Vaccinia virus strain Lister Elstree (VACV) is a test virus in the DVV/RKI guidelines as representative of the stable enveloped viruses. Since the potential risk of laboratory-acquired infections with VACV persists and since the adverse effects of vaccination with VACV are described, the replacement of VACV by the modified vaccinia Ankara strain (MVA) was studied by testing the activity of different chemical biocides in three German laboratories.The inactivating properties of different chemical biocides  were tested in a quantitative suspension test according to the DVV/RKI guideline. All tests were performed with a protein load of 10% fetal calf serum with both viruses in parallel using different concentrations and contact times. Residual virus was determined by endpoint dilution method.The chemical biocides exhibited similar virucidal activity against VACV and MVA. In three cases intra-laboratory differences were determined between VACV and MVA - 40% (v/v) ethanol and 30% (v/v) isopropanol are more active against MVA, whereas MVA seems more stable than VACV when testing with 0.05% glutardialdehyde. Test accuracy across the three participating laboratories was high. Remarkably inter-laboratory differences in the reduction factor were only observed in two cases.Our data provide valuable information for the replacement of VACV by MVA for testing chemical biocides and disinfectants. Because MVA does not replicate in humans this would eliminate the potential risk of inadvertent inoculation with vaccinia virus and disease in non-vaccinated laboratory workers. The global eradication of variola virus is one of the greatest achievements of modern medicine. Following this eradication, routine childhood vaccination against the causative agent was halted because of a declining probability of the importation and spread and occasional serious side effects of the vaccination . In EuroDue to the incidence of serious side effects in immunocompetent individuals about 1/500,000) a more attenuated vaccinia virus with a high safety profile for mammalian species was developed - the so-called modified vaccinia Ankara strain (MVA) ,5. In th00,000 a Presently the vaccinia virus Lister Elstree is still used as model virus for testing the virucidal activity of chemical disinfectants. In Europe the norm EN 14476:2007 exists for determining virucidal activity , which mIn veterinary medicine vaccinia virus strain Lister Elstree, ECBO-virus strain LCR-4, reovirus type 1 and Newcastle-Disease-Virus strain Montana are required for virucidal testing of chemical disinfectants, pursuant to the guideline of the German Veterinary Medical Society (DVG) .The DVV/RKI guidelines state that laboratory workers handling vaccinia virus should be vaccinated . HoweverIn the USA it is not necessary to report laboratory-acquired VACV infections - nevertheless between 2005 and 2007, five cases were reported to the CDC . These iWithin this context it is important to highlight that in a recently published study comparable results could be shown when using MVA instead of vaccinia virus strain Lister Elstree when testing chemical disinfectants for virucidal activity. This study was conducted according to the DVG guidelines with quantitative suspension and qualitative carrier tests .In order to investigate the possibility of substituting VACV with MVA under the guidelines of the DVV/RKI, different biocides , which are often ingredients used in disinfectants, were tested with quantitative suspension tests using VACV and MVA in parallel. The tests were performed in the presence of organic load. Furthermore, the aim of the study was to evaluate the robustness of the testing method when performing an inter-laboratory comparison ). Therefore, the results of three laboratories were compared using statistical analysis as required by the DVV/RKI guidelines .Three German laboratories participated in this EQA program (a list of participants is given in the acknowledgements section) - lab. 1, lab 2, lab. 3, respectively. Participation was open and free of charge to all laboratories.2 in minimum essential medium (MEM) supplemented with 10% foetal calf serum (FCS). These cells were infected with a multiplicity of infection of 0.1. After cells exhibited a cytopathic effect, they were subjected to a twofold freeze/thaw procedure followed by a low speed centrifugation (10 min and 1000 × g) in order to sediment cell debris. After aliquoting, test virus suspension was stored at -80°C.MVA  were propagated in BHK-21  and VACV (ATCC VR-1549) in African green monkey kidney cells . For virus propagation cells were grown at 37°C and 5% COFive biocides were used in the study: formaldehyde , glutardialdehyde , ethanol , isopropanol , and peracetic acid (PAA) . The pH values of the biocides were measured if necessary.Test samples for the proficiency panel (stock solutions) were generated by an independent provider . All samples were sent out by a commercial parcel service and arrived within 24 h of dispatch, according to the date of receipt provided by the participants. The participants were asked to analyze the material according to the method described in the German DVV/RKI guidelines .-8. One part by volume of water of standardized hardness (instead of virus suspension) was mixed with one part by volume of organic load and eight parts by volume of the biocide. Aliquots of 50-100 μl from each test concentration and each dilution was then inoculated into seven to eight wells of a 96-well microtitre plate containing 50 μl cell suspension of Vero or BHK-21 cells, respectively. The cell cultures were observed for cytotoxic effects for the same incubation time as afterwards used for the suspension tests.In order to determine cytotoxicity biocides were serially diluted tenfold in MEM up to a dilution of 107-108 tissue culture infectious dosis 50% (TCID50)/ml) and one part by volume of the organic load were mixed with eight parts by volume of the biocide. The tests were carried out in ambient temperatures of 20-22°C. Infectivity was determined by means of end point dilution titration in microtitre plates. At the end of the chosen exposure time, activity of the biocides was immediately stopped by serial dilutions with ice-cold cell culture medium. 50 μl from each dilution were placed in seven to eight wells of a sterile polystyrene flat-bottomed 96-well microtitre plate containing 50 μl cell suspension (10-15 × 103 cells per well) of Vero or BHK-21 cells. Cultures were observed for cytopathic effects (CPE) after seven to ten days of inoculation. All tests were conducted in two independent tests run on different days. Virus controls were incorporated after the longest exposure time. All tests were performed with an organic load of 10% FCS.Tests were carried out in accordance with the DVV/RKI guidelines at 20°C . One par50/ml including standard deviation. Titre reduction is presented as the difference between the virus titre after contact time with the substance and control virus titre. This difference is given as reduction factor (RF) including its 95% confidence interval (CI). A reduction of infectivity of ≥4 log10 steps (inactivation ≥99.99%) was regarded as evidence of sufficient virucidal activity. The calculation was performed according to the DVV/RKI guidelines [10 step considering the lower respectively upper bounds of the 95% CI.The virus titres were determined using the method of Spearman and Kaerber ,23 and eidelines . Biologi3.6 TCID50/ml was observed after 1 min with both - VACV  a maximum titre reduction of at least 10CV Table . TestingCV Table . At loweA time-dependent increase of virucidal activity of the biocides was detected when testing lower concentrations (not shown in table). Concerning glutardialdehyde a time - and concentration-dependent increase of virucidal activity were detectable for the 0.1% and 0.05% concentrations for both viruses Table . In cont10 TCID50/ml (not shown in tables).The standard deviations for the control virus titrations were below 0.5 logIn many cases the RF is marked with the prefix "≥" indicating that the determination of the RF was limited by cytotoxic reactions and/or the amount of infectious viruses in the virus stock. Furthermore, the different levels on test virus suspension are to some extent responsible for inter-laboratory and inter-virus RF differences.10 step considering 95% CI) were seen. The different results concerned in both cases MVA (for 40% ethanol and 30% isopropanol)  Figure , 3.VACV is still used in many laboratories - e.g. as a model virus for a large enveloped DNA virus. The virus is also required in the German guidelines of DVV/RKI and DVG as a model virus for testing the virucidal efficacy of chemical disinfectants ,9.Before eradication of variola the risk of human laboratory infection with VACV was negligible because most of the population was vaccinated with VACV . As a loTo diminish this risk one possibility would be to vaccinate these laboratory workers, but there are concerns about the adverse effects of vaccination with VACV because such side effects are about 10 times more common in primary vaccinees than in those who are revaccinated . TherefoThe present study was undertaken with two aims in mind: first to evaluate a possible substitution of VACV with MVA and second to investigate the robustness of the test in evaluating the laboratory proficiency in a multi-center study. Three different laboratories participated in this EQA.By testing five chemical biocides from different groups  in a quantitative suspension test  we were 10 step considering 95% CI) intra- and inter-laboratory differences of viable VACV and MVA. Marked inter-laboratory RF differences were only observed in two cases; in three cases intra-laboratory differences between VACV and MVA were demonstrated.In our study we chose the concentration of the biocides and the length of the exposure time in order to see a kinetic and the transition from non-efficient to efficient virus inactivation. For the different biocides all RF data were compared with regard to relevant . In contrast, when testing with 0.05% glutardialdehyde, MVA seems to be more stable than VACV. One reason for these differences could be that in the selection of concentration and contact time-relation the kinetic was in the ascending proportion of the sigmoid area of the inactivation curve. These conditions were chosen in order to see a kinetic and the transition from non-efficient to efficient virus inactivation. On the other hand this would mean that even the slightest change in conditions or the test procedure could cause a maximum variance and lead to measurable changes in the results. Therefore, the RF variations do not appear to be attributable to relevant differences in the tenacity of both viruses. Furthermore, differences in the tenacity of both viruses are only found at concentrations far below the standards applied in practice.10 TCID50/ml. Observed RF discrepancies are found at a concentration of low activity and might be caused by different preparation of the virus test suspension, usage of different cell lines (Vero or BHK-21 cells), limitations by cytotoxic reactions and the aforementioned strong influence of experimental conditions due to the inactivation kinetic.This is the first multi-center German EQA study on testing the virucidal activity of chemical biocides according to the DVV/RKI guidelines. Comparative testing of defined samples is the most efficient method to identify weaknesses in a laboratory or in certain methodological components. Our data shows that the test accuracy across the three participating laboratories is high. In all cases the standard deviations for the control virus titrations were below 0.5 logAs stated by Hartnack et al.  our dataThe authors declare that they have no competing interests.HR: author of the publication, also provided analysis and interpretation of data, responsible for study design. IR and JS: Co-authors of the publication and responsible for study design, also contributed analysis and interpretation of data. All authors have read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/185/prepub


We isolated an apparently new virus in the family Paramyxoviridae from stillborn piglets with deformities at a piggery in New South Wales, Australia. In 1997, the pregnancy rate and litter size at the piggery decreased markedly, while the proportion of mummified fetuses increased. We found serologic evidence of infection in pigs at the affected piggery and two associated piggeries, in humans exposed to infected pigs, and in fruit bats. Menangle virus is proposed as a common name for this agent, should further studies confirm that it is a newly recognized virus.


