Numerous tickborne viruses, including Dhori virus and foot-and-mouth disease virus, were isolated. Rhipicephalus pulchellus (56%), Amblyomma gemma (14%), R. appendiculatus (8%), A. variegatum (6%), and others. A total of 56 virus isolates were obtained, 26 from A. gemma, 17 from R. pulchellus, 6 from A. variegatum, and 7 from other species. Virus isolates included Dugbe virus (DUGV), an unknown virus related to DUGV, Thogoto, Bhanja, Kadam, Dhori, Barur, and foot-and-mouth disease (FMDV) viruses. This is the first report of Dhori virus isolation in East Africa and the first known isolation of FMDV associated with tick collection. Our results demonstrate the potential for tickborne dissemination of endemic and emergent viruses and the relevance of A. gemma in the maintenance of tickborne viruses in this region.To identify tickborne viruses circulating in Kenya and the surrounding region, we conducted surveillance at abattoirs in Nairobi, Kenya. Species of ticks collected included  Viruses transmitted by blood-feeding arthropods (arboviruses) are responsible for some of the most serious emerging infectious disease problems facing the world today. Arthropodborne viruses constitute the largest biologic group of vertebrate viruses. Their considerable number and diversity suggest that arthropod vector transmission offers distinct survival benefits for the virus. Approximately 50% of arbovirus isolations from field-collected arthropods are from mosquitoes, and 25% are from ticks; however, this difference may represent a sampling bias, since many more mosquitoes are collected and tested for virus than ticks. To investigate the abundance of tickborne arboviruses in Kenya and the surrounding region, we collected and tested ticks infesting livestock driven to market at 2 major abattoirs in Nairobi, Kenya. These abattoirs receive the bulk of animals slaughtered for Nairobi and its environs, which is the largest livestock market in the country. Approximately 30% of animals slaughtered in these abattoirs come from within Kenya; the rest are from neighboring countries, including Ethiopia, Sudan, Somalia, and Tanzania.Among pastoral communities in this region, livestock are frequently maintained in enclosures close to human habitation, and small ruminants sometimes sleep inside houses overnight for security reasons. Such practices increase the potential for zoonotic virus transmission between animals and humans. Poor husbandry and grazing practices put great pressure on land resources, which results in the need to continuously move large numbers of animals, especially cattle, in search of pasture. In some parts of East Africa, these pastoral communities exist near wildlife parks, and wildlife and livestock sometimes mix, which allows transfer of ticks and possibly viruses between these animal groups. Additionally, livestock marketing practices allow movement of animals across borders in the region, which allows ticks and tickborne viruses to move between countries.Bunyaviridae, Flaviviridae, Rhabdoviridae, Reoviridae, and Orthomyxoviridae , isolated in Kenya; Barur virus (family Rhabdoviridae), isolated in Somalia; and Kadam virus (KADV)  and Chenuda virus , confirmed serologically in cattle in South Africa , KADV, and Barur virus. In addition, 2 viruses related to DUGV were isolated. An unexpected result of this study was the isolation of foot-and-mouth disease virus (FMDV) from tick pools.Ticks were collected from the hides of flayed animals between September and November 1999 at the Njiru and Dagoretti abattoirs, on the outskirts of Nairobi, Kenya. Attached ticks were pulled off manually and placed in sterile plastic vials, which were loosely capped and transported to the laboratory. The origin of individual sampled animals could not be determined. All animals to be slaughtered for the day were put in 1 enclosure, irrespective of origin.,Hyalomma species, the primary vectors of CCHFV, each pool was screened by reverse transcription–polymerase chain reaction (RT-PCR) for CCHFV before tissue culture injection was conducted , with antimicrobial agents . Ticks were identified by sex and species by using appropriate identification keys , 2% glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 μL/mL amphotericin B. Cells were rinsed with sterile saline, and 0.2 mL clarified tick homogenate was added followed by injection at 37°C for 45 minutes to allow virus adsorption. After incubation, cells were rinsed with saline and maintenance medium  was added. Cells were incubated at 37°C and observed daily for signs of cytopathic effects (CPE). The pooled infection rate program  was used to compare virus infection rates in the tick species collected and processed in this study.For virus isolation in cell culture, Vero cells were grown in 25-cmAgents causing CPE in tissue culture were initially identified to virus group by using the indirect immunofluorescent antibody assay (IFA) on spot slides of infected Vero cells with polyvalent mouse hyperimmune ascitic fluids obtained from the National Institutes of Health Reference Reagents Program. Fluorescein isothiocyanate–conjugated goat-anti-mouse immunoglobulin G was the secondary antibody.http://www.ncbi.nlm.nih.gov/BLAST).RT-PCR was also used to identify most of the virus isolates obtained from tissue culture. RNA was extracted from cell culture supernatants with the QIAamp Viral RNA kit  according to the manufacturer's recommended protocol. RT-PCR was performed with the Titan One Tube RT-PCR kit  with primers mainly targeting the known African tickborne viruses . Referenhttp://www.megasoftware.net).Additional methods used to identify selected isolates included complement fixation (CF) and hemagglutination-inhibition tests , followed by Amblyomma gemma, R. appendiculatus, and A. variegatum. Other species were sampled in smaller numbers. However, the calculated virus pooled infection rate was higher for A. gemma than for R. pulchellus . These species are the primary vectors of CCHFV; this agent was not among the viruses isolated.A total of 15,851 ticks were collected and processed in 1,071 pools for virus isolation. Species of ticks collected and their proportions in the collection are shown in lchellus . The numA total of 56 virus isolates were obtained from 51 tick pools; 52 of the 56 viruses were identified . Five poA. gemma (62%), whereas the most commonly sampled tick, R. pulchellus, yielded only 2 DUGV isolates (8%) . Most DUGV isolates were recovered from tes (8%) .A. gemma pools, and 1 (17%) was from a pool of R. pulchellus. One additional isolate, obtained from a pool of A. lepidum, was identified as being DUGV-like by CF test. RT-PCR conducted on RNA extracted from this isolate with the primers designed for the DUGV-like virus described above produced a weak band. Sequence of this product was ≈80% homologous to the other isolates of DUGV-like virus and 70%, 63%, 57%, and 55% homologous to DUGV, NSDV, CCHFV, and Hazara virus, respectively, which suggests that it may be a different DUGV-like isolate.Two of the virus isolates that were IFA-positive with Congo group antiserum were RT-PCR negative when primers specific for DUGV, CCHFV, Hazara virus, or BHAV, all of which were represented in the antiserum, were used. However, RT-PCR using NSDV nucleocapsid–specific primers and RNA extracted from these isolates produced 3 major bands, including one ≈880 bp in size; the expected band size for the NSDV-specific fragment was 887 bp. The 880-bp fragment was sequenced, and an alignment of 513 nt (nt) of this sequence with nucleocapsid sequences from DUGV, CCHFV, and NSDV showed 71%, 58%, and 60% homology, respectively, which suggests that these isolates were most closely related to DUGV. Alignment of the sequences from the 2 isolates showed them to be 95% homolgous. Specific primers were designed for this DUGV-like virus from sequence of the fragment described above. RT-PCR conducted with these primers produced bands of correct size and sequence with RNA from the DUGV-like virus isolates, while RT-PCR results using these primers with RNA from DUGV and BHAV were negative. RT-PCR and sequencing of all of the virus isolates using the primers designed from the DUGV-like virus sequence showed 4 additional isolates of this DUGV-like virus; 2 were from pools that also contained DUGV. Sequence homology between all 6 DUGV-like isolates was 95%–100%, based on a 508-nt alignment of the S segment of the virus RNA. Of the 6 isolates of this virus, 5 (83%) were from A. gemma, l from A. lepidum, and 1 from R. pulchellus. Since the THOV genome is segmented, a portion of each of the 6 genome segments from each isolate was sequenced and compared with available sequence from other THOV isolates to determine if reassortment of virus genome segments had occurred. No evidence was found for reassortment of virus segments. Phylogenetic analysis showed that the THOV isolates were most closely related to other African isolates from Uganda (1996), Kenya (1960), and Nigeria (1969) (data not shown).Six isolates of THOV were obtained: 4 from pools of R. pulchellus. A single isolate of KADV, the only African member of the tickborne flavivirus group, was recovered from a pool of R. appendiculatus. Six isolates were found by CF test to be indistinguishable from Barur virus, a rhabdovirus. Further characterization of these isolates was not conducted.The single isolate of DHOV, another member of the tickborne orthomyxovirus group, was obtained from a pool of Three isolates of FMDV were identified by using panviral microarray-based technology. RNA isolated from viral culture of 1 isolate was reverse transcribed, randomly amplified, and hybridized to panviral DNA microarrays as described  and shrub steppe and is much more restricted than A. lepidum to very dry areas. DUGV and DUGV-like viruses were the most frequently isolated viruses in this study , and 64% of these isolates were from pools of A. gemma. Four of the 6 THOV isolates were obtained from pools of this species as well. Our results suggest that viruses are being actively transmitted in the drier parts of East Africa where A. gemma is more common. The pastoral regions, which supply many of the animals slaughtered at abattoirs in Nairobi, are predominantly dry and therefore likely to harbor this tick species in abundance.In this study, R. appendiculatus collected in this study was small when one considers the distribution of this tick in Kenya and its importance as a pest, a finding that suggests that most sampled cattle came from climatic zones where this species is not abundant. In Tanzania and Kenya, R. appendiculatus is most abundant in areas receiving >1,000 mm mean annual rainfall. It is absent from xerophytic and dry thicket zones with overgrazed pastures and little grass cover (R. appendiculatus, and the virus is found only in areas where this species is abundant (R. appendiculatus, however, did yield single isolates of BHAV and KADV. BHAV has been isolated previously in Kenya and Nigeria (,–R. pravus ticks taken from a cow in Uganda (,A. variegatum and R. pulchellus (The proportion of ,,,,,DUGV is commonly isolated in surveillance studies conducted in Africa (Rhipicephalus species and Boophilus decoloratus in the 1930s (A. gemma (A. lepidum (R. pulchellus (Thogotovirus genus in the Orthomyxovirdae family, has been previously isolated in Europe, Asia, and the Middle East (–,,THOV was first isolated in Kenya from R. pulchellus. FMDV is endemic in many parts of Africa; however, it has not previously been identified in association with tick surveillance or transmission studies (Rhipicephalus ticks or blood-feeding flies (,An unexpected finding in this study was the isolation of FMDV from 3 pools of Our study illustrates the potential for tickborne dissemination of endemic and emergent viruses, some of which are human pathogens, among livestock as well as the potential for transmission of these pathogens to humans. Regular surveillance is warranted to monitor the presence and spread of these and other viruses facilitated through livestock rearing, marketing, and movement in Africa.


