Embryonated chicken eggs (ECE) are sometimes used for the primary isolation or passage of influenza viruses, other viruses, and certain bacteria. For small-scale experiments with pathogens that must be studied in biosafety level three (BSL3) facilities, inoculated ECE are sometimes manipulated and maintained in small egg incubators within a biosafety cabinet (BSC). To simplify the clean up and decontamination of an egg incubator in case of egg breakage, we explored whether ethylene breather bags could be used to encase ECE inoculated with pathogens. This concept was tested by determining embryo survival and examining virus yields in bagged ECE.Virus yields acceptable for many applications were attained when influenza-, alpha-, flavi-, canine distemper-, and mousepox viruses were propagated in ECE sealed within ethylene breather bags.For many small-scale applications, ethylene breather bags can be used to encase ECE inoculated with various viruses. Rickettsia [Embryonated (embryonating) chicken eggs (ECE) have long been used for isolating or propagating influenza and other viruses and certain bacteria such as ckettsia -5. AlphaNo differences were detected in the survival of chicken embryos in bagged vs non-bagged 7 - 12 day old ECE after five days of incubation without rotation as performed for virus-inoculated ECE. Noteworthy, especially during summer months, up to 20% attrition (death of non-inoculated ECE) occurred with some batches, regardless of whether the ECE were bagged or not bagged. Since the ECE are checked and culled if dead upon receipt from the supplier and again immediately prior to use, the deaths have been attributed to failure to thrive under normal circumstances. Since the ECE are not rotated, a factor contributing to attrition may be attachment of the embryo to the egg-shell and its subsequent deleterious deformation/improper development.50) in Madin-Darby canine kidney (MDCK) cells and 50% egg infectious dose (EID50) in ECE of two influenza viruses strains grown in the chorioallantoic sac (CAS) of ECE  are given in Table 50/ml) obtained for various other influenza A and B viruses are given in Tables Various type A and B influenza viruses were grown to levels acceptable for our applications in ECE in ethylene breather bags. It was not necessary to add water to humidify the interiors of sealed bags. Compared to bags containing eggs without extraneously added moisture, virus yields and embryo development were similar when up to one ml of sterile water or a moistened filter were placed with eggs in bags (data not shown). Virus growth occurred regardless of the inoculation route/site and storage orientation  of the egg (data not shown). An example of a virus-inoculated egg in a breather bag is shown in Figure Canine distemper virus (CDV) strain Lederle (American-1 lineage) obtained from the American Type Culture Collection  grew readily in bagged ECE, evidenced by RT-PCR detection of CDV RNA in isolated chorioallantoic membrane (CAM) five days post-infection (p.i.). Changes in the general appearance (of the CAM) were also visible without staining and microscopic evaluation of isolated CAM. In contrast, wild-type CDVs from canine specimens required two to three serial passages before facile detection . Titers of 106 - 107 pfu/ml were obtained from isolated CAM with CDV-Lederle upon first passage with a starting inoculum of 102 pfu/ECE in both bagged and non-bagged ECE, whereas 101 - 102 pfu/ml were obtained with wild-type American-2 lineage CDVs  in bagged and non-bagged ECE. Similarly, after three serial passages of wild-type CDVs with a starting inoculum of 102 pfu/ECE in either bagged or non-bagged ECE, the yields of cell-associated virus (CAM-associated virus) were around 103 pfu/ml, and were not detectable to 101 pfu/ml for CDV isolates in allantoic fluid (details to be presented elsewhere).Egg-adapted "Pocks" were visible on the CAM of bagged ECE at 3 days post-infection (data not shown).3 pfu of VEEV-Td per ECE. Virus yields in bagged ECE were: CAM > brain (head) > body > allantoic fluid > yolk sac (YS) membrane. Virus yields from homogenized CAM were generally around 3 × 108 pfu/mL whereas homogenized brain tissue yielded about 4 × 107 pfu/mL as measured by plaque assays in African green monkey kidney (Vero) cells. Though a direct comparison with VEEV-Td grown in non-bagged ECE was not allowed due to biosafety rules imposed by our institute, the virus yields from the CAM are within the expected range for alphaviruses grown in ECE based on historic data [Chicken embryos were usually killed (by the virus) within 24 hrs after infection with 103 pfu of JEV-Nak per ECE. Virus yields in bagged ECE were: brain (head) > body > allantoic fluid > CAM > YS membrane. Virus yields from homogenized brain tissue were generally around 4 × 105 pfu/mL whereas homogenized body tissue yielded about 4 × 104 pfu/mL by plaque assay in Vero cells. A direct comparison of our stock of JEV-Nak grown in non-bagged ECE was not allowed due to biosafety rules imposed by our institute.Chicken embryos usually died between 48 - 72 hrs after infection with 10E. coli when the bags were immersed in flasks containing the bacteria for 24 hrs (starting out with log-phase bacteria). In contrast, the phage infected E. coli when pin-holes were made in the bags prior to immersion in flasks containing log-phase bacterial cultures. Though exhaustive tests were not performed, chicken embryos past seven days of development did not thrive in various other types of plastic bags.Various viruses were successfully cultivated in ECE in ethylene breather bags. No defective bags (defined as bags with obvious holes) were observed during this work. Whereas additional safety is inferred since the gas-permeable ethylene bags retain water , this has not been extensively tested at MRI. However, a preliminary test supports this thesis: T4 phage inside intact bags did not infect 6 to 109 pfu/ml (infectivity measured in MDCK cells) are common for primary or low-passage virus isolates provided contaminating microbial flora normally