To investigate West Nile virus (WNV) circulation in rural populations in Gabon, we undertook a large serological survey focusing on human rural populations, using two different ELISA assays. A sample was considered positive when it reacted in both tests. A total of 2320 villagers from 115 villages were interviewed and sampled. Surprisingly, the WNV-specific IgG prevalence was high overall (27.2%) and varied according to the ecosystem: 23.7% in forested regions, 21.8% in savanna, and 64.9% in the lakes region. The WNV-specific IgG prevalence rate was 30% in males and 24.6% in females, and increased with age. Although serological cross-reactions between flaviviruses are likely and may be frequent, these findings strongly suggest that WNV is widespread in Gabon. The difference in WNV prevalence among ecosystems suggests preferential circulation in the lakes region. The linear increase with age suggests continuous exposure of Gabonese populations to WNV. Further investigations are needed to determine the WNV cycle and transmission patterns in Gabon. Flavivirus, family Flaviviridae. Although human WNV infection is generally asymptomatic or causes a 'flu-like illness, life-threatening neurological complications such as meningoencephalitis and flaccid paralysis have been reported [West Nile virus (WNV) is a mosquito-borne RNA virus belonging to the genus reported . WNV is reported ,3. WNV hreported ,4. Howevreported , Camerooreported  and the reported , with Igreported  during creported , as wellreported , led us reported . We consA total of 2320 villagers were interviewed, of whom 1869 (80%) lived in forested regions, 243 (11%) in savannas, and 208 (9%) in the lakes region (Table Surprisingly, the overall WNV-specific IgG prevalence was high, at levels similar to those found in countries hit by outbreaks. During the WNV outbreaks that occurred in Sudan in 1898  and in tThese preliminary results strongly suggest that WNV circulation is widespread in Gabon. The linear increase in WNV IgG prevalence with age suggests continuous exposure of Gabonese populations to this virus. Moreover, although serological cross-reactions due to non specific WNV antibodies could not been ruled out, the larger difference in the prevalence rates between the different ecosystems suggests preferential WNV circulation in the lakes region. This may be explained by specific ecological features such as mosquito vector species or a higher density of residental and migratory birds .In conclusion, this first serological survey of West Nile virus in Gabonese human populations shows widespread circulation of this virus. Further investigations are needed to determine the WNV cycle and transmission patterns in Gabon.The authors declare that they have no competing interests.Performed the experiment: DN, XP, JP. Analyzed the data: XP, DN, EL. Wrote the paper: XP, EL. All authors read and approved the final manuscript


Cldn13), a member of the Claudin family of tight junction proteins using RNA expression, microarray and phylogenetic analysis. We present evidence that Cldn13 appears to be co-ordinately regulated as part of a stress induced erythropoiesis pathway and is a mouse-specific gene mainly expressed in tissues associated with haematopoietic function. CLDN13 phylogenetically groups with its genomic neighbour CLDN4, a conserved tight junction protein with a putative role in epithelial to mesenchymal transition, suggesting a recent duplication event. Mechanisms of mammalian stress erythropoiesis are of importance in anaemic responses and expression microarray analyses demonstrate that Cldn13 is the most abundant Claudin in spleen from mice infected with Trypanosoma congolense. In mice prone to anaemia (C57BL/6), its expression is reduced compared to strains which display a less severe anaemic response (A/J and BALB/c) and is differentially regulated in spleen during disease progression. Genes clustering with Cldn13 on microarrays are key regulators of erythropoiesis , erythrocyte membrane proteins (Rhd and Gypa), associated with red cell volume (Tmcc2) and indirectly associated with erythropoietic pathways . Relationships between genes appearing co-ordinately regulated with Cldn13 post-infection suggest new insights into the molecular regulation and pathways involved in stress induced erythropoiesis and suggest a novel, previously unreported role for claudins in correct cell polarisation and protein partitioning prior to erythroblast enucleation.Mammals are able to rapidly produce red blood cells in response to stress. The molecular pathways used in this process are important in understanding responses to anaemia in multiple biological settings. Here we characterise the novel gene Claudin 13 ( The Claudins are a family of more than 23 small (20–27 kDa) tetraspan transmembrane proteinsStress induced erythropoiesis is a process invoked under conditions of anaemia and requires significant proliferation of progenitor cells before terminal differentiation processes are invoked. In adult mammals the usual site of erythropoiesis is bone marrow, however, under conditions of anaemic stress (e.g. caused by acute bleeding or parasitic infection) the spleen can become a major site of red blood cell (RBC) production. This is observed as increased numbers of erythropoietic islands, the functional units of erythropoiesis, comprising a central macrophage surrounded by erythrocytic cells at various stages of maturation. In addition, in adult mice (but not humans) a significant proportion of extra-medullary erythropoiesis normally occurs in spleen Trypanosoma congolense is a tsetse fly-transmitted intravascular protozoan parasite causing severe, acute or chronic disease (trypanosomosis) in mammals including cattle and other livestock and consequently affects development and economic growth in sub-Saharan Africa. In cattle, consistent features of trypanosomosis are anaemia and sporadic episodes of fever. Infected animals exhibit leukopenia, weight loss and enlargement of some organs (spleen and liver). Chronic infection is characterised by appetite loss, lethargy and emaciation and often death due to congestive heart failureT. congolense initially causes an increase in medullary erythropoiesis and a reduction in the myeloid: erythroid cell ratio. Subsequently erythropoiesis declines, while granulopoiesis, megakaryopoiesis, plasma cell production and erythrophagocytosis increase T. congolense infection: C57BL/6 mice are relatively tolerant but develop a severe anaemic response, whereas BALB/c and A/J strains, although regarded as susceptible (exhibit higher levels of parasitemia and early death), have relatively mild and transient anaemia It is hoped that understanding of trypanotolerance, as seen in some indigenous breeds of cattle, will advance disease control. Inbred strains of laboratory mice are used as a model system to study susceptibility to Cldn13). Functionally, we have evidence from expression patterns and microarray data that Cldn13 may play a role in a stress erythropoietic response to T. congolense induced anaemia. Our data implicate Cldn13 and other previously unassociated genes in an erythropoietic pathway constitutively downregulated in mice prone to anaemia, thus providing potential new insights into the molecular regulation of erythropoiesis. It is probable that Cldn13 is a mouse specific gene that arose as a result of a local gene duplication event.Here we report the characterization of a member of the Claudin family, Claudin 13 (Cldn13. Screening an in-house mouse embryo cDNA library with segments from a BAC clone (gi: AC079938) identified a new gene, Cldn13 (submitted to Genbank in 2002 gi: AF516681) with a PMP22_Claudin motif. Cldn13 spans a genomic region of 1277 bp and is located 29579 bp centromeric to Cldn4  region on human chromosome 7q11.23 to Cldn4 . The cDNto Cldn4  encodingto Cldn4 . CLDN13 to Cldn4 . Other pCldn13 sequences were not identified in any additional species, including humans, suggesting that Cldn13 may be specific to Mus musculus and possibly other closely related rodents. In Rattus norvegicus, a 225 bp sequence with 78% sequence identity to the 3′ UTR of Cldn13 was identified on chromosome 12 (AC091616). The overlapping genomic clone (AC091752) contains the rat Cldn4 gene indicating that this is the region syntenic to the mouse chromosome 5 region containing Cldn3, Cldn4 and Cldn13. There does not appear to be a full-length rat Cldn13 ortholog since we failed to identify any sequences sharing significant identity to the first exon, suggesting that regardless of any earlier functional role, it has degenerated into a non-functional remnant in the modern rat (dern rat .Cldn4-like’ sequence was detected in rat (XP_001066865) which maps between Cldn4 and the degenerate Cldn13-like sequence on rat chromosome 12 and shares sequence similarity with a mouse ‘Cldn4-like’ genomic sequence between Cldns4 and 13 on mouse chromosome 5G2 (Accession number AC079938) (Cldn4-like and rat Cldn13-like) indicating that they are unlikely to be transcribed.In addition, a predicted ‘C079938)  and S2. Cldn13 specific probe identified a single mRNA transcript (>1 kb) in adult mouse thymus, femur and spleen; no bands were observed in brain, kidney, lung or liver  showed different profiles. Both were detected in adult spleen, bladder, intestine, lung, kidney and teeth, and in lung and skin of newborn mice. Cldn3 is also expressed in adult liver, muscle and ribs and newborn liver and ribs and Cldn4 in adult eyes and tongue  from a global mouse gene expression analysis in 91 tissues including >20 haemopoietic cell lineages. Importantly, these data demonstrate exceptionally strong expression in mega-erythrocyte progenitor cells, bone marrow and bone The expression of Cldn13 appears to be mouse specific, we explored its phylogenetic relationship with other Claudin family members. Eighty-nine sequences were collected .As ollected  and a roollected . Many reDanio rerio is interesting because it appears to have followed two different routes during Claudin family development and, in addition to the orthologs it shares with other chordates, it possesses a group of paralogous alphabetically named Claudins Danio rerio genome Ciona intestinalis sequences behaved as outliers.From this data we suggest that mammalian members of the Claudin protein family are orthologous, sharing