The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity. The liver has long been known as the garbage dump of the body, capable of rapidly removing hazardous pathogens and useless particles from the blood stream, thereby protecting the host. The only cell doing the removal has been thought to be the liver's macrophages. This is likely true for larger particles such as bacteria. But for smaller particles the size of virus or small antibody-antigen complexes, macrophages are probably not the cell responsible for the bulk of removal. We suggest, rather, it is the endothelial cell of the liver's blood circulatory system that takes up and destroys the majority of virus, doing so quickly (minutes) and extensively (>90%), leaving only a small fraction of circulating virus to infect the body in ways peculiar to each virus. To test this possibility, we infused mice intravenously with a harmless common cold virus and tracked its destination by molecular and microscopy methods. Affirming our conjecture, we found that ∼90% of the infused virus homed to the endothelium of the liver and ∼10% went to its macrophages. These data support a unique role, generally underappreciated, for the liver endothelium in viral clearance. It has been known for more than 50 years that viruses of many sorts when injected into the blood stream of an animal are cleared with astonishing rapidity and efficiency Of the many viruses studied, one has received special attention. Workers using adenovirus as vectors for gene transfer for genetic vaccine and gene therapy have long known that while these vectors are effectively targeted to chosen tissues, the process is highly inefficient because most of the dose is cleared quickly by the liver. Effective countermeasures to clearance have been elusive, the usual strategies being to increase the dose, to bypass the liver circulation, and to modify the capsid As the initial step in studying the viral clearance mechanism, we have undertaken to test whether, as is generally assumed, the KC is the major cell responsible for the rapid and efficient clearance of virus from the blood stream, or, as we suspect, that the long-overlooked LSEC is the major scavenger. Using the well-known mouse model of adenovirus clearance we document, employing two physicochemically different methods of virus detection, that adenovirus is rapidly and efficiently cleared, and with 4-color fluorescence confocal microscopy we show the virus to be localized chiefly to the LSEC and to a much lesser extent to the KC. We confirmed our results with electron microscopy.t½ 1 and 10 minutes) of virus from the bloodstream with ∼70% cleared in 5 minutes and >90% in 20 minutes. Sampling blood frequently during the initial 5 minutes after infusion defined the steep first decay phase  and anti-MR (red) gave largely coincidental sinusoidal patterns in green and red and in the merged image, although the cell-associated intensities of the two colors varied independently  and S3, Having documented that nearly all blood-borne virus is cleared by the liver, we asked the whereabouts of liver rAd5 at 1 and 10 minutes after infusion, examining fixed liver sections by fluorescence confocal microscopy. rAd5 was detected either by the use of a rabbit anti-Ad5 antibody (red) applied to the liver sections  or by coFrom images such as those in Confirming by electron microscopy our immunofluorescence results, we examined sections of 39 sinusoidal lumens from livers of 2 mice infused with rAd5 1 min prior to hepatectomy, identifying 34 putative virus particles  situated within LSEC but none in KC , whereas 30 sinusoidal lumens from livers of 2 uninfused mice evidenced 2 such particles in LSEC and none in KC (1 KC identified).We interpret our data to indicate that in the mouse it is the LSEC and not the KC that clears the bulk of blood-borne human adenovirus. This conclusion is based first on our clearance studies documenting that >90% of infused virus has disappeared from the blood within 20 minutes. Second, it is clear from our data and those of others that the liver is responsible for most of the rapid and extensive clearance th century biologists who described the reticuloendothelial system (RES) th century most biologists dismissed the ‘endothelial’ portion of the RES. Rather, RES function was reconceptualized in terms of a system of mononuclear phagocytes (MPS) Thus our data support the contention that the LSEC and not the KC is the major cell type involved in clearing adenovirus from blood during the early minutes after virus infusion. Early 20th century Only recently has the concept of sinusoidal scavenging been amended: Smedsrod and colleagues, using modern techniques to repeat the vital stain clearance studies of early 20Whether serum factors are required for clearance of rAd5, we know only that immunologically naïve animals were used and that clearance rate was unchanged in Rag2-deficient mice, which would indicate that antibodies were not required. Prior workers have not dealt with this issue systematically and those who have approached the question are at odds. One laboratory noted that Newcastle disease virus was cleared ten-times more rapidly when combined with antiserum Although all of the relevant studies on the clearance of blood-borne virus utilized the contrived modality of intravenous infusion (cited above), these data should nevertheless be applicable, one would infer, to the clearance of blood-borne virus during any viremic phase of a natural viral infection. Free virions during viremia should be cleared quickly and effectively by the liver just as when infused unless the clearance mechanism is modified by the adaptive immune system. What fraction of total blood-borne virus during a viremic episode of natural infection remains free of immune system modification has not been systematically studied, insofar as we can discern, and remains a worthy challenge. Thus, it appears currently impossible to estimate directly the magnitude of the ‘free virus’ clearance mechanism during the viremia of natural infection. Nevertheless, for these reasons it would seem desirable to explore the mechanism by which the liver clears blood-borne virus after intravenous infusion. The mechanism may yield new strategies for accelerating clearance for therapeutic purposes. Our data suggest that studies should begin with the LSEC.Beyond our conclusion that virus is being removed from blood largely by LSEC we can only infer the mechanism of clearance. Note that it is commonly thought that viruses are small enough to be pinocytosed, a process dedicated to small particles (<0.2 µm), and do not require a phagocytic mechanism generally reserved for large particles (>0.5 µm) Using the strategy described herein for showing LSEC uptake of virus we should be able to test the prediction that other small particles such as small immune complexes, nanoparticles, and others, are taken up in a like manner by the LSEC. Further, now that we have identified the virus-clearing cell, studies investigating the mechanism of clearance and the fate of endocytosed virus should readily follow.Wild type male Balb/C mice of age 12–15 weeks were obtained from Taconics Laboratory. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by The Ohio State University Institutional Animal Care and Use Committee. All surgery was performed under Isoflurane anesthesia, and all efforts were made to minimize suffering.2 and 200 mM NaCl). Adenoviral particles from the supernatant were purified from defective viral particles using two CsCl density gradients. First, a discontinuous step gradient was prepared by loading 20 ml of the clarified viral lysate into a 34 ml polyallomer ultracentrifuge tube, underlaying with 6 ml of a 1.2 g/ml CsCl solution, followed by 8 ml of a 1.4 g/ml CsCl solution. Tubes were centrifuged in a SW28 rotor at 23,000 rpm for 90 min at 4°C. The banded virus (∼3 ml) at the 1.2/1.4 g/ml CsCl interface was collected and transferred to a 13 ml ultracentrifuge tube, and approximately 10 ml of 1.35 g/ml CsCl solution added to completely fill the tube. An isopycnic gradient was generated using a NVT65 rotor at 60,000 rpm for 4 hours. The lowest bluish white band containing the purified infectious virus was collected and dialyzed over night against TMN 200 buffer with a minimum of 4 volume exchanges. The rAd5 viral particles were stored in TMN 200 containing 0.1 volume of 50% sucrose at −80°C.Recombinant adenoviral particles, E1 and E3 deleted Ad5.CMV.eGFP vector (Q-Biogene), were propagated and amplified in HEK 293 A cells (Microbix), cultured in Corning hyper flasks with 10% CCS DMEM. The cells were lysed after 72 hr or once they reached the maximum cytopathic effect by 4x freeze-thaw in the presence of 0.1% Triton X-100 and 25 units/ml Benzonase in TMN 200 buffer ; Reverse primer- 5′ CAA GGC CAA GAT CGT GAA GAA; Tagman probe 5′ (6∼FAM) TGC ACC ACA TTT CGG CCC CA (TAMRA-Q) 3′ (150 nM). Concurrently, an adenovirus DNA plasmid standard curve (pHelp adenovirus plasmid containing E2A gene) was set up in triplicate using 104–1010 rAd5 genome equivalents. Tagman quantitative PCR assay was performed on the ABI PRISM Sequence Detector 7900HT (PerkinElmer) using Sequence Detector version 1.7 software. Reaction conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Fluorescence emission was plotted as a function of increase in reporter fluorescence (Δ Rn) versus copy number. The data were analyzed using the default settings of the software for determining baseline and the threshold value. For every assay a standard curve for the primer set was generated and used by extrapolation to estimate the copy number of target sequences in unknown samples. As a general confirmation of the DNA containing DRP vector particle titer, purified rAd5 particles were lysed in lysis buffer  and the optical density (OD) at 260 nm measured where an OD of 1 corresponds to 1.1×1012 virus particles. As expected, the DRP and OD260 titers of Ad5.CMV.eGFP