in LE and all 2097152 possible haplotypes of 21 SNPs in LD (including a trait locus).To show the validity of the replicates generated by FastSLINK/SUP, haplotype frequencies were estimated from 1000 replicates of family 2F, generated under the scenario of a single susceptibility locus, in LD with the markers, forming the haplotypes found in Table In summary, FastSLINK is one of the most flexible simulation tool that exists: the complexity of the algorithm described in Ott  is lineaWith SIMLA and SLINK or SLINK-based approaches available, investigators have access to a wide variety of options, including the possibility of simulating linkage disequilibrium with one or more trait loci, to conduct efficient and realistic family-based simulation studies.Project name: SUP: Slink Utility ProgramProject home page: Operating systems: Unix/LinuxProgramming language: C/C++Other requirements: FastSLINK , gcc 2.95.3 or higher, perl, csh/tcshLicense: GNU General Public Licence, a linux emulator. It compiles using gcc version 2.95.3 and higher. To install, type at the prompt % gunzip sup.tgz % tar -xvf sup.tar Go to the src/directory and compile by typing % make or % g++ *.cc -o sup The documentation can be found in the doc/directory. In there, scripts are included to help in the creation of input files. These scripts require perl and csh/tcsh.Included with this manuscript are the source code for SUP and documentation on both steps of the implementation described here. It is intended to be compiled on a UNIX/Linux system. On a Windows environment, the simplest way is to install CYGWIN Click here for file


Information about the costs of recurrent breast cancer is potentially important for targeting cost containment strategies and analysing the cost-effectiveness of breast cancer control programmes. We estimated these costs by abstracting health service and consumable usage data from the medical histories of 128 patients, and valuing each of the resources used. Resource usage and costs were summarised by regarding the recurrence as a series of episodes which were categorised into five anatomical site-based groups according to the following hierarchy: visceral, central nervous system (CNS), bone, local and other. Hospital visits and investigations comprised 78% of total costs for all episodes combined, and there were significant differences between the site-based groups in the frequency of hospital visits and most investigations. Total costs were most accurately described by separate linear regression models for each group, with the natural logarithm of the cost of the episode as the dependent variable, and predictor variables including the duration of the episode, duration squared, duration cubed and a variable indicating whether the episode was fatal. Visceral and CNS episodes were associated with higher costs than the other groups and were more likely to be shorter and fatal. A fatal recurrence of duration 15.7 months (the median for our sample) was predicted to cost $10,575 . Reduction of the substantial costs of recurrent breast cancer is likely to be a sizable economic benefit of adjuvant systemic therapy and mammographic screening. We did not identify any major opportunities for cost containment during the management of recurrences.