a common ancestor prior to the human/mouse split. This is also true for many fish Claudins. H. sapiens, M. musculus, T. rubripes and D. rerio. CLDN 25–33 are specific to T. rubripes. With the exception of CLDN13, mice and humans share the same complement of Claudins (Takifugu rubripes Claudin gene has been named Cldn13 (AY554386) Mus musculus CLDN13  we analysed microarray data derived from mice exposed to the pathogenic protozoan T. congolense to determine whether Cldn13 levels were affected. In spleen, Cldn13 is the most abundant of the twenty Claudins analysed. Log2 relative expression levels of Cldn13 averaged across all three strains prior to infection are 9.12 , compared to 7.41  of Cldn5 (p = 0.0009), the second most abundant Claudin in spleen  mice, across five time points  (Cldn13 is 2.4–3.3 fold less abundant (p≤0.03) in C57BL/6 than in A/J and BALB/c (strains in which anaemia is transient). Cldn13 levels remain significantly lower  at days 3 and 7 in C57BL/6 compared to BALB/c. C57BL/6 Cldn13 expression is also reduced  compared to AJ mice, however the difference only reaches significance at day 7 (p = 7.2×10−5). At later stages of infection (days 9 and 17), although expression in C57BL/6 still appears lower (1.2–1.9 fold), the strain differences do not reach significance (p≥0.08).To explore early changes initiated in response to infection, fection) . Prior tCldn5, which demonstrated 1.5–1.6 fold higher expression in AJ mice on day 7 (p = 0.04), and 1.5–1.6 fold higher expression in both AJ and BALB/c relative to C57BL/6 mice on day 9 (p = 0.01)   . Levels  points) . Differe points)  and S5. Cldn13 expression also varied significantly post-infection over time  then increasing 3.6 fold between days 3 and 17 (p = 0.0009) to reach levels marginally higher (1.3 fold) than in uninfected mice at day 17. Expression in BALB/c and C57BL/6 mice reached a minimum (2.0 and 2.2 fold decreased respectively) at day 7 followed by recovery to pre-infection levels by day 17 in BALB/c and a substantial rise of Cldn13 to above pre-infection levels (2.2 fold at day 17 versus day 0) in C57BL/6. However, this final increase was not significant (p = 0.19) and levels remained lower than those in AJ and BALB/c strains.ver time . In A/J Cldn5 also demonstrated significant changes in transcription levels over the course of infection in all three strains with initial decreases of between 1.6 and 2.7 fold . However, in contrast to Cldn13, Cldn5 expression remained low  throughout the experiment  Gypa, Rhd) Tmcc2 is the homologue of human TMMCC2 which has recently been strongly associated with mean corpuscular volume , a measure of anaemia, in a genome wide association study Cdca8, Cenpk and Cdkn2d have more indirect associations with erythropoietic pathways. Cdca8  and CenpK  are thought to be involved in mitotic chromosome segregation. Cdca8 encodes a member of the centromeric chromosomal passenger complex (CPC) required for correct alignment and segregation of chromosomes To ascertain potential pathways in which CenpK has been proposed as the homolog of the Schizosaccharomyces pombe protein Sim4 and, in common with the CPC, is thought to localize to centromeric kinetochores and be involved in chromosome segregation Cdkn2d  is a member of the Ink4, ‘Inhibitor of cyclin dependant kinase (CDK) 4’ family of repressors which prevent inappropriate progression into the cell cycle T. congolense infection. The remaining clustered gene is Cdr2 (cerebellar degeneration-related 2 or Yo), a protein which has recently been shown to be important for mitotic spindle formation and proliferation of dividing cells CenpK and Cdca8.All nine genes act in pathways known to be downstream of either EPO, BMP4 or KITL, suggesting that a possible role in the stress response to anaemia induced by Cldn13 were identified in a separate publicly available array experiment Trim10, Gypa and Rhd), also identified in our T. congolense arrays, and a further six with known associations to either erythrocyte-specific processes  or myeloid progenitor cell proliferation (Hemgn)  Mus musculus Cldn13 and Cldn4/CLDN4. Cldn4 has been implicated in a variety of processes as diverse as osmotic response in the kidney CLDN4 has been shown to be both up and downregulated in various malignancies (reviewed in Cldn4 is downregulated in EMT in response to SNAI1 CLDN3 and 4 on chromosome 7 appear to have undergone duplication. The presence of a predicted ‘Cldn4-like’ sequence in rat (between Cldn4 and the degenerative Cldn13-like sequence on rat chromosome 12) but with no transcript evidence, suggests a gene duplication prior to mouse/rat speciation events.Phylogenetic profiling is useful for inferring functional and evolutionary relationships and previous work has positioned Cldn13 is involved in erythropoiesis, a recent report identified it as one of a number of genes whose expression is significantly higher in erythroid cells D. rerio mutant model of haematopoiesis, cloche, which identified Cldng  as differentially expressed between mutant and wild type zebrafish and predominantly found in erythroid cell lineages In support of our hypothesis that 4β1 and αv participate in these interactions and it is possible that CLDN13 may also contribute in some way.Haemopoietic cells are not generally thought to connect via intercellular junctions such as tight junctions although in a rare form of human acquired dyserythropoiesis, erythroblastic synartesis, septate-like membrane junctions  and invaginations between erythroblasts which are tightly linked together have been observed Cldn13, form part of a macromolecular erythrocyte membrane structure incorporating the Band-3 complex The erythrocyte specific membrane proteins GYPA and RHD, also encoded by genes clustering with Cdca8, Cenpk and Cdr2 clustered with Cldn13 in our microarray data. The clustered gene Trim10 is an important regulator of erythropoiesis downstream of the key transcriptional regulator of haematopoiesis RUNX1  Cldn13, E2f2, has been shown to form a complex with RB1, a key player in the regulation of the G1-to-S-phase transition of the cell cycle Tal1, Trim10, E2f2, Gypa, Rhd) the involvement of others  has not been proven. However, our data, along with evidence of association between Cdkn2d, Cdca8 and Cenpk with factors essential in erythrocyte maturation strongly suggest a role for these genes in this process. Cldn13 in T. congolense infected mice, may interact in developing erythroid cells.The role of EPB4.1 in the regulation of mitotic chromosome segregation in erythroid cells One of the final steps in red blood cell maturation is enucleation. Chromatin becomes condensed and the nucleus migrates to one pole of the erythroblast before separating surrounded by plasma membrane and a small rim of cytoplasm Cldn13 regulation observed over the course of T. congolense infection is consistent with a stress induced erythropoietic response to parasite induced anaemia. We speculate that the downregulation of CLDN13 at the early stages of the stress erythropoietic response may influence the transition to a proliferating phenotype among splenic RBC pre-cursors. It is possible that this is mediated through reduced contacts with supportive stromal cells undergoing EMT as observed in fetal liver Cldn13 levels in later stages of infection may indicate populations of erythrocytic cells undergoing terminal differentiation, including enucleation, preceding the recovery from anaemia observed in strains A/J and BALB/c. The fact that expression of Cldn13 and clustered genes are constitutively lower in C57BL/6 mice than in A/J or BALB/c mice is consistent with them representing a snapshot of an erythropoietic pathway permanently depressed in this anaemia prone strain. Further, levels of expression of these genes in C57BL/6 mice fail to reach those seen in A/J or BALB/c mice, consistent with the previous suggestion of a suppressed stress erythropoietic response contributing to the chronic anaemia observed in this strain The pattern of Cldn13 in most haemopoietically active tissues  we do not exclude the possibility that it may also function in the generation of other blood cell lineages. Indeed, Claudin positive cells have been observed at the margins of distinct structures within the medulla of the thymus known as Hassall's corpuscles (HCs)Cldn expressing cells have functional TJs, although these structures were not detected between Cldn expressing cells in mouse Given the expression pattern of Cldn13 has previously been reported in mouse intestine Cldn13 expression in mouse bladder Cldn5 during T. congolense infection in all three inbred strains. Rat CLDN5 has been observed as part of atypical TJs within the splenic sinus endothelium, a structure associated with removal of senescent RBCs Cldn5 transcript in the spleens of all three inbred strains may be demonstrative of disrupted splenic architecture in the face of the massive haemophagocytosis in this organ in response to infection and/or the transformation of the spleen to erythropoietic function.In agreement with our RT-PCR expression data, expression of Cldn13, and three other genes with no previous link to erythropoietic function , in a known stress response pathway with specific relevance in resistance to trypanosome infection and potential importance in many other contexts including acute anaemia and erythroleukaemia. Protein sequence analyses indicate that Cldn13 is a member of the Claudin family and as such may well contribute to TJ formation. However, Cldn 13 is an unusual gene; its distribution is apparently limited to the murinae, and belongs to a clade encompassing recently duplicating genes evolutionarily linked to human CLDN3 and CLDN4. The most parsimonious explanation for the absence of CLDN13 in humans, and all other species surveyed, is a gene duplication event specific to the murinae which lead to the evolution of a functional Cldn13 gene in Mus musculus, but appears to have been lost in Rattus norvegicus leaving only the degenerate sequence we identified. Since Cldn13 appears to be mouse specific, we speculate that other members of the Claudin family (possibly CLDN4) may be involved in erythropoiesis in other mammals and, given the widespread use of animal models to predict biological functions in humans, it will be important to clarify if this is the case.In conclusion, our data provides evidence of a role for Animals were housed at the International Livestock Research Institute (ILRI), Nairobi, Kenya. Mice used were 5–8 weeks of age. Animals received food and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee  at ILRI.RNA was isolated from mouse tissues for northern blots and RT-PCR by extraction with total RNA isolation reagent (ABgene) according to manufacturer's instructions. mRNA was purified from total RNA using the Nucleotrap kit (ABgene) according to manufacturer's instructions. For microarray analysis, RNA was prepared from mouse liver and spleen using Trizol Reagent (Invitrogen) as described previously st strand synthesis kit (ABgene). PCR was carried out using the ReddyMix PCR system (ABgene) according to manufacturer's instructions. PCR primers are detailed in cDNA was prepared from 2 µg total RNA (pre-treated with DNaseI) using the ReverseIT 132 labelled PCR probes (Random Primers Labelling System GibcoBRL). The filters were washed at 60°C (0.2XSSC+0.1%SDS) and exposed to X-ray film at −70°C. Cldn13 probe was generated by PCR amplification of a 588 bp fragment from mouse genomic DNA (mRNA from each tissue (1 µg) was added to 4 µl RNA loading buffer without ethidium bromide (Sigma) and heated to 65°C for 10 min before loading on a 1% MOPS Formaldehyde gel (electrophoresis for ∼3 h at 5–6 mA/cm). 