vector were in general agreement .The DNase resistant viral particle (DRP) rAd5 titer  was determined by a method previously described for AAV particle titers by our group 50 infectious unit (IU) endpoint titration assay was performed. The high throughput method consists of detecting replicating rAd5 DNA using E2A qPCR following a 3 day infection of HEK 293 cells. Briefly, 10-fold serial dilutions of a rAd5 stock in replicates of 12 were prepared and used to infect 293 cells in a 96-well plate format. After a 72 hr incubation period, cells were directly lysed with a detergent buffer  and sequentially incubated at 37°C for 1 hr, 55°C for 2 hr, and 95°C for 30 min. The plate was centrifuged briefly to clarify the crude cell lysate , and a 1∶4 dilution of the lysate in 1X Taqman PCR Buffer was prepared (this step dilutes out cellular PCR inhibitors). The diluted sample (2.5 ul) was then added to a standard 25 µlE2A qPCR reaction and Taqman E2A qPCR performed to detect the presence of rAd genomic DNA as described above. Genomic rAd5 copies are extrapolated from a plasmid DNA standard curve (pHelp plasmid), and total copies present in the well calculated as follows: total lysate volume per well is 185 µl and 2.5 µl of a 1∶4 lysate dilution was used in each PCR reaction (185/(2.5×0.25)  = 296). Coupled with a Limit of Detection (LOD) of 10 rAd5 genomic copies per qPCR reaction (based on the lowest standard on the curve), a minimum endpoint input value corresponds to 2,960 copies/lysate. Therefore at the assay endpoint of 1 infectious unit in an individual well, greater than 2,960 genomic copies/lysate are required to score the well as positive based on our assay sensitivity. Once scored, the data are then applied to the Karber equation to determine the TCID50 of the sample. Significantly, the human Ad5 Reference Material (ARM) 11 IU/ml ARM vs. 8.2×1011 IU/ml E2A qPCR method). Per the ATCC's Product Information Sheet for the Ad5 ARM (Cat. No. VR-1516), the IU titer on HEK 293 cells is 7.0×1010 Normalized Infectious Units (NIU)/mL, with 95% certainty that the infectious titer on HEK 293 cells lies within the range of 7.0×1010 to 8.0×1010 NIU/mL. The average Ad5 ARM TCID50 IU titer determined by us using the E2A qPCR method was 8.0×1010 IU/ml (assay performed in duplicate). The IU titer of the Ad5.CMV.eGFP was determined to be 1.6×1012 IU/ml using the TCID50 assay method above, and this yielded a DRP to infectivity ratio of 2.6, indicative of a highly infectious rAd5 preparation. We acknowledge that the calculated IU titer is greater than the calculated OD260 titer, which may reflect the inherent variability in all titration methods measuring single particle events, but note that all experiments use the DRP titer to calculate particle inputs.Lastly, to confirm that the particles isolated by double CsCl banding were highly infectious, a TCID9, 1.6×1010 and 1.6×1011 DRP 10 DRP and bled via the retroorbital plexus at 0.5, 1, 2, 3, 4, and 5 min. Total blood DNA was extracted using DNeasy Blood & Tissue kit (Qiagen). Adenovirus genomes were quantified using qPCR (adenovirus E2A gene as the target) and plotted as the decay of plasma virus vs. time.rAd5 DNase Resistant Particles (DRP) at 3 doses  was 82% of the parent stock (2.8×1012 DRP). Clearance of Cy3 labeled rAd5 was tested in the same manner as the clearance of unlabelled rAd5  were removed and weighed. DNA was extracted from weighed portions of each organ using DNeasy Blood & Tissue kit (Qiagen) and quantified for rAd5 by E2A qPCR as described earlier.Mice (n = 3) were infused intravenously with 10The livers were removed as rapidly as possible and small pieces (∼5 mm) were cut and transferred to 4% paraformaldehyde-PBS and fixed for 2 hrs at room temperature. Tissue was washed several times in PBS and then infused in 20% sucrose-PBS overnight at 4°C. The tissue was then embedded in a freezing medium for cryostat sectioning and stored at −80°C until used. Cryostat sections, 5 m thickness, were collected on Superfrost microscope slides . The sections were blocked in 5% milk-PBS prior to incubation with primary antibodies overnight at 4°C. The details of all the primary antibodies are given in 11 rAd5 viral particles and processed as above. 5 µm liver sections were incubated with the rat mab 2.4G2 and the rabbit polyclonal anti-whole Ad5 antibody  The above protocol was modified as follows for 4 color imaging and to accommodate two rat antibodies. Livers were removed at 1 minute from mice infused with 1.6×10We determined by inspection of 4-color confocal images that rAd5 was associated only with LSEC and KC, and not with hepatocytes or endothelium in larger vessels. To quantify the relative proportion of virus associated with KC and LSEC we employed Image J software developed in JAVA, using a 3-step procedure. 1. After thresholding the entire image to separate viral pixels from background pixels, we determined the total fluorescence intensity of virus in the entire image. 2. Each KC, identified using anti-CD68 or anti-F4/80 in a microscopic image, was manually segmented by liberally outlining the margins; thresholding was applied and the fluorescence intensity of rAd5 associated with each KC was recorded; the data were summed to obtain the total intensity of KC-associated virus of an image. 3. The total intensity of rAd5 associated with KC was subtracted from the total intensity of rAd5 of the entire image to give the total intensity of rAd5 in LSEC alone. The values for all images were summed. The number of KC analyzed in To illustrate in 3-dimensions the association of virus with each of the two relevant cells (LSEC and KC), 7 videos were created using Olympus Fluoview Software (version 2.1.39) from a single 3-color fluorescence image of a liver section prepared as described in the legend to 11 viral particles or un-infused were removed after 1 min and cut into 1–2 mm3 pieces and fixed in 2% glutaraldehyde in 100 mM cacodylate buffer (pH 7.2) containing 5% sucrose for 1 hr at 22°C and subsequently washed with cacodylate buffer. The tissue was subsequently post- fixed in 2% OsO4 in 100 mM cacodylate buffer pH 7.2 followed by dehydydration in graded series of ethanol and incubation with propylene oxide prior to infiltration and embedding in Epon 812 resin. Sections of ∼70 nm were cut using a Leica EM UC6 Ultramicrotome, mounted on grids and contrast-stained with lead citrate and uranyl acetate. Thin sections were then examined using a FEI Tecnai G2 Spirit Transmission Electron Microscope. LSEC were identified as very thin gap-disrupted cells, thick at the nuclei, defining the sinusoidal lumens, overlying the space of Disse. KC were identified as cells in the sinusoidal lumens closely adhering to the LSEC surface.Electron microscopy sample preparation was based on our previous method with some modifications Figure S1Ab-labeled rAd5 localizes predominantly to LSEC-using CD68 as marker for KC and RIIb for LSEC. Liver from a mouse infused 1 min earlier with 1.6×1011 DRP rAd5 and analyzed by 4-color fluorescence using mab anti-CD68, rabbit anti-Ad5, mab 2.4G2, and DAPI. A. Magenta color delineates KC. B. Red puncta identify rAd5 particles. C. Green 2.4G2 marks LSEC. D. Merge of A, B, and C shows virus confined to LSEC and KC. E. Panels A, B, C and DAPI showing cell nuclei plus DIC defining tissue structure including sinusoidal lumens. The bar in panel C signifies 10μm. Quantification of this experiment is included in (PDF)Click here for additional data file.Figure S2Cy3-labeled rAd5 confined predominately to LSEC shown using CD68 as marker for KC and RIIb for LSEC. Liver from a mouse infused 1 min earlier with 1011 Cy3-labeled rAd5 viral particles (1011 DNAase-resistant particles) analyzed by 4-color fluorescence microscopy using mab CD68 to label KC, mab 2.4G2 to identify LSEC, and DAPI. A. Magenta color delineates the KC. B. Red puncta of Cy3 identify rAd5 particles. C. Green mab 2.4G2 marks the LSEC. D. Merge of A, B and C. E. Merged panels A, B, C with DAPI showing cell nuclei (blue) plus DIC defining tissue structure including sinusoidal lumens. The bar in panel D signifies 10 μm. Quantification of this experiment is included in (PDF)Click here for additional data file.Table S1Details of all antibodies used in this study (complementary to ).(DOC)Click here for additional data file.Video S1Virus (red) alone.  Viruses appear as red puncta often coalescing into a tubular pattern that manifests some directionality, all suggestive of the walls of LSEC-lined sinusoids. Details of image acquisition and 3D video creation are given in Materials and (MOV)Click here for additional data file.Video S2LSEC (pseudo-green) alone.  Sinusoids cut transversely and longitudinally are lined with thin green LSEC encircling empty sinusoidal lumens. Uncolored hepatocytes fill the spaces between sinusoids. Details of image acquisition and 3D video creation are given in Materials and (MOV)Click here for additional data file.Video S3KC (pseudo-magenta) alone.  Several KC are seen, most with long projections. KC are more sparse than LSEC. Details of image acquisition and 3D video creation are given in Materials and (MOV)Click here for additional data file.Video S4Virus (red) + LSEC (pseudo-green).  The great majority of red virus is either in, on, or very near the green LSEC image suggesting close association of the two. Details of image acquisition and 3D video creation are given in Materials and (MOV)Click here for additional data file.Video S5Virus(red) + KC (pseudo-magenta).  A minority of virus puncta appear associated with KC. To enhance contrast, we have colored KC green in the next video (S6). Details of image acquisition and 3D video creation are given in Materials and (MOV)Click here for additional data file.Video S6Virus (red) + KC (duplicate of video 5 using green for contrast with red virus).  In this image alone KC are colored green to heighten the contrast with red virus. It shows that only a minority of virus are associating with KC, some of which are merged yellow. Most viruses are distant from KC. Details of image acquisition and 3D video creation are given in Materials and (MOV)Click here for additional data file.Video S7Virus (red) + LSEC (green) + KC (pseudo-magenta).  This moving 3D image of all three colored elements confirms that most of the virus associates with the LSEC and a minority with KC. The association of virus with each of the two cell types is better discernable in the 2-color videos (S4–S6) above. It is also apparent that the LSEC is the predominant cell type of the two. The section of a sinusoid marked by a white arrow is enlarged in (MOV)Click here for additional data file.Video S8Zoom of a single sinusoidal lumen from   Note here a section of a hepatic sinusoid defined by walls of green LSEC, densely speckled with red virus, some of which appear yellow having merged with green. The dimensions of the reconstructed stack in x, y and z are 34.5, 28.5 and 9.6 µm, respectively. Details of image acquisition and 3D video creation are given in Materials and (MOV)Click here for additional data file.