3 µl RNA markers (Sigma), in 2 µl ethidium bromide (75 µg/ml), were used on each gel. Gels were blotted overnight in 20× SSPE. Hybridization was in ‘Rapid-Hyb’ (Clontech) using Pomic DNA ; this reHomo sapiens, Mus musculus, Danio rerio and Takifugu rubripes full length Claudin sequences were retrieved from GenBank (http://www.ncbi.nlm.nih.gov). This sequence set was supplemented with Ciona intestinalis sequence ). To simplify the phylogenetic analysis, a single representation of each entity was used and only the longest isoforms selected. This yielded a dataset totalling 89 sequences . Gap-containing sites were removed from the alignment and Maximum Likelihood trees were inferred using ProML from the PHYLIP package (http://evolution.genetics.washington.edu/phylip.html) http://www.tree-puzzle.de/) http://mrbayes.csit.fsu.edu/) A total of 121 equences . ClaudinT. congolense infection of mice is an established and valuable model of anaemia and is appropriate to use for investigating the association between gene expression and anaemia 4 parasites in 200 µl of phosphate-buffered saline (pH 8.0) containing 1.5% glucose).http://www.affymetrix.com) were used to determine expression of genes in each of A/J, BALB/c and C57BL/6 mice. For each strain RNA was prepared from spleens of thirty mice. The samples were then mixed into five pools of five samples for each strain. This pooling strategy is predicted to give the same power to detect differential expression as fifteen individual samples hybridised separately Cldn13 during infection were identified using a distance metric , 100 µl of supernatant transferred to a new plate and the relative haemoglobin concentrations measured by spectrophotometrical determination of optical density at 540 nm in an ELISA plate reader . Measurements were carried out in triplicate. The data were analysed by ANOVA  using strain, time and their interaction. Subsequently mice were euthanised to allow tissue collection for microarrays, therefore observed changes in haemoglobin levels were not due to repeated sampling. All mice were euthanised after day 35 of the experiment.Figure S1ClustalW alignment of rat predicted CLDN4-like protein sequence with rat CLDN4, mouse CLDN4 and 13, and human CLDN4. Rat sequence XP_001066865 is unlikely to be an ortholog of mouse Claudin 13 as it shares more sequence similarity with mouse, rat and human Claudin 4.(0.71 MB TIF)Click here for additional data file.Figure S2tBLASTx alignments using Rat Cldn4-like protein sequence as the query. A genomic sequence with a high degree of similarity to rat ‘Cldn4-like’ is located on mouse chromosome 5 between Wbscr27 and Cldn13.(0.28 MB TIF)Click here for additional data file.Figure S3Putative Fugu Claudin 13. A. ClustalX alignments highlighting the relatively weak alignment of between mouse Claudin 13 (AY554386) and Fugu Claudin 13 (AF516681). B. Contrasting strong alignments between mouse Claudin 3 (NP_034032) and Fugu Claudin 3 (AAT64047). Combined with phylogenetic evidence this indicates that the suggested Fugu Claudin 13 is unlikely to be the true ortholog of mouse Claudin 13.(0.53 MB TIF)Click here for additional data file.Figure S4T. congolense infection in three different strains .Data is expressed as mean Log2 intensity Click here for additional data file.Figure S5T. congolense infection in three different strains .Data is expressed as mean Log2 intensity Click here for additional data file.Figure S6Cldn13 in microarray analysis of T. congolense infection in three different strains . All of these are either known to function in erythropoiesis or have more indirect associations with erythropoietic pathways.Data is expressed as mean Log2 intensity (±) standard error. A–I: Expression profiles of nine of the ten genes with expression patterns correlating most closely to those of (0.43 MB TIF)Click here for additional data file.Table S1(0.03 MB DOC)Click here for additional data file.Table S2(0.03 MB DOC)Click here for additional data file.Table S3(0.12 MB DOC)Click here for additional data file.Table S4(0.03 MB DOC)Click here for additional data file.


Of primary concern when evaluating macaque bites are bacterial and B-virus infections. B-virus infection is highly prevalent (80% to 90%) in adult macaques and may cause a potentially fatal meningoencephalitis in humans. We examined seven nonoccupational exposure incidents involving 24 persons and eight macaques. Six macaques were tested for herpes B; four (67%) were seropositive. A common observation was that children were more than three times as likely to be bitten than adults. The virus must be assumed to be a potential health hazard in macaque bite wounds; this risk makes macaques unsuitable as pets.


Flavivirus that was isolated from field-collected Ae.  macintoshi mosquitoes in 1999, and is closely related to cell fusing agent virus. Both of these viruses belong to the family Flaviviridae, which also contains other viruses of medical importance, such as yellow fever virus, West Nile virus and dengue. Because Ae. macintoshi is the only known natural host to KRV, the main objective of this study was to establish the possibility that other mosquito hosts of the virus exist, by determining its ability to infect Ae. aegypti mosquitoes under laboratory conditions. The study also sought to determine the rates of infection and, subsequently, vertical transmission as a possible means of its maintenance and propagation in nature, given that it neither grows in vertebrate cells or mice. The mosquitoes were infected by the virus either as larvae or adults. Virus assay was done by re-isolation in tissue culture and indirect immunofluoresce assay methods. KRV infected Ae. aegypti mosquitoes, with the observed rates as high as 74 to 96 %. The virus was also transmitted vertically in these mosquitoes. Vertical transmission rates of 3.90 % were observed for the 2nd and 3rd ovarian cycles combined. These results suggest that Ae. aegypti mosquitoes are likely to be infected with KRV in nature, and that vertical transmission is the natural means by which it is maintained and propagated in this host, and possibly others.Kamiti river virus (KRV) is an insect-only  Flavivirus that was isolated from field-collected Ae. macintoshi mosquitoes that were collected as larvae from a flooded dambo on the shores of Kamiti river in the Ruiru division of Central Province, Kenya in 1999 (Flavivirus (Flavivirus to be isolated in nature as the cell fusing agent virus was isolated from laboratory-cultured Ae. aegypti mosquito cells (Kamiti river virus (KRV) is an insect-only  in 1999 . Assays avivirus . KRV is to cells .Ae. aegypti mosquitoes transmit the dengue-2 virus only if the mosquitoes are maintained at 30 °C, and the extrinsic incubation period is shortened if the extrinsic incubation temperature is increased to between 32 and 35 °C , and for Japanese encephalitis virus in Culex. tritaeniorhynchus, Cx. annulus, Cx. quinquefasciatus, and Armigeres subalbatus , Nairobi Kenya.Since KRV does not grow in vertebrate (Vero) cells, we used C6/36 cells, which are derived from The C6/36 cells were grown in growth medium (Dulbecco's modified Eagle's medium) containing 5 % heat-inactivated fetal bovine serum, 2 % L-glutamine and 1 % antibiotic/antimycotic solution . Infected cells were held in maintenance medium (Dulbecco's modified Eagle's medium) in which the fetal bovine serum had been lowered to 2%.Ae. macintoshi mosquitoes collected in the Sukari range. This virus, which had been preserved as a stock at the Viral Hemorrhagic Fever Laboratory at -80 °C and whose previous passage history was 4, was amplified by passaging twice in C6/36 cells to prepare stock virus for use in this study. Two 75-cm2 flasks of confluent C6/36 cells were infected with 600 μl of the virus suspension each and incubated for adsorption for 1 hr at 28 °C. The infected cells were thereafter maintained in 20 ml maintenance medium and incubated at 28 °C. Eight days post-infection the virus was harvested by scraping the cells from the flask surface and centrifuging the cell-virus-medium suspension at 1500 rpm for 15 minutes to remove cell debris. The supernatant containing the virus was stored in 0.5 ml aliquots at -80 °C. This was the source of the virus for infectious blood meals, and which was also used as the immunogen for the production of antiserum.The SR-75 strain of KRV, which was used in this study, was isolated from adult 6 cells per ml, and incubated at 28 °C in a humidified chamber until development of a cytopathic effect. The viral titer was calculated using Karber formula , near Karura forest in Nairobi, Kenya. These mosquitoes were reared to F3 in the insectary maintained at approximately 28 °C and 75–80 % RH with a photoperiod of 16:8 L:D. The larvae were reared in white enamel pans and fed on Tetramin® fish food and liver powder. Adult mosquitoes were continuously given 10 % sucrose solution as a carbohydrate source and fed on normal laboratory mice to stimulate egg production.The F3 generation of the