Traumatization in childhood can result in lifelong health impairment and may have a negative impact on other areas of life such as education, social contacts and employment as well. Despite the frequent occurrence of traumatization, which is reflected in a 14.5 percent prevalence rate of severe child abuse and neglect, the economic burden of the consequences is hardly known. The objective of this prevalence-based cost-of-illness study is to show how impairment of the individual is reflected in economic trauma follow-up costs borne by society as a whole in Germany and to compare the results with other countries’ costs.From a societal perspective trauma follow-up costs were estimated using a bottom-up approach. The literature-based prevalence rate includes emotional, physical and sexual abuse as well as physical and emotional neglect in Germany. Costs are derived from individual case scenarios of child endangerment presented in a German cost-benefit-analysis. A comparison with trauma follow-up costs in Australia, Canada and the USA is based on purchasing power parity.The annual trauma follow-up costs total to a margin of EUR 11.1 billion for the lower bound and to EUR 29.8 billion for the upper bound. This equals EUR 134.84 and EUR 363.58, respectively, per capita for the German population. These results conform to the ones obtained from cost studies conducted in Australia (lower bound) and Canada (upper bound), whereas the result for the United States is much lower.Child abuse and neglect result in trauma follow-up costs of economically relevant magnitude for the German society. Although the result is well in line with other countries’ costs, the general lack of data should be fought in order to enable more detailed future studies. Creating a reliable cost data basis in the first place can pave the way for long-term cost savings. Traumatization of children (the United Nations Convention on the Rights of the Child defines a "child" as "a human being below the age of 18 years") occurs in many ways. Due to their often very pronounced aftereffects, sexual, physical and emotional abuse in the home environment play a central role. For example, Maercker et al.  in up toAll in all, childhood traumatization is not a rare event. In two German studies on juveniles and young adults, 25.5% of the male and 17.7% of the female participants  had alreA recent representative population survey in Germany  has showA characteristic feature of childhood traumatization is that children are often affected by several types of traumatization. This has been shown for Germany ,10 and iThe consequences of childhood traumatization (hereinafter referred to as trauma-related disorders) are – apart from acute injuries and reactions – additionally mirrored in restricted social, emotional and physical development, which are associated with an increased risk of mental disorders, alcohol- and drug abuse as well as physical diseases in adulthood . The “FiBesides Post-Traumatic Stress Disorder (PTSD) – a trauma-related disorder by definition – increased risks after childhood traumatization have been shown and connections proven, respectively, in the following selected mental illnesses in retrospective and partly also prospective studies: depressive disorders ,13,16-19Trauma-related disorders are thus by no means limited to the time of traumatization; they often accompany patients throughout their lives, with the time lag to the trauma being highly variable. While PTSD is defined over a close time connection of several weeks or months , trauma-However, childhood traumatization can by no means be regarded as the sole cause of the development of diseases or disorders . TraumatNational Center of Early Assistance  [Up to now, there are only few sources examining the cost side of childhood traumatization and the relevant social- and health policy questions. The total societal costs incurring due to childhood traumatization (hereinafter referred to as trauma follow-up costs) are unknown. A few studies from English-speaking countries have attempted to give at least approximate estimates of trauma follow-up costs. Authors of a cost-benefit analysis by the  Hilfen)  express What all studies known to the authors have in common is great uncertainty and incompleteness of the available data – starting from the prevalence of traumatization over the definition of cost areas to the calculation and allocation of costs. The variability of individual progressions between complete resilience and lifelong trauma-related disorders must be estimated as well, since they directly determine the long-term follow-up costs. By means of their conservative calculation methodology, studies on trauma follow-up costs explain in detail that the results of this puzzle are throughout underestimating -58. In sOne US-American study estimates societal costs related to reported cases of child abuse and neglect at USD 103.8 billion per year – without taking intangible costs into consideration . For AusSources about federal costs in Germany are not known to the authors. The objectives of the present study are to estimate societal trauma-follow up costs (including direct and indirect costs) in Germany for the first time and to compare the results to costs in Australia, Canada and the USA.This prevalence-based cost-of-illness study is performed from the societal perspective. This perspective comprises not only individual costs, but most of all costs borne by society, caused by expenses in cost sectors such as health insurance, social service or losses in added value. The costs include those directly linked to traumatization as well as short- and long-term costs occurring due to aftereffects (indirect costs). The insufficiency of the available data did not allow for any estimation of opportunity costs. In order to estimate annual trauma follow-up costs in Germany, already published, aggregated data was used. The cost derivation follows a bottom-up approach based on the following formula:Number of "A" units x costs per unit "A" = Total cost of "A" units.In this context, unit "A" represents a case of (former) child abuse and/or neglect, respectively, where trauma-related disorders incur additional, economical costs for a lifetime.Prevalence data for the determination of the number of "A" units were taken from the most recent survey on the prevalence of child abuse and neglect in Germany . The surExpertise Kosten und Nutzen Früher Hilfen") by Meier-Gräwe and Wagenknecht [The costs per unit "A" were obtained from the "Expertise on Cost-Benefit of Early Assistance" , whereas in the age group of 13 to 16 years the single matter of expense is considered in relation to the corresponding age.Finally, the case costs were converted into annual costs by dividing them by the age range of 50 years (15 up to including 64 years). The case scenarios are presented on the 2008 cost level . ConsequFor a comparison of German costs with results from other countries, three prevalence-based cost studies from Australia , the USAComparison is made on the basis of purchasing power parity. While the German cost study  calculatAdditionally, the international costs were calculated as notional total costs for Germany by multiplying per capita costs of the respective country by the German population. These figures serve as a complementary way of illustrating the results, in order to point out the cost dimension of traumatization in relation to other societal expenses.By means of these methods, differences between the individual countries with regard to purchasing power, population size and currency shall be balanced, so that results can be compared in the form of single figures on one level.The prevalence rate of at least one form of child abuse or neglect classified as "severe/extreme" is 14.5% . This 14On the basis of the indications available in literature, only a 21% share of the 7.8 million individuals affected by “severe/extreme” child abuse or neglect has been included in the derivation of costs. This percentage equals 1.6 million (or 3.0% of the population aged 15 to 64 years), which represent the number of units "A".The costs of the moderate scenarios average to EUR 432,951  for the age range of three to 67-year-olds; of the pessimistic scenarios, the average costs are EUR 1,159,294  for the age range of six to 67-year-olds [Substituting the variables of the above described formula by figures of the cost margin’s lower bound (moderate scenario):the resulting total annual costs amount to EUR 11.1 billion, which incur as follow-up costs of child abuse and neglect, respectively, for German society. In other words, the annual per capita trauma follow-up costs would amount to EUR 134.84 .Applying the costs of the pessimistic scenario to the formula:the upper bound of the annual trauma follow-up cost frame is EUR 29.8 billion in total or EUR 363.58 per capita.The international, comparative values of per capita trauma follow-up costs (without intangible costs) are EUR 106.20 according to the Australian, EUR 22.14 according to the US-American, and EUR 368.16 according to the Canadian calculation each year .The objective of the present study was to estimate trauma follow-up costs for Germany. A margin of total annual trauma follow-up costs was calculated in the amount of EUR 11.1 billion for the lower bound and EUR 29.8 billion for the upper bound, respectively. The correspondence of the Australian result with Germany’s lower bound should be considered with utmost caution, since both cost studies are based on completely different methods and also include different cost areas. In contrast to the Australian study , the GerIn both studies, prevalence is based on a population survey with similar results for the lifetime prevalence of physical and sexual abuse ).The Canadian result  is very With a loss of productivity of over 70% in the moderate case scenarios and over 50% in the pessimistic ones, the German cost-benefit-analysis  ranks clThe total costs in the United States  are consThe two results from Australia  and the a priori due to structural conditions.Generally, the comparison of the four aforementioned results of trauma follow-up costs is limited due to the time lag of altogether fifteen years between the individual studies, which have certainly influenced prices, services and their utilization. Additionally, national service organization and funding structures, e.g. of the health care systems, are fundamentally different . These vThe calculated amount of trauma follow-up costs clearly has economic relevance, constituting 0.44% (lower bound) and 1.20% (upper bound) of Germany’s 2008 Gross Domestic Product  . The figBasically, trauma follow-up costs were determined by following a conservative approach. This is reflected in several details, for example in the restriction to a 21% share of only severely affected cases ,10. ResuMoreover, total costs have only been taken into account for the age group from 15 up to including 64 years. Consequently, direct costs are only considered to a small extent and indirect costs of older ages remain completely excluded. With existing trauma-related disorders, it can be assumed that the age-related, increasing instability of life situation leads to further health problems, which again incur additional costs in higher age. In general, trauma-related disorders do not tend to decrease in higher age , but eldLast but not least it should be noted that types of traumatization other than sexual, physical and emotional abuse and neglect are left unconsidered in the present study, so that no statements can be made on their prevalence or on follow-up costs. However, it appears reasonable to limit the derivation of trauma follow-up costs to child abuse and neglect, since other current data are not available and international cost studies -58 referCTQ. Since trauma-related disorders tend to be more severe the earlier traumatization was experienced [When trying to estimate whether the true costs may tend towards the lower or the upper bound one has to keep in mind that the cost scenarios are based on early childhood traumatization, whereas the prevalence data include the entire childhood and adolescence as defined by the erienced ,70, thisThe international comparison supports both the lower and the upper bound of the cost margin – depending on the respective study. Due to methodological differences the results have to be interpreted rather as crude reference points, though. Despite all limitations, the comparison shows that the cost margin calculated for Germany is well associated with other countries’ results.Limitations associated with the use of already existing data are particularly given by the fact that these data have been collected with different objectives and are not well-matched. The question arises, in particular, to what extent the cost scenarios – determined under the assumption of child endangerment  – can beThe prevalence of traumatization has been determined by Häuser et al.  retrospeAnother significant uncertainty lies in the cost data themselves. The authors of the cost study explain in detail that due to the lack of data, several parts had to be completed by expert knowledge and international literature . The comThe problem of low availability and unsatisfactory quality of the data with regard to the estimation of trauma follow-up costs does not only exist in Germany but it is criticized in all cost studies -58. ConsRealizing numerous questions and imponderabilities in the assessment of results, creating a reliable data basis must be of highest priority in Germany, in order to answer the question how expensive it is not to provide sufficient and timely assistance to traumatized children and juveniles. The gathering of reliable cost data seems to be a highly challenging task in the light of an extremely fragmentary information basis. Serious efforts should therefore be undertaken to collect reliable data, in the first place. Only on the basis of results that are accepted by all sides due to their validity can steps be ground in order to sustainably improve the status quo of prevention and post-traumatic care.Starting points for the improvement of care and thus assumingly also for long-term cost savings are indicated in numerous literature sources, which, for example demand a stronger interconnectedness of the respective institutions ,73-77 orTotal costs of EUR 11.1 billion or EUR 29.8 billion, respectively, for the consequences of childhood traumatization by various types of severe child abuse as well as neglect are undoubtedly relevant for German economy. Considering the paucity of data, especially of cost data, the result cannot be seen without restrictions. Therefore serious efforts should be undertaken to generate reliable data, in the first place.Besides the question of personal suffering, political decision-makers should pay much more attention to the economic perspective of childhood traumatization and its comprehensive dimension of long-term consequences. By improving trauma-related care and prevention, the societal economic burden might be reduced.Nationales Zentrum Frühe Hilfen); PTSD: Post Traumatic Stress Disorder.CTQ: Childhood Trauma Questionnaire; NZFH: National Center of Early Assistance . Currently, SH is working on different health economics-related projects.SH has worked as a medical doctor before additionally graduating in Public Health. She specialized in DRG-based hospital reimbursement while working in the German DRG-Institute , and since 2010 he is deputy chairman of this board.JMF is child and adolescent psychiatrist and psychotherapist. He is Medical Director and founder of the Hospital for Child and Adolescent Psychiatry and Psychotherapy at the Ulm University Hospital. He is member of the Scientific Board for Family Affairs at the


High selection pressure on domestic cattle has led to an undesirable increase in inbreeding, as well as to the deterioration of some functional traits which are indirectly selected. Semen stored in a cryobank may be a useful way to redirect selection or limit the loss of genetic diversity in a selected breed. The purpose of this study was to analyse the efficiency of current cryobank sampling methods, by investigating the benefits of using cryopreserved semen in a selection scheme several generations after the semen was collected.The theoretical impact of using cryopreserved semen in a selection scheme of a dairy cattle breed was investigated by simulating various scenarios involving two negatively correlated traits and a change in genetic variability of the breed.Our results indicate that using cryopreserved semen to redirect selection will have an impact on negatively selected traits only if it is combined with major changes in selection objectives or practices. If the purpose is to increase genetic diversity in the breed, it can be a viable option.Using cryopreserved semen to redirect selection or to improve genetic diversity should be carried out with caution, by considering the pros and cons of prospective changes in genetic diversity and the value of the selected traits. However, the use of genomic information should lead to more interesting perspectives to choose which animals to store in a cryobank and to increase the value of cryobank collections for selected breeds. In its Global Plan of Action, the FAO [ex situ programmes to complement in situ conservation of animal genetic resources. It was also suggested that cryopreserved bio-specimens could be used as a backup material to redirect the selection scheme of a given breed, if needed [ex situ genetic resources to optimise the management of genetic diversity in endangered breeds [Within the context of farm animal biotechnologies, cryopreservation is one of the most useful tools for selection improvement, dissemination of genetic progress and  the FAO  recommenf needed ,3. Consef needed ,5 and med breeds . Breeds d breeds . In thesDepending on the country, different strategies have been implemented to sample individuals for national collections. In the Netherlands, most of the tested bulls are sampled for preservation in the gene bank , while iThe purpose of this study was to analyse the efficiency of current cryobank sampling methods by investigating the benefits of using cryopreserved semen in a selection scheme several generations after the semen was collected. Based on simulations, we examined two situations in which cryopreserved sperm was used (1) to redirect the selection goal, by including a trait which, in the past, had shown a negative correlated selection response (e.g. fertility in dairy cattle), and (2) to limit the loss of genetic diversity in the breed. The impact of using cryopreserved sperm was measured by estimating the evolution of two negatively correlated traits and the evolution of the breed's genetic