Mosquitoes were orally exposed to the virus either as adults (adult infection), or as larvae .7 to 3.2 × 107 per ml were used, with no significant change over the feeding period.One hundred and fifty 3-day-old adult mosquitoes held in 4-liter plastic cages were starved of sucrose and deprived of water for 24 hours prior to blood feeding. The mosquitoes were orally exposed to an infectious blood meal prepared by mixing a 1:1 ratio of the stock virus suspension with defibrinated rabbit blood. The blood meal was placed in membrane feeders covered with mouse skin. Blood meals were maintained at a constant temperature of 37 °C throughout the 45 minutes feeding period. Fully engorged mosquitoes were selected with an aspirator and held at a constant temperature of 28 °C and 70–80 % RH, during a 16:8 L:D, and provided with a 10 % sucrose solution in cotton wicks for an extrinsic incubation period of 14 days. Sampled mosquitoes were killed by chilling at -70 °C before being used for the virus assay. Samples of the infectious blood meal were taken before and after the feeding period and immediately titrated in C6/36 cells to determine infecting virus titer. In all mosquito infections, infective blood meals of virus titer ranging from 3.0 × 102 flasks, and left at the same temperature for 30 minutes for maximum feeding. The larvae were then removed using a Pasteur pipette, thoroughly rinsed in distilled water and transferred to normal rearing conditions in the insectary. The virus assay was performed on individual mosquitoes 14 days after emergence.Second instar larvae of mosquitoes were exposed to KRV-infected cells eight days after infection at 28 °C in 25-cmAe. aegypti mosquitoes orally exposed to KRV were subjected to single-pair mating experiments. A virgin male and female mosquito (20 pairs) were placed together in individual cylindrical (20 × 20 cm) cardboard containers, covered with a fine nylon mesh at the top. Each container had a 5 × 4 cm plastic jar partially filled with water and lined with brown paper for oviposition. 3 to 5 days later, the male mosquito was aspirated from the containers and the female was fed on a mouse. After oviposition, the eggs were hatched and the offspring reared separately. Individual parent mosquitoes were then assayed for KRV infection. Only F1 offspring of the parent mosquitoes that tested positive for KRV were tested for KRV-infection as adults by isolation in tissue culture.www.labnetlink.com) at 4 °C for 1 min. 50 μl of each suspension was inoculated onto 96-well, flat-bottom-cell culture plates (50 μl per well) and seeded with 150 μl of C6/36 cell suspension containing 106 cells per ml. The plates were incubated at 28 °C and cells monitored daily for development of characteristic cytopathic effects. KRV-infected and non-infected C6/36 cells incubated for the same period of time were included as negative and positive controls respectively.Mosquitoes were selected 14 days after virus exposure and sacrificed by chilling. Individual mosquitoes were then placed in chilled Eppendorf vials and triturated in 1 ml growth medium using sterile pestles. Mosquito suspensions were clarified by centrifugation at 3,000 rpm  and separated by centrifugation at 2,000 rpm  at room temperature for 10 minutes. A PBS-cell suspension was prepared and 20 μl of it spotted on each well of the 12-well Teflon coated slides and air-dried. The cells were fixed in cold acetone at -20 °C for 30 minutes and stained with KRV antiserum diluted 1:20 in PBS. The slides were incubated for 1 hr at 37 °C in a humidified chamber and then washed three times in PBS for 5 min each. 20 μl of a commercial anti-mouse Immunoglobin G (IgG) conjugated with fluorescein-isothiocyanate (FITC), diluted 1:32 in PBS, was added to each well. Evans blue was also added to the conjugate at the ratio of 1:10. The slides were again incubated for 1 hr at 37 °C in a humidified chamber and washed as above in the dark and mounted with cover slips using mounting solution consisting of Mcllvaine's Buffer: Glycerol (M-7534) in a ratio of 1:1. The slides were examined under an Olympus BH-2 epifluorescence microscope equipped with a 20x objective lens, an HBO lOO-W high-pressure mercury burner, and an IF-49O exciter filter.7 to 3.18 × 107 per milliliter, tested positive for KRV by isolation in tissue culture, while 74.5 % infections were detected by IFA. There was no significant change in the KRV titers over the feeding period. For those females exposed to KRV-infected cells as larvae, 90.2% infections were detected by isolation in tissue culture and 96.3 % by IFA (p = 0.028 for those exposed as adults and p = 0.012 for those exposed as larvae). There was also a statistically significant difference (p = 0.039) in the rates of infection between mosquitoes exposed as adults and those exposed as larvae, by IFA. However the difference was not significant (p = 0.056) by isolation in tissue culture.About 62 % of adult female mosquitoes orally exposed to infectious blood meals of virus titer range 3.02 × 10% by IFA . In bothAe. aegypti female mosquitoes to their progeny was experimentally demonstrated by isolation in tissue culture. Infected progeny were detected in the F1 generation from the combined 2nd and 3rd ovarian cycles of the 13 parent mosquitoes that had been found to be infected with KRV. Total filial infection rate for the two cycles was 14:410, representing 3.90 %. The rates were also higher in females (4.53 96) than in males (2.76 %)  , althougAe. aegypti mosquitoes orally exposed to KRV are reported using laboratory-reared mosquitoes. These observations suggest that Ae. aegypti mosquitoes are likely to be infected with KRV in nature. This hypothesis is supported by previous isolation of KRV-like virus from Ae. aegypti mosquitoes . However, cross-sectional field studies are needed to confirm this, as well as if other species of mosquitoes are likely to be infected with KRV or related viruses in nature.High infection rates (74 and 96 %) of KRV in Higher infection rates (90.2 and 96.3%) were observed in mosquitoes exposed as larvae. This is in agreement with expectation, as in the adult female the blood meal initiates the formation of a peritrophic membrane within mosquitoes  that phyAe. aegypti to infection  obtained by IFA is indicative of the fact that this method is more sensitive compared to that of virus isolation in tissue culture. The lower rates detected by the latter method do not therefore rule out the possibility that some mosquitoes that tested negative were actually infected at the time of assay but the viremic level may have been too low to cause detectable cytopathic effects in the tissue culture, yet high enough to be detected by IFA. The difference in infection rates observed between the two methods was significant, Ae. aegypti mosquitoes can vertically transmit KRV to their progeny. The high overall first filial infection rates (3.41 %) were attributed to the fact that KRV is an insect-only Flavivirus with enhanced infection. There was no statistically significant difference between vertical transmission rates in females vs. those in males, p= 0.076, although the rate was twice as high in females. Significant differences in infection rates between male and females were found by Ae. aegypti mosquitoes. These investigators explained that the higher rates in females than males was due to venereal transmission of the virus to non-infected females by infected males during mating.We have observed that orally infected Ae. aegypti (Ae. cooki mosquitoes transmitted Dengue 1 and 3 viruses to 6.7 % of their offspring, while 4.6 % of filial infection was observed in Ae. polynesiensis for the same virus.Although our study did not specifically demonstrate venereal transmission, it is a process that may increase the prevalence of this virus in nature. As  aegypti  have bee aegypti , demonstAe. aegypti mosquitoes are highly susceptible to KRV, and that infected mosquitoes are able to vertically transmit the virus to their progeny. This is the likely mechanism by which the virus is maintained and propagated among its natural hosts. These observations contribute to the existing knowledge about KRV. Studies should be carried out to determine the susceptibility of other mosquito species to KRV, and to compare the filial infection rates of mosquitoes infected as adults and those infected as larvae.Previous studies with KRV showed that it is an insect-only Flavivirus most closely related to the cell fusing agent virus. We have observed that


West Nile Virus in Birds, Argentina To the Editor: West Nile virus (WNV), genus Flavivirus, family Flaviviridae has been rapidly dispersing through the Americas since its introduction in 1999 in New York  of 1,845 serum specimens from 117 bird species collected from January to June 2006 tested positive when using the blocking ELISA; 30% inhibition was the threshold for a positive test. SLEV infections were confirmed in 105 birds by PRNT; WNV infections were confirmed in 43 birds. Anti-WNV antibody titers ranged from 40 to 2,560 in birds collected as early as January 2005 in Córdoba City and as late as June 2006 in Mar Chiquita . Recent Furnariidae (a family absent from temperate North America) prove to be competent hosts, they could play an important role in WNV transmission in Argentina because of their frequent exposure to WNV. Twelve (12.5%) of 96 F. ruffus sampled in 2005 and 2006 tested positive.In 2006, WNV was isolated from equines in Buenos Aires province  could be due to 1) false positives; 2) cross-reactions between WNV- and SLEV-reactive antibodies that prevented definitive diagnosis by PRNT; 3) cross-reactive antibody and multiple, heterologous flavivirus infections; 4) previous infections by both WNV and SLEV; and/or 5) presence of other flaviviruses circulating in Argentina. SLEV is endemic throughout Argentina and, like WNV, belongs to the Japanese encephalitis virus serocomplex. Hemagglutination-inhibiting antibodies against several Brazilian flaviviruses  have been reported in the neotropical region of extreme northern Argentina (Our serologic data suggest that WNV has established itself in 4 ecologic regions in Argentina in a brief period. Additional studies are needed to define the reservoir hosts and vectors of WNV in Argentina, and most importantly, to define the public health risk this virus represents.