diversity, assessed through pedigree information.A simplified cattle breed was simulated with 13 discrete generations, each consisting of 100 males and 10000 females. In each generation, 10 bulls and 50 cows were chosen as parents of the male progeny, and 20 bulls and 10000 cows were chosen as parents of the female progeny (with no selection on the dam to dam path). Mating was random resulting in random variation of progeny size among parents, i.e. the sire and dam of a given newborn were randomly chosen in the corresponding lists of parents.A and σB, respectively) was set to 1 and the correlation between traits (ρ) was set to -0.3.We considered two traits A and B. Trait A corresponded to a production trait which had been recently and intensively selected and improved (such as milk production in dairy cattle). Trait B corresponded to a functional trait which had deteriorated because of a negative correlation with trait A (e.g. fertility or longevity). The genetic standard deviation of each trait  distribution. For a given individual (i), the genetic value for trait B (iB) was generated from its value for trait A (iA):For each trait, an additive polygenic model was assumed and the simulation of correlated genetic values was based on the bivariate normal distribution . At geni is a N  random number independent of iA.where βi were simulated from the genetic values of its sire (p Aand pB) and its dam (m Aand mB), taking into account the parent's coefficients of inbreeding  [In the following generations, genetic values of individual , resp.) ,13:i and δi are two numbers randomly drawn from a N  bivariate normal distribution with a correlation equal to ρ.In these equations, γCD = square of the correlation between the EBV and the true genetic value) equal to 0.6 for bulls and 0.4 for cows, whatever the trait and the generation considered. Therefore, the EBV of a given individual for trait A (iEBVA) and for trait B (iEBVB) were computed as follows:EBV were directly simulated from genetic values, assuming an evaluation procedure leading to an accuracy  distribution. Finally, a Total Merit Index (iTMI) was computed, weighting the two EBV by A wand B w= 1 - Aw, respectively:where εSimulations comprised two stages. During stage 1 (generations 0 to 8), the lists of parents were selected based on their EBV for trait A only, without considering the evolution of the genetic mean for trait B or the average coefficient of inbreeding. During stage 2 (generations 9 to 12), the bulls were also used to improve trait B or to introduce genetic diversity in the breed.During stage 1, the semen of some bulls was sampled and cryopreserved if the animals fulfilled one of the three following conditions, which correspond to the current sampling rules of the French National Cryobank for type "II"  :EBVA is three standard deviations above or below the mean of the generation,- (i) EBVB is two standard deviations above the mean of the generation ,- (ii) - (iii) the bull is a sire of sires with no male offspring selected after the evaluation process .To check the validity of this elaborate sampling method, we tested a simpler sampling method (similar to the one used in the Netherlands), where the semen of all young bulls is stored in the cryobank.In the simulations performed here, we investigated the impact of a one-time use (i.e. during a single generation) of cryopreserved semen.i TMIor (ii) they have the lowest average kinship with the existing population . We studied the impact of various selection orientations  only on the male path, because applying the above conditions on the female path would be much more restrictive, less effective, and would require a larger amount of semen, all the more since the number of doses is generally limited in cryobanks  [At generation 9, four bulls with cryopreserved semen were selected (hereafter referred to as 'cryobank bulls'), these bulls fulfilling one of the following conditions either (i) they are the best cryobank bulls for the th generation of young bulls was then generated using either the bulls from the cryobank or the group of 10 sires selected as described in previous sections. Depending on the scenario (see following section), 0, 40 or 80 individuals  were sired randomly by one of the four selected cryobank bulls.For these reasons, we considered that cryobank bulls were used only to procreate young bulls for progeny testing. The 9Six simulation scenarios were completed with two main options Table .Firstly, in scenario "b", emphasis was put on the selection of both traits B and A. To achieve this goal, three methods were compared:TMI (B w= 0.5) were used to sire 40% of the young bulls, while the selection criterion during stage 2 remained unchanged (improving EBVA). The other young bulls were sired by bulls randomly sampled within the group of 10 sires;- b1: at generation 9, the four bulls with the highest TMI (B w= 0.5) was used as the selection criterion instead of EBVA;- b2: at generation 9, no cryobank bull was used, and during stage 2, TMI were used to sire 40% of the young bulls, and during stage 2, TMI was used as the selection criterion instead of EBVA. To test more or less drastic selection changes, scenario b3 was tested with an increasing weight given to trait B (B wincreasing from 0.5 to 1).- b3: at generation 9, the four cryobank bulls with the highest Secondly, in scenarios "d", emphasis was put on genetic variability while trait A remained the breeding goal. Three methods were also compared:- d1: at generation 9, the four cryobank bulls having the lowest kinship with the existing population (scenario b1) were used to sire 40% of the young bulls;EBVA became the sires of dams and that with the best EBVA became a sire of sires;- d2: at generation 9, no cryobank bull was used, while during stage 2, the progenies on the sire to sire path were given the same size i.e. for each sire of sires, 10 male offspring were created among which those with the two best - d3: at generation 9, the four cryobank bulls having the lowest kinship with the existing population (scenario b1) were used to sire 40% of the young bulls, while during stage 2, selection was used to equalise progeny sizes on the sire to sire path.Simulations were performed with 1000 runs for each scenario. For each generation, individual inbreeding coefficients and genetic values were computed and averaged for the entire male and female populations. The individual coefficients of kinship were also computed and averaged over males only and over the entire populations. The proportion of genes originating from cryobank bulls was computed on the basis of the gene dropping procedure (one locus averaged over the 1000 runs).As expected, the results of the different scenarios did not differ significantly for generations 0 to 8 given that in stage 1, the conditions were the same whatever the option chosen, (here we present results averaged over the 1000 runs of one scenario only). With the parameters chosen for the simulation, each sire of sires had on average 10 male offspring (across sires standard deviation s.d. = 2.9) and each sire of dams had on average 500 female offspring (across sires s.d. = 21.6). As expected  had a temporary impact on traits A and B as well as on the diversity indicators of the young bulls. At the whole population level, the impact was negligible, since young bulls sired by cryobank bulls were rarely subsequently selected as sires: three generations after introduction (generation 12), the cryobank contribution to genetic diversity was less than 3% , without using cryobank bulls (scenario b2), there was a per generation increase in the mean of trait B from generation 9 on (b1: -0.3 vs b2: +0.4), while the genetic gain for trait A decreased  resulted in a slight but significant (P < 0.001) reduction in average kinship , while the genetic gain for trait B increased slightly . These tendencies increased slightly when 80% of the males from generation 9 were sired by cryobank bulls , while average kinship reached 8.9% at generation 12 (versus 11.9% when B w= 0.5).As a result of the increased weight of trait B within Φ = 12% for scenario d1 and Φ = 11% for scenario d2), with an almost negligible impact on genetic progress. Combining this option with the introgression of cryobank bulls (scenario d3) resulted in a significant reduction in average kinship (-2% in comparison to d1). Under such a scenario, the genetic mean of trait B also increased slightly , while that of trait A and the average of both traits decreased slightly . It should be noted that most of the cryobank bulls used originated from the founder population, their average birth generation being 0.3 . It should be noted that in this case, the average birth generation of the cryobank bulls used was 7, in scenario b3 , and 0, in scenario d3 .In this study, we assessed the impacts of using cryopreserved bull semen either to redirect selection or to improve the genetic variability of a selected cattle breed. Simulation parameters were chosen as a compromise between realism in the scenarios, their applicability, and the simplicity of the model. For instance, with respect to the choice of population size, a breed with 20 breeding males and 10000 potential dams could be considered quite small, especially with reference to the FAO endangerment status . In our Concerning sampling conditions in the simulations, as mentioned above, the procedure chosen to select bulls for cryopreservation is similar to that currently applied in France. This choice was made to test if bulls selected this way could be effectively used in a selected breed. Compared to the case in which all young bulls are sampled for cryopreservation (which corresponds more or less to the current procedure in the Netherlands), the results were basically the same. This shows that the French sampling procedure is reasonably efficient to select useful bulls, and could be applied in situations when only a limited number of semen samples can be stored in a cryobank .One of the main conclusions of this study is that using cryopreserved semen is relevant for a breed for which major changes in selection objectives or practices are considered. Since genetic progress is rapid in dairy cattle breeds (e.g. ), a bullIf the objective is to introduce genetic diversity into the breed, using cryobank bulls appears to be a valid choice. However, it is imperative that other measures are also taken to guaranty that genes are spread within the breed i.e. either conserving male lines (scenario d3), when their use is promoted among breeders, or setting up more restrictive and effective breeding schemes. Several methods of varying complexity have been proposed to minimise kinship , or to mOn the one hand, decreasing inbreeding in a selected breed may improve selected traits; for instance, it has been shown that in Holstein cattle, milk production (over 305 days) can decrease by about 20 litres per 1% inbreeding increase . On the Based on our results, using semen from cryobank bulls should be useful either to introduce drastic changes in selection goals or to reintroduce genetic diversity within a given population. However, it is important to carefully assess the pros and cons of the potential changes in genetic diversity and values of the selected traits.Our simulations were based on a classic quantitative selection scheme. Recent progress in genomic tools should make it possible to identify semen from cryobank bulls that share specific alleles or QTL of interest for selection. This could then be taken into account when choosing cryobank bulls as well as how they will be used. Using such reproducers should be investigated in further studies, which opens exciting perspectives for an improved exploitation of cryobank collections.The authors declare that they have no competing interests.EV, CDB and GL jointly conceived the design of the study and discussed the results. GL wrote and checked the simulation program. GL wrote the first draft of the manuscript, which was then modified by CDB and EV. All authors read and approved the final manuscript.Changes in genetic values (a) and in genetic diversity (b) (scenario b2). The data represent the simulation results for scenario b2. Dotted lines: young bulls; solid lines: whole population; red: trait A; blue: trait B; green: average between A and B; purple: inbreeding F; pink: kinship Φ.Click here for fileChanges in genetic values (a) and in average kinship (b) when trait B was added to selection goals. The data represent the simulation results when selection is redirected with a new trait accounting for 50% of the total merit index and when the use of semen from cryobank bulls is increased. Scenario b3 and whole population are considered with the weight B wgiven to trait B accounting for 50% of the total merit index and an increased use of the semen from cryobank bulls. Brown: no cryobank bull is used (scenario b2); red: cryobank bulls are used to produce 40% of sons (scenario b3); yellow: cryobank bulls are used to produce 80% of sons; o: genetic value for trait A; ♦: genetic value for trait B; dotted line: average genetic value between A and B; x: kinship Φ.Click here for file


To the Editor: Birge and Sonnesyn report the first death of a Minnesota resident caused by Powassan virus (POWV) (1]) .The 17 POWV infections detected in Minnesota residents from 2008 through 2011   were found through enhanced surveillance. Health alerts to Minnesota medical providers described POWV as a possible etiologic agent for viral meningitis and encephalitis. Providers consulted with MDH on suspected cases and submitted serum and cerebrospinal fluid specimens to MDH. MDH conducted serologic testing for endemic arboviruses (including POWV) and reverse transcription PCR (RT-PCR) for flaviviruses and POWV. MDH would not have detected any POWV infections without enhanced surveillance. Limited field studies also identified POWV-infected ticks in 4 Minnesota counties  is preferred (Ixodes scapularis ticks (blacklegged ticks), the tick species associated with deer tick virus transmission, and viruses from all POWV-positive tick pools were confirmed as deer tick virus by sequencing. The distribution of the 2 lineages in North America is poorly understood, and most cases likely go undetected without specific POWV surveillance efforts.Few POWV infections are identified by lineage (prototype vs. deer tick virus); Minnesota’s first case in 2008 was identified as a deer tick virus infection, but the lineage was unknown for the other 16 cases. However, many case-patients had likely exposure to


To analyze the validity and feasibility of the Juvenile Arthritis Disease Activity Score  (JADAS)-1124 visits in 255 JIA patients were evaluated. Correlations of JADAS-27 (range 0-57) with JADAS-10, JADAS-71, core-set criteria, Disease Activity Index (DAS-28), and Clinical Disease Activity Index (CDAI) were analyzed in the total group and treatment groups.Responsiveness to minimal clinically important difference (MCID) was analyzed longitudinally. Worsening was defined as a flare with at least 20% PGA and 15% ESR increase, as well as starting therapy, or drug dosage escalation. Improvement was defined as reaching ACRped50 or as reduction of medication.The distribution of JADAS-27  was skewed to the left. The JADAS-27 score could not be calculated in 261 visits (23%), due to missing parent/patient global assessment of well-being (n=119) or missing ESR (n=157). The JADAS-27 correlated highly with JADAS-71 (R=1.00), JADAS-10 (R=1.00) and CDAI (R=0.98), moderately with core set criteria (R=0.65 to 0.85) and DAS-28 (R=0.63), and poorly with ESR (R=0.30). JADAS-27 was higher in patients using MTX , and in patients prior to starting anti TNF-alpha treatment .The minimal clinically important improvement was represented by a median change in JADAS of -5.2 (IQR= 6.6), and deterioration by +3.4 (IQR= 4.3). Improvement was detected in 95% and worsening in 74% of visits.. However, the high frequency of missing values in the criteria that are needed to calculate the JADAS-27, may hamper its feasibility.The JADAS-27 is a reliable tool for assessing JIA disease activity given its high correlation with other disease activity parameters, its markedly higher scores in patients requiring biologicals, and its ability to detect MCID in the majority of patients


Discovery of new viruses has been boosted by novel deep sequencing technologies. Currently, many viruses can be identified by sequencing without knowledge of the pathogenicity of the virus. However, attributing the presence of a virus in patient material to a disease in the patient can be a challenge. One approach to meet this challenge is identification of viral sequences based on enrichment by autologous patient antibody capture. This method facilitates identification of viruses that have provoked an immune response within the patient and may increase the sensitivity of the current virus discovery techniques. To demonstrate the utility of this method, virus discovery deep sequencing (VIDISCA-454) was performed on clinical samples from 19 patients: 13 with a known respiratory viral infection and 6 with a known gastrointestinal viral infection. Patient sera was collected from one to several months after the acute infection phase. Input and antibody capture material was sequenced and enrichment was assessed. In 18 of the 19 patients, viral reads from immunogenic viruses were enriched by antibody capture . Enriched reads were also determined in an identity independent manner by using a novel algorithm Xcompare. In 16 of the 19 patients, 21% to 100% of the enriched reads were derived from infecting viruses. In conclusion, the technique provides a novel approach to specifically identify immunogenic viral sequences among the bulk of sequences which are usually encountered during virus discovery metagenomics. Virus infections are a continuous threat to the human population; e.g. HIV, hepatitis viruses, and influenza viruses constitute a large proportion of morbidity and mortality each year. Apart from infection with well-described viruses, outbreaks with previously undescribed viruses occur regularly  Discovery of new viruses in the last decade has been boosted by large improvements in sequencing technology. These methods form the basis for improved virus discovery processes capable of generating 10e5–10e7 sequence reads directly from a clinical sample. A virus discovery method to amplify RNA and DNA virus sequences directly in patient material (VIDISCA-454) without prior knowledge of the viral genome sequence has been developed One limitation of the current technique is that a substantial amount of non-viral RNA and DNA derived from the host or from other agents in the sample can dominate the resulting sequences. Especially in respiratory samples, ribosomal RNA is massively present, over 80% of all sequence reads derived from a clinical sample originate from this material Another limitation of the current techniques is that detection of reads derived from a known virus does not necessarily indicate that this virus is a pathogen. Recently, many new viruses have been identified in human samples without clear association with disease, necessitating further detailed investigations to determine the pathogenicity of the virus To facilitate the detection of immunogenic viruses and to reduce the detection of non disease-related viruses (bacteriophages and plant viruses) and host cellular RNA, a technique was developed that uses convalescent serum of the patient to concentrate viruses that have elicited and immune response prior to sequencing. Comparing the sequences derived from input and antibody captured material identifies immunogenic agents and can provide an important first step in identifying a disease-related virus.Respiratory samples were collected during the GRACE study. Flocked nasopharyngeal swabs (Copan) were collected in universal transport medium (UTM). In