Specific binding of virus to oysters can selectively concentrate a human pathogen. The primary pathogens related to shellfishborne gastroenteritis outbreaks are noroviruses. These viruses show persistence in oysters, which suggests an active mechanism of virus concentration. We investigated whether Norwalk virus or viruslike particles bind specifically to oyster tissues after bioaccumulation or addition to tissue sections. Since noroviruses attach to carbohydrates of the histo-blood group family, tests using immunohistochemical analysis were performed to evaluate specific binding of virus or viruslike particles to oyster tissues through these ligands. Viral particles bind specifically to digestive ducts  by carbohydrate structures with a terminal N-acetylgalactosamine residue in an α linkage (same binding site used for recognition of human histo-blood group antigens). These data show that the oyster can selectively concentrate a human pathogen and that conventional depuration will not eliminate noroviruses from oyster tissue. Three oysters were added to the sea water and incubated at room temperature for 12 or 24 h. Sea water was continuously aerated to maintain adequate oxygen levels. A negative control was used under similar conditions but without the addition of Norwalk virus or recombinant VLPs.Norwalk virus  in phosphate-buffered saline (PBS) at room temperature (Oysters (uncontaminated or after bioaccumulation) were shucked, and the flesh was fixed in 10% formaldehyde for 48 hours. The digestive gland was then dissected, embedded in paraffin, and sliced into thin sections (5 μm). After preparation of tissue sections, sections from uncontaminated oysters were covered with 4 × 10Treatment with periodate was performed as previously described (Helix pomatia (HPA), Dolichos biflorus (DBA), and Ulex europaeus (UEA-1) , were used for analysis of binding to tissues and inhibition of VLP binding. UEA-1 recognizes the H type 2 trisaccharide and shows some cross-reactivity with Ley. HPA and DBA recognize α-linked N-acetylgalactosamine residues. For the binding assay, lectins were applied to shellfish tissues for 30 min at different concentrations .Three biotinylated lectins, derived from b (LM-137); anti-H type 2 (19-OLE); anti-Ley (12-4LE); and anti-H type 1. The primary antibodies were serially diluted, incubated with tissues overnight at 4°C, and subjected to standard immunohistochemical analysis , which recognizes all A epitopes; anti-A types 3/4 ; anti-A type 2 (III-2A5); anti-H/Le6 particles/mL or recombinant VLPs at final concentrations of 2 × 109 and 2 × 106 particles/mL. After incubation for 12 h, virus was detected in digestive diverticula. Immunostaining showed particles in the lumen of tubules and ducts or in phagocytes between epithelial cells and in the surrounding connective tissue .Oysters were immersed in sea water seeded with Norwalk virus at a final concentration of 10The binding of virus or recombinant VLPs was tested directly on oyster gut sections. Both recombinant VLPs and native virions bound to the midgut, the main and secondary ducts of the digestive diverticula, and tubules, although labeling of tubules was weaker , Ley (12-4LE), or A type 2 epitopes (III-2A-5) showed little staining of shellfish tissues and when positive, staining did not correspond to cells to which VLPs bind. These results indicate that oyster tissues have carbohydrates that resemble human histo-blood group antigens, but structures recognized by VLPs were not identical to those recognized by these antibodies.Antibodies to carbohydrates on cells in the human digestive tract can inhibit binding of recombinant VLPs to human tissue . Anti-H Lectins have been used to determine the distribution of carbohydrate residues in tissue. Since lectins have a broader reactivity than monoclonal antibodies, we used lectins to identify recombinant VLP-specific carbohydrate ligand in oyster tissue. UEA-1 bound to shellfish tissues, but only at high concentration (50 μg/mL), whereas 2 other lectins, DBA and HPA, bound at a lower concentration (1 μg/mL) . Tissue ,,,,,Virus-mediated disease can be transmitted when contaminated shellfish are eaten. Oysters are believed to act as filters or ionic traps, passively concentrating particles. A simple depuration process should be sufficient to rid oysters of virus, as observed for bacteria (,Our data demonstrate specific binding of viral particles from a genogroup I norovirus to the oyster digestive tract and suggest a specific mechanism for concentration of virus particles. We tested recombinant VLPs of prototype genogroup I Norwalk virus. VLPs are stable in the marine environment and the disinfection processes (Different results were seen between bioaccumulation experiments and particle binding to tissues sections. After bioaccumulation, some viral particles were detected in phagocytes in either epithelium or connective tissue. This finding could reflect elimination of virus during digestion. The time required for food to pass through the entire shellfish intestinal tract varies from 90 to 150 min. We do not know whether immunoreactive material detected in phagocytes corresponds to particle degradation and digestion or if particles can escape digestion. Binding to main ducts may provide a mechanism for viral particles to avoid entering the digestive system and being degraded. Specific attachment of virus to oyster cells and capture by phagocytes may explain why depuration in oysters is not an effective mechanism for eliminating virus.Virus accumulation in oysters may depend on factors such as water temperature, mucus production, glycogen content of connective tissue, or gonadal development (,Human susceptibility factors for norovirus infections that depend upon carbohydrates of the ABH, secretor, and Lewis histo-blood group family have been observed (H. pomatia, which recognizes α-linked N-acetylgalactosamine terminal residues of glycans, the oyster ligands are similar to those of histo-blood group A. The DBA lectin did not inhibit attachment of recombinant VLPs, which indicates that it does not bind oligosaccharide structures recognized by VLPs and HPA. Although DBA binds to N-acetylgalactosamine residues similar to HPA, the 2 lectins have different carbohydrate specificities that depend on the subjacent sugar residues (Similar to human histo-blood group structures, mutant VLPs showed that an alanine substitution at positions H329A and W375A prevented binding to oyster tissue. However, a mutant with a substitution at position N331A did not affect binding. This binding specificity identified the amino acids required for binding and further confirmed the similarity with the mechanism of recognition of human tissues (,,,,,,Genogroup I and II strains of norovirus show various binding patterns with different carbohydrate structures of the histo-blood group family, which suggests coevolution of this group of viruses and their host or carrier vector. The ability of Norwalk virus to bind to oysters tissues at the same binding site as that used to bind to human tissues suggests a possible coevolution mechanism involving the oyster as an intermediary vector. This mechanism would favor selection of some viruses, such as Norwalk virus, over other viruses that are unable to bind to the oyster and would not be transmittable by this intermediary host. Epidemiologic data suggest a predominance of genogroup I strains in oyster-related gastroenteritis outbreaks, whereas genogroup II strains are predominant in other food-related outbreaks (As knowledge increases in understanding the binding of these viruses in humans, we will likely further understand more about their behavior in shellfish. This knowledge may help identify species that could be less sensitive to contamination. Since the oyster can actively and specifically bind a human pathogen, this knowledge has practical consequences because conventional depuration cannot eliminate noroviruses from oyster tissues.


The lame sire, unsound for breeding, can cause substantial economic loss due to reduced pregnancies in the beef-producing herd.To test the hypothesis that joint disorder is a possible cause of infertility in beef sires, right and left hind limb bones from 34 beef sires were examined postmortem to identify lesions in the femorotibial, femoropatellar (stifle), tarsocrural, talocalcaneus, and proximal intertarsal  joints. The bulls were slaughtered during or after the breeding season due to poor fertility results. Aliquots of the cauda epididymal contents taken postmortem from 26 bulls were used for sperm morphology evaluation. As a control, hind limbs (but no semen samples) from 11 beef bulls with good fertility results were included.Almost all infertile bulls (30/34) had lesions in at least one joint. Twenty-eight bulls  had lesions in the stifle joint, and 24  of these were bilateral. Fourteen bulls  had lesions in the tarsal joint, and 10  of these were bilateral. Four bulls  had no lesions, three bulls  had mild osteoarthritis (OA), 5  moderate OA, 17  severe OA and 5  deformed OA. Almost all OA lesions (97%) were characterized as lesions secondary to osteochondrosis dissecans. All the bulls with satisfactory sperm morphology (n = 12/34) had joint lesions, with mostly severe or deformed bilateral lesions (83%). Consequently, the most likely cause of infertility in these 12 bulls was joint disease. Almost all control bulls (10/11) had OA lesions, but most of them were graded as mild (55%) or moderate (36%). None of the control bulls had severe lesions or deformed OA.We suggest that joint lesions should be taken into consideration as a contributory cause of reproductive failure in beef sires without symptoms of lameness. The lame herd sire, unsound for breeding, can cause substantial economic loss , especiaOsteochondrosis of the articular-epiphyseal-cartilage complex (AECC) is characterized by focal disturbed enchondral ossification of the epiphyseal growth cartilage. The aetiology is not fully understood but there is strong evidence that focal failure of blood supply in the growth cartilage causes local ischemia, which in turn leads to focal necrosis of the cartilage, named OC latens with subsequent cartilage retention into the subchondral bone (OC manifesta) . The disEarlier reports on musculoskeletal disorders, as a cause of infertility in bulls, have mainly focused on spondylosis of the vertebrae in dairy bulls . To our The aim of the present study was to test our hypothesis that hind limb joint disorder of the bull can contribute to infertility in beef herds.In the present study, an investigation of hind limb joints from 34 non-lame beef bulls, slaughtered due to infertility, was performed. These bulls were compared with 11 fertile beef bulls, slaughtered due to risk of inbreeding in the herd. The 34 infertile bulls were slaughtered during or after the breeding season due to a total reproductive failure. The bulls had not produced any calves during the last breeding season. All bulls had a normal mating behaviour according to the owners. Lameness had not been observed. Femorotibial, femoropatellar (stifle), tarsocrural, talocalcaneus, and proximal intertarsal  joints from right and left hind limbs of these beef sires were examined postmortem. Semen samples were obtained by cauda epididymal dissection post mortem from 26 of the 34 bulls. The bulls were of five different breeds, Charolais (n = 15), Simmental (n = 7), Limousin (n = 6), Hereford (n = 5) and Angus (n = 1), and the mean age was 2.5 years (range 1–7 years). As controls, hind limb bones from 11 beef bulls with good fertility result were included. The control bulls were of three different breeds (Charolais (n = 6), Limousin (n = 3), Simmental (n = 2)), with a mean age of 4.5 years (range 2–9 years).The joints were disarticulated and examined macroscopically. The articular cartilage, synovial membrane/capsule, ligaments, menisci and subchondral bone were inspected for lesions such as cartilage fraying, wear lines, erosion, ulceration, osteochondrosis dissecans (OCD), cartilage retention, osteophytes and villiformation of synovial membranes. Osteochondrosis (OC) was diagnosed when cartilage retention into the subchondral bone was found and OCD when a cartilage rupture with a cartilage flap or loose osteochondral body could be seen. The lesions were recorded as unilateral or bilateral and graded as normal and with mild, moderate, severe or deformed osteoarthritis (OA).Mild OA in the femorotibial and femoropatellar joints was characterized by superficial cartilage fraying of less than 30% of the articular cartilage with single