addition, a single nasopharyngeal specimen was obtained at the Academic Medical Center from a patient with an upper respiratory tract infection.. Fecal samples were suspended in broth .Fecal samples were selected from a sample bank from 196 HIV-1-infected patients with and without diarrhea, aged above 18 who visited the out-patients clinic at the Academic Medical Center in the years 1994–1995Ethics review committees in each country approved the study, Cardiff and Southampton (United Kingdom): Southampton & South West Hampshire Research Ethics Committee A; Utrecht (Netherlands) Medisch Ethische Toetsingscommissie Universitair Medisch Centrum Utrecht; Barcelona (Spain) Comitè ètic d'investigació clínica Hospital Clínic de Barcelona; Mataro (Spain): Comitè d'Ètica d'Investigació Clínica (CEIC) del Consorci Sanitari del Maresme; Rotenburg (Germany) Ethik-Kommission der Medizinischen Fakultät der Georg-August-Universität Göttingen, Antwerpen (Belgium): UZ Antwerpen Comité voor Medische Ethiek; Lodz, Szeczecin, and Bialystok (Poland): Komisja Bioetyki Uniwersytetu Medycznego W Lodzi; Milano  IRCCS Fondazione Cà Granda Policlinico; Jonkoping (Sweden): Regionala etikprövningsnämnden i Linköping; Bratislava (Slovakia): Etika Komisia Bratislavskeho; Gent (Belgium): Ethisch Comité Universitair Ziekenhuis Gent; Nice (France) Comité de Protection des Personnes Sud-Méditerranée II, Hôpital Salvator; Jesenice (Slovenia): Komisija Republike Slovenije za Medicinsko Etiko. Written informed consent was provided by all study participants.Collection of fecal material was performed in accordance with the ethical principles set out in the declaration of Helsinki and written informed consent has been obtained prior to data collection. The study was approved by the Amsterdam Medical Center institutional medical ethics committee.Respiratory and fecal samples were centrifuged  and 150 µl of the supernatant was mixed with 50µl Dynabeads protein A and G . After 20 minutes incubation, 10 µl of autologous convalescent serum of the patient was added to the mixture . After aReal time PCR for ribosomal RNA was performed as described 7 ng/µl. Samples were pooled and Kapa PCR (Kapa Biosystems) was performed to determine the quantity of amplifiable DNA in each pool. Subsequently, the Bioanalyser  was used to determine the average nucleotide length of the libraries and the pools were diluted until 106 copies/µl to be used for a titration  in an emulsion PCR according to the suppliers’ protocol (LIB-A SV emPCR kit). Sequencing was done on a 2 region GS FLX Titanium PicoTiterPlate (70×75) with GS FLX Titanium XLR 70 Sequencing kit (Roche).VIDISCA-454 was performed as previously described http://blast.ncbI.nlm.nih.gov/Blast.cgi) tool Primers, MIDs and ribosomal RNA sequences were trimmed from the reads. Sequences were compared with all available sequences in the nonredundant Genbank database The percentage of viral sequences was calculated by dividing the number of virus derived reads in the sample by the total numbers of reads in the same sample. The enrichment index was calculated by dividing the percentage of viral sequences in the captured sample by the percentage viral sequences in the input sample. A value above 1 indicates antibody capture of the virus, which suggests a immunogenic course of infection.xcompare (source available on request). The algorithm is comprised of the following steps: the identification of identical or nearly-identical sequences within the input subset was performed by creating a custom BLAST database To identify sequences which are enriched by antibody capture, input and enriched sequences were compared using a Python algorithm The second BLAST analysis yields the percentage of sequence space occupied by each fragment in the original sample  and the enriched sample . The ratio between these percentages is calculated and all fragments with a ratio higher than 1.0 were extracted and further analyzed.Serum collected a few weeks to a few months after respiratory or gastrointestinal infection generally contains a substantial amount of pathogen-specific immunoglobulin type G (IgGs) with a proportion of these antibodies binding to virus surface exposed epitopes Of the 13 respiratory samples and the 6 fecal samples, a total of 110,752 reads were obtained from the input material and 93,779 reads were obtained from the antibody captured material with a median read length of 159 nucleotides. For each patient the enrichment index was determined by calculating the ratio between the percentage of viral reads in the captured sample versus the input sample .For all respiratory samples the enrichment index was above 1.0, indicating that in every sample tested the number of viral reads increased after capture. The human coronaviruses OC43, NL63 and 229E were detected with an enrichment index of 169, 343 and 23 respectively. In a second NL63 case (patient 6), the virus was enriched 20-fold. The para-influenza viruses 1, 2 and 4 infections were detected with an enrichment index of 1.5, 54 and 19 respectively. The enrichment index for the metapneumovirus was 7.2, while it was 1.6 for the respiratory syncytial virus. Human rhinovirus infection was observed in two different patients and the enrichment index was 1.9 and 3.1 respectively. Influenza B virus was detected with an enrichment index of 3.3 and influenza A virus with an enrichment index of 25.Fecal samples were analyzed by the same process. Norovirus infection was observed in two patients and enriched 20-fold and 11-fold. Adenovirus was also detected in a patient with norovirus infection and no enrichment for adenovirus was scored (0.9). Enterovirus reads were detected with an enrichment factor of 5.2 in one case but enterovirus reads were not enriched (enrichment factor of 0.0) in a second case. Viral reads derived from sapovirus were detected in samples from two patients with an enrichment factor of 4.9 and 1.4 respectively. Hepatitis B viral reads were detected in patient 19 with an enrichment factor of 53. As controls, a plant virus (cucumber mosaic virus) and an enterobacteriophage were tested. These viruses had an enrichment index of 0.08 and 0.01 respectively, indicating that there presence was reduced in the captured material.Consistent with capture working as anticipated, reads derived from human ribosomal RNA showed a consistent reduction in the antibody captured fractions with an average decrease of 1,000 fold . Reads present in higher quantity in the captured samples compared to the input samples were selected. Reads originated from archaea, bacteria or eukaryota were excluded and the remaining reads, , were further investigated.EAR values ranged from 0 to 3019 . In respA new method is described that facilitates virus identification by enriching viral material with convalescent autologous patient antibody capture followed by deep sequencing. For all samples tested here, an increase in percentage of viral reads was detected when compared to the input sample. In 9 out of the 21 viruses the amount of viral reads after capture increase 10-fold or more.By using the Xcompare script, enriched reads in the antibody captured sample could be determined in an identity independent manner. In 11 out of the 13 patients, between 98% and 100% of the enriched analysis reads were of mammalian viral origin. In fecal samples between 21% and 100% viral reads were detected in the EAR in 4 out of 6 cases. Surprisingly, in two patients with enterovirus infection, no viral reads were detected in the EAR.Of note are patient 1 and 2. For both patients viral reads were increased after capture , but theAlso enteroviruses were poorly captured. A possible explanation might be that the bacteria or bacteriophage background in fecal samples is high which makes it more difficult to enrich a virus with a relatively low load. Nonetheless, we were able to reduce the amount of reads originating from bacteriophages and plant viruses (enrichment index of 0.01 and 0.08), indicating that this method can efficiently reduce the background in fecal samples. Furthermore, it could be that the immune response to enteroviruses is poor, especially in immunocompromised patients, which is the case in our study patients with diarrhoea. These patients were HIV-1 positive with relatively low CD4 counts (0.18 and 0.05×10E9 cells/L in the two patients with enterovirus infection). On the other hand, it has been published that HIV-1 infected patients are not hampered in their immune response to enteroviruses, and chronic shedding is as rare as in the normal population Antibody response to a virus infection can vary, depending on the virus, but also age and immune state of patients. Also, antibodies subclasses can differ; some viruses elicit a strong IgG response, while others induce a response dominated by IgA. In our antibody capture experiment, a combination of protein A and G coupled beads was used. Both proteins strongly bind to IgGs, but not or less efficient to IgA. It has been shown that serum antibodies, for instance against PIV-1 are not neutralizing efficiently, and that especially secretory IgA plays an important role in preventing re-infection To date many new viruses have been identified In summary, a new selective approach for virus discovery is presented that enables detection of viruses that have recently elicited an immune response resulting in antibody production. The method appears to be an effective tool for reducing host DNA/RNA and bystander virus sequences and provides a selection for viruses that are able to elicit an antibody response, thus agents that are possibly pathogenic for the host. Moreover, the possibility of comparing reads obtained after antibody capture with reads obtained from the input samples is useful for identifying sequences not yet labelled in Genbank. Further analysis of those sequences  will allow the identification and characterization of viruses which are highly divergent from the currently known virus families.