erosion <1 cm in diameter and superficial wear lines. Mild OA in the tarsocrural, talocalcaneus, and proximal intertarsal joints was characterized by single erosion or single OCD  Fig , multiplSevere OA in the femorotibial and femoropatellar joints was characterized by single or multiple OCD Fig , multiplDeformed OA in both the stifle and the tarsus was characterized by severe OA with periarticular osteophytes Fig .Semen samples were collected by cauda epididymal dissection in accordance with a previous study on Swedish beef bulls . The tesLocations of joint lesions in all bulls studied are displayed in Table Four of the bulls 4/34; 12%) with impaired fertility had no lesions; three bulls were classified as having mild OA , 5 moderate OA , 17 severe OA  and 5 deformed OA . See Table % with imTen 91%) of the 11 control bulls had OA lesions in one or both joints. Eight  of these bulls had lesions in the femorotibial joint, of which 7  were bilateral. Six  bulls had lesions in the femoropatellar joint; of which 4  were bilateral and only 2 bulls  had lesions also in the tarsal joint, of which 1 (50%) was bilateral. Lesions were most prevalent in the plateau of the proximal tibia , followed by the lateral trochlear ridge of femur  (Table % of the Fourteen (54%) of the 26 infertile bulls that were assessed had unsatisfactory sperm morphology, while the rest of these 26 bulls (12) had satisfactory sperm morphology. Ten (71%) of the 14 bulls with unsatisfactory sperm morphology also had joint lesions. Of these 10; 3 had mild OA, 4 had severe OA and 3 had deformed OA. All the 12 bulls with satisfactory sperm morphology had joint lesions. Of these 12; 2 had moderate OA, 9 had severe OA and 1 had deformed OA.The present results indicate that lesions compatible with osteoarthritis (OA) are common postmortem findings in beef sires, regardless of clinical history. Judged on shape and location  most of The severe and deformed OA with denuded bone and, often secondary, synovitis can cause pain with subsequent gait asymmetries. However, bilateral lesions result in lameness from both hind limbs, which may be very difficult to observe without a lameness examination including flexion test, joint palpation and radiological examination. Hence these bulls may appear non-lame in the field. Clinical signs of lameness associated with OCD are not frequently reported in cattle . Almost The lateral ridge of the trochlea, with a proximal position in the femoropatellar joint, is not a weight bearing articular cartilage, but is under load when the bull is mounting, which can result in pain from denuded bone. We suggest that the main clinical problem arises when the bull mounts and the patella slides over the trochlea. This pain may prevent the bull from completing the mounting and therefore lead to no pregnancies in the herd.The present findings of severe and deformed OA in most of the (22/34) infertile bulls suggest that these bulls may have difficulties completing mounting, which would explain the lack of pregnancies in the herd. The control bulls were presented with normal joints or with mild or moderate OA, probably without clinical relevance.The bulls in this study had been culled due to infertility with no pregnancies the last year in the herd, whereas the control bulls had served satisfactory in the herd for one or more years and were sent to slaughter due to risk for inbreeding in the herd. Hence the controls were older (mean 4.5 years) than the infertile bulls (mean 2 years), which in theory would account for higher probability of articular cartilage degeneration and development of OA. However, the control bulls were classified with fewer and milder joint lesions than the infertile bulls.All the bulls with satisfactory sperm morphology had joint lesions with mostly severe or deformed bilateral OA lesions of the femoropatellar joint (10/12). Consequently, we suggest that the most likely cause of infertility in these 12 bulls was leg weakness and not poor sperm morphology. The high incidence of sperm abnormalities found in the 14 bulls with unsatisfactory sperm morphology was probably the main cause of the infertility seen in the 7 bulls with normal joints or only mild OA. In the remaining 7 bulls however, the unsatisfactory sperm morphology could not alone explain why these bulls failed to reproduce completely. However, in this group we recorded a moderate to severe OA that together with the deteriorated sperm quality may have contributed to the total reproductive failure among these bulls. The reason for the poorer sperm quality in this group is unknown, but a negative effect of pain, caused by moderate or severe OA, on the spermatogenesis can not be excluded.In conclusion, the present results suggest that hind limb OA can contribute to lower breeding results, probably mainly by rendering the bulls difficulties when mounting, but also by indirectly affecting the spermatogenesis negatively. Hence, joint lesions should be taken into consideration as a contributory cause of reproductive failure in beef sires with or without symptoms of lameness. Special attention should hence be paid to the bulls' hind limbs and gait when performing the bull breeding soundness evaluation.YP participated in the design of the study, carried out the macroscopic examination of joints, performed the analysis of the joint and sperm data and drafted the manuscript. LS participated in the design of the study and in the analysis of the sperm data and helped to draft the manuscript. SE participated in the design of the study and in the macroscopic examination and evaluation of joints and helped to draft the manuscript. All authors read and approved the final manuscript.


Vesiculovirus genus. The second, designated Rhode Island virus, is unclassified antigenically, but its ultrastructure and size are more similar to those of some of the plant rhabdoviruses. Both viruses infect birds and mice, as well as monkey kidney cells in culture, but their importance for human health is unknown.Two novel rhabdoviruses were isolated from birds during surveillance for arboviral encephalitis in the northeastern United States. The first, designated Farmington virus, is a tentative new member of the  West Nile virus (WNV) in North America in 1999 Corvidae, is a sensitive method for detecting WNV activity in a region  activity in the northeastern United States. This finding serves as a reminder that WNV and EEEV are not the only viruses that may be associated with bird deaths in this region. Since the appearance of Columba livia) collected at two localities in Rhode Island in summer 2000, as part of WNV surveillance activities. The two dead pigeons were collected on September 15 and 16 in Barrington, Bristol County (#175), and East Providence, Providence County (#166), respectively. No trauma or obvious gross pathology was noticed in the brain of either bird at necropsy. Brain tissue, including nearly equal portions of cerebrum and cerebellum, was collected and immediately frozen at –80oC until processed for culture. Frozen tissue was thawed, and a small portion was completely homogenized in 3.0 mL of medium 199 . Homogenized brain tissue was centrifuged at 3,500 × g for 20 minutes at 2oC in a refrigerated centrifuge; then 100 μL of the supernatants was immediately added to 25-mL flasks containing Vero cell monolayers. Tissue cultures were incubated at 37oC and 5% CO2 and examined for cytopathic effect (CPE) on days 3–7 postinoculation.We examined three virus isolates from birds. Virus strains RI-166 and RI-175 were both isolated from brain tissue of dead pigeons .Complement fixation (CF) tests were performed by a microtechnique Aedes albopictus cells Culex quinquefasciatus cell line oC and 28oC, respectively. Culture slides with Vero cells were fixed in cold acetone when the cells showed 2+ to 3+ viral CPE; the mosquito cells were fixed after 6 days of incubation. The IFA tests were performed by using HMAF at dilutions of 1:10 and 1:20 and a commercial fluorescein isothiocyanate-conjugated goat antimouse immunoglobulin G  Indirect immunofluorescent antibody (IFA) tests were done on Vero and mosquito cells grown in eight-chamber Lab Tek tissue culture slides . The mosquito cells tested were the C6/36 clone of 2 were added, as described 4 in the same buffer. They were stained en bloc with 1% uranyl acetate in 0.1 M maleate buffer at pH 5.0, dehydrated in ethanol, and embedded in Poly/Bed 812 . Ultrathin sections were cut on a Reichert/Leica Ultracut S ultramicrotome , stained with 2% aqueous uranyl acetate and 0.4% lead citrate, and examined with Philips 201 or Philips CM-100 electron microscopes at 60 kV .Immediately after removal of the medium, Vero cell monolayers infected with RI-175 and CT-114 viruses were fixed in a mixture of 1.25% formaldehyde and 2.5% glutaraldehyde in 0.05 M cacodylate buffer at pH 7.3, to which 0.03% trinitrophenol and 0.03% CaClHighlands J virus, Jamestown Canyon virus, La Crosse virus, Saint Louis encephalitis virus, and Flanders virus antigens, with specific monoclonal and polyclonal antibodies; results were negative. Both viruses RI-166 and RI-175 were subsequently sent to UTMB for further study and characterization. When added to Vero cell cultures, both viruses produced extensive CPE within 48 hours. Newborn Institute for Cancer Research outbred mice that were injected intracerebrally with both RI-166 and RI-175 viruses became sick and moribund within 96 hours. RI-166 virus was also added to cultures of C6/36 and Cx. quinquefasciatus cells; it did not produce CPE, and no viral antigen could be detected in the mosquito cells when examined by IFA 6 days later.Viruses RI-166 and RI-175 were initially isolated in cultures of Vero cells at the Center for Vector-Borne Disease (CVBD), University of Rhode Island. Media from the positive cultures were tested by immunoassay for WNV, EEEV, Cx. quinquefasciatus cells injected with CT-114 virus, but not in C6/36 cells.Virus CT-114 was initially isolated by Robert Wallis at the Department of Epidemiology and Public Health, Yale University School of Medicine, following intracerebral injection of a homogenate of bird tissue into newborn mice. The virus was subsequently transferred to UTMB. Virus CT-114 produced illness and death in newborn mice 24–48 hours after intracerebral injection, as well as massive CPE in Vero cells within 48 hours; however, it did not produce CPE in the mosquito cells. Specific viral antigen was detected by IFA in Virions of isolate CT-114 were bullet shaped and were found budding mostly into the intracytoplasmic vacuoles, either as single virions into a small vacuole, or as several virions budding into the same large vacuole , A and BVirions of the isolate RI-175 were seen budding predominantly into the extracellular space from the plasmalemma of the Vero cells , C and DCarajas virus;Chandipura virus; Cocal virus;Isfahan virus; Maraba virus; Piry virus; vesicular stomatitis virus, types Alagoas, Indiana, and New Jersey; Vesiculovirus species Boteke, Jurona, Klamath, La Joya, Malpais Spring, Radi, and Yug Bogdanovac; Iriri virus, Flanders virus, Mosqueiro virus, Mossuril virus, Kern Canyon virus, Nkolbisson virus, Le Dantec virus, Connecticut virus, New Minto virus, sawgrass virus, Chaco virus, Timbo virus, Bangoran virus, Inhangapi virus, Joinjakaka virus, Kannamangalam virus, Kotonkan virus, Marco virus, Tibrogargan virus, and Yata virus  and RI-175 viruses were indistinguishable ; but RI-Vesiculovirus  as well as Vero cells, are strong evidence that it is a vertebrate rhabdovirus. Yet its ultrastructure and relatively large size more closely resemble some of the plant rhabdoviruses


Healthcare costs, particularly pharmaceutical costs, are a dominant issue for most healthcare organizations, but it is unclear if randomized controlled trials (RCTs) routinely discuss costs. Our objective was to assess the frequency and factors associated with the inclusion of costs in RCTs.We randomly sampled 188 RCTs spanning three years (2003-2005) from six high impact journals. The sample size for RCTs was based on a calculation to estimate the inclusion of actual drug costs with a precision of +/−3%. Two reviewers independently extracted cost data and study characteristics. Frequencies were calculated and potential characteristics associated with the inclusion of costs were explored. Actual drug costs were included in 4.7% (9/188) of RCTs; any actual costs were included in 7.4% (14/188) of RCTs; and any mention of costs was included in 27.7% (52/188) of RCTs. As the amount of industry funding increased across RCTs, from non-profit to mixed to fully industry funded RCTs, there was a statistically significant reduction in the number of RCTs with any actual costs  and any mention of costs . Logistic regression analysis also indicated funding was associated with the inclusion of any actual cost  or any mention of costs . Journal, study conclusions, study location, primary author's country and product age were not associated with inclusion of cost information.While physicians are encouraged to consider costs when prescribing drugs for their patients, actual drug costs were provided in only 5% of RCTs and were not mentioned at all in 72% of RCTs. Industry funded trials were less likely to include cost information. No other factors were associated with the inclusion of cost information. The costs of pharmaceuticals in almost all countries of the Organisation for Economic Co-operation and Development (OECD) are increasing faster than any other aspect of healthcare budgets Physicians have indicated they want more cost information Our objective was to determine how often actual drug costs and healthcare costs in general were included in randomized controlled trials (RCTs) and to identify the factors that may be associated with the inclusion of cost information in RCTs.Although there are no established templates for studies assessing reporting in the literature, PRISMA Our study was limited to RCTs, the gold-standard of therapy evidence. To address the inclusion of drug costs, only RCTs comparing a pharmaceutical to no treatment, placebo or active control were eligible.Similar to past studies that assessed reporting in the literature We used PubMed to identify all RCTs. A search was performed in May 2006 using the abbreviated journal title restricted to the date range January 1 to December 31 for each individual year  and Randomized Controlled Trial for type of article. For example, to search for RCTs published in the N Engl J Med in 2005, we entered N Engl J Med in the search bar and limited the search to RCTs under type of article and the dates to January 1, 2005 to December 31, 2005. To confirm these searches captured all the RCTs published in journals, we reviewed each issue from two of journals for 2005 and found none were missed. The PubMed search did capture a few articles that were not RCTs but these were identified during review for inclusion.The total number of articles found in each search of article type, journal and year  was used to generate the randomization for that group. Randomization was performed using Excel.The sample size was based on our objective to obtain an accurate and precise estimate of reporting of actual drug costs in RCTs. Initially, 10 articles were randomly selected from each of the six journals published in the year 2005. An interim analysis of those 60 RCTs found that actual drug costs were reported in less than 5% of the RCTs. To attain a precision of +/− 3% with a 95% Confidence Interval (CI), 188 RCTs had to be reviewed, assuming the reporting of actual cost was ≤5%. In addition to 10 RCTs from 2005 from each journal, 10 RCTs were randomly selected from each journal for the years 2003 and 2004. Eight more studies were randomly selected from any of the included years or journals.The abstract of each article randomly selected was reviewed to confirm it met the eligibility criteria: an RCT that assessed pharmaceutical therapy. If it did not, a random number was again generated to choose a replacement RCT.To identify cost information, two authors (GMA & KL) independently searched each RCT with the Adobe Reader Search function using the key words “cost”, “pric”, “$”, “dollar”, “pound”, “£”, “money”, “fee”, “fund”, “econom”, “financ”, and “expens”. One author (KL) also read each RCT for any mention or discussion of costs. We classified costs in to three categories: actual drug costs, any actual costs, and any mention of costs. ‘Actual drug costs’ were any specific drug cost provided as a numeric value in dollars, pounds or other currency. ‘Any actual costs’ were any drug cost and/or any healthcare cost provided as a numeric value in dollars, pounds or other currency. ‘Any mention of costs’ was considered any of the above plus any discussion of health care costs, no matter how general (e.g. the management of this condition is very expensive). We limited counting ‘any mention of costs’ to once per paragraph when costs were discussed without numeric values. This was because a single paragraph may have frequently included words such as “cost”, “expense”, or “economic”, but the rest of the article had no other discussion of costs. We did not count costs relating to expenses of the trial (e.g. “we paid participants $5 to complete the testing”), funding sources or financial conflict of interest. Finally, we did not count costs-associated words appearing in reference sections.Two authors (GMA and CK) independently abstracted data on RCT characteristics: experimental drug, age of the experimental drug (novel or established), the comparator, funding of the trial, study country, primary author's country and conclusion. We defined the age of the experimental drug as “novel” if it was still on-patent at the time of the study and as “established” if it was off-patent at the time of the study. Funding was divided into categories of industry, non-profit or mixed. Funding was classified as mixed if the study had both non-profit and industry funding sources. If a study had any industry funding, even if only supplying the pharmaceutical product, it was classified as mixed. We found that classifying conclusions as simply “positive” or “negative” did not reflect the range of language used by authors to explain their results. Therefore, we modified the classification used by Kjaergard and Als-Nielsen Kappa values for areas with a higher potential for disagreement: the age of the experimental drug (novel or established), funding of the trial and conclusion.Similar to previous studies We summarized the results to provide a descriptive analysis including overall mean percent of studies which had actual drug costs, any actual costs and any mention of costs, with 95% CI. We originally intended to compare the inclusion of actual drug costs or any mention of costs across different study characteristics . However, RCTs with actual drug costs were so few in number that we opted to analyze any actual costs  and any mention of cost. We used Fisher-Freeman-Halton test for nominal data to compare the number of RCTs that included actual costs and any mention of costs associated with different journals, primary author's country or study location. We used Cochran-Armitage test for ordinal data to compare the number of RCTs that included actual costs and any mention of costs associated with funding and study conclusions. We used Fisher's Exact Test to compare the number of RCTs that included actual costs and any mention of costs associated with product age (novel or established). We also did a logistic regression to compare the dependant variables of actual cost and mention of cost simultaneously with the independent variables of funding, conclusion, primary author's country, and product age.Kappa values for the age of the experimental drug (novel or established) was 0.87, funding of the trial 0.74, and the conclusion 0.75. All disagreements were resolved with discussion.A total of 188 RCTs were reviewed , there was an average of 3.2 mentions per RCT and a median of 1.5 mentions per RCT. Half (26 of 52) of the RCTs including costs only mentioned them once. Six of the 188 RCTs had 10 or more (maximum 21) mentions of costs per article and account for 90 of the 166 mentions overall. Therefore, 3% of the articles account for 54% of the total number of times that costs were mentioned.Logistic regression analysis indicated that funding was the only factor associated with the inclusion of any actual cost  or any mention of costs . None of the other independent variables  were significantly associated with the inclusion of any actual cost or any mention of costs. The significant result of product age associated with the inclusion of any actual cost disappeared in regression analysis, suggesting that the inclusion of any actual costs with product age may be attributed to the correlation between product age and funding . The correlation coefficient between these two variables was high at 0.54, while no other two variables in the regression analysis had a correlation higher than 0.13.Healthcare costs were mentioned in only 28% of RCTs of pharmaceutical therapy and actual drug costs were rarely (5%) included. The User's Guide to the Medical Literature The total number of times costs were mentioned (166 times) appears to be relatively high, but this number is inflated by a few studies with cost as their primary focus. For example, a Lancet article includes costs 20 times which is equal to or more than all the articles from three of the other journals. We felt that these differences did not warrant statistical testing among the journals as the number of RCTs with any mention of cost did not differ among journals. Additionally, any differences between the total number of mentions could be due to chance since only 3% (6/188) of the RCTs provided 54% (90/166) of the total number of times costs were mentioned. Cost was a focus of these articles in contrast to the other 97% (182/188) of the RCTs.Inclusion of healthcare costs did not vary by journal, primary author's country, study location, product age or conclusion. Although the inclusion of any actual cost was statistically less common in established (or older) drugs compared to novel (or newer), this finding disappeared on logistic regression. The association to product age and the inclusion of any actual cost was due to the correlation of funding and product age. Funding appears to be the only factor associated with the inclusion of cost information, as identified by the inclusion of any actual cost or any mention of cost in bi-variant analysis and multi-variant analysis of logistic regression. Industry funded studies were significantly less likely to mention costs than studies funded solely from non-profit sources. In fact, the likelihood that cost information was included in RCTs increased as the degree of industry funding declined. RCTs with any mention of costs increased across funding groups: from industry (18%) to mixed (31%) to non-profit (36%). This pattern was consistent with the inclusion of any actual costs: from industry (1%) to mixed (7%) to non-profit (15%) funding.In light of previous studies showing that funding was associated with study conclusion Kappa ranged from 0.87 to 0.74 while Pitkin et alKappa and Moher et alKappa values between 1 and 0.53.A potential limitation of the study is the four-year delay from search to submission as cost reporting may have changed in that time. This is, however, highly unlikely as no incentive or initiative has been introduced to trigger such as change. Our journal selection may have impacted our results, particularly compared to journals with a health economic focus. Additionally, we included only RCTs — other types of articles may have included more cost information. That said, our goal was to examine clinically relevant RCTs in high impact clinical journals that are more often read and used by clinicians. As data extraction required some interpretation, particularly for the subjective areas such as study conclusion, there is the potential that different raters would categorize the conclusions differently. Our level of agreement was similar to investigators in previous studies assessing reporting in the literature. Our Future research should examine other medical information resources, such as internet evidence-based summarized sites, other types of articles and perhaps other journals. If other resources provide cost information, the accuracy of the information should be examined, although defining true cost and accounting for cost difference over time and different locations will be challenging.In summary, actual drug costs or any actual costs are rarely included in RCTs and costs are not mentioned at all in 72% of RCTs. Trials funded from non-profit sources were more likely to include cost information compared to trials with solely industry based funding. No other factors were associated with cost inclusion. More work is needed to provide physicians with the cost information that can inform their decision-making and potentially reduce healthcare costs.Appendix S1Data Abstraction Form(0.06 MB DOC)Click here for additional data file.Appendix S2Articles Included(0.11 MB DOC)Click here for additional data file.