present in clinical specimens are suppressed by antibiotic treatment. The variability in virus yield is also dependent on viral strain. The growth and yield of other viruses  in bagged ECE was also consistent with expectations. Whereas some JEV strains that have been adapted for growth in ECE attain higher viral yields than was attained here, it is not considered aberrant to attain the lower yields with fresh or low-passage JEV stocks. Similarly, wild type CDVs may require adaptation and serial passage in ECE before vigorous growth is detected [Influenza virus yields in bagged ECE were consistent with expectations; for example, titers within the range of 10For small-scale operations using relatively few ECE for the propagation of BSL3 pathogens, breather bags are convenient for containing spillage from broken eggs. Use of the bags for the application described here offers one solution over the need to purchase or construct additional barrier and containment enclosures. There are two drawbacks: bagging ECE imposes additional labour constraints, and candling of the eggs through the bags can be challenging especially when numerous eggs are present in a bag. However, this technique is only intended for small-scale experiments requiring few ECE. The same idea may be useful for tissue culture plate systems since they have the same problem if dropped; i.e. as an added safety feature.For larger-scale needs, other primary containment devices could achieve the same goal of increased containment such as tradition egg incubators placed inside a primary containment device such a BSC or high-efficiency particulate arrestor (HEPA) - filtered vinyl enclosures. One alternative would be the use of an egg incubator with removable self-sealing modular containers for eggs. A large stand alone incubator could thus be positioned somewhere outside the BSC, and inoculated ECE safely transported in and out of the BSC and the containers with ECE repositioned into designated racks in the incubator. Midwest Research Institute is currently building such a device.Mousepox virus, JEV-Nak, and VEEV-Td as a precaution to contain possible spillage from broken eggs. We predict these bags will work for ECE infected with other viruses as well, and may be useful for tissue culture plate systems as well. Intended for small scale projects, the procedure is not practical for medium- to large-scale operations.For small-scale operations, ethylene breather bags can be used to enclose ECE infected with influenza viruses, CDV, Ethylene breather bags were obtained from Kordon LLC . For up to three ECE, 5.5 × 8 inch bags were used, whereas 7.5 × 12 inch bags were used for small-batch cultivation of up to six ECE inoculated with virus.Chicken anemia virus (CAV)-free ECE were obtained from Charles River Laboratories .Specific pathogen-free (SPF) Various methods for the growth of influenza viruses in ECE were tested. The primary method was by inoculation of the CAS. Amniotic sac (AS) inoculation (solely or in combination with CAS inoculation), YS, and CAM routes of inoculation were also tested for some virus strains. Inoculation into the AS is technically demanding and primarily reserved for isolating influenza viruses from humans when standard methods are shown to be insufficient or for high-priority efforts. Inoculations into the YS and CAM are performed for the primary isolation of some avian influenza viruses from non-chicken species and some swine influenza viruses [, and J. Top- side inoculation of the AS was performed by: (a) direct inoculation while candling to guide the needle beside the embryo, or (b) by sending the needle through the natural air sac until the needle touched the embryo; upon contact with the embryo, the needle's opening was in the AS. Sideway inoculation of the AS was also evaluated after first creating a false air-space beside the embryo. Similar manipulations were used for YS and CAM inoculations. The incubation period varied according to virus type and strain; in general, up to 24 hrs were used for highly pathogenic H5N1 viruses, 48 - 72 hrs (or longer) for seasonal and type B influenza viruses.Post-inoculation, the eggs were placed in ethylene bags, the top part of the bag rolled over individual or multiple eggs, and the rolled plastic secured using a rubber band, paper clamp, or laboratory tape. Attempts were made to collect virus just before death of the embryo. Prior to harvest, the eggs were refrigerated for up to 18 hrs to kill the embryo and constrict the blood vessels (and thus reduce bleeding into the AS during harvest). Virus was then harvested as appropriate to the inoculation site and aliquots of the virus stored at -80°C for up to one year or in liquid nitrogen for long-term (>one year) storage. Influenza virus genomic sequences were analyzed by isolating viral RNAs from allantoic fluid  and performing two-step reverse transcription-PCR with synthetic universal and other oligonucleotide primers ,13. The Egg-adapted CDV-Lederle was purchased from the ATCC. Six to eight day old ECE were used, with inoculation to the CAM. The inoculated eggs were observed daily to monitor embryo viability, and chilled to 4°C after 5 days' incubation and the CAMs harvested and homogenized to a 10% w/v suspension in phosphate-buffered saline with 0.5% w/v purified BSA fraction V. The homogenate was clarified with a low speed spin for 10 min at 4°C, and the supernatant used either as an inoculum for the succeeding passage or stored at -80°C. Wild-type CDVs were isolated from lung and/or brain homogenates of dogs with distemper (data to be presented elsewhere). CDV isolates were analyzed by RT-PCR and nucleotide sequence analyses as previously described ,15, and Mousepox virus in PBS (0.1 - 0.5 ml) were inoculated onto the CAM of 10 - 12 day old ECE. The ECE were incubated for 3 days at 37°C; virus growth was evidenced by the presence of "pocks" on the CAM [50) were determined in BSC-1 cells.Suspensions of  the CAM . Viral tVEEV was inoculated into the YS of 6 - 8 day old ECE, and incubated at 35 -37°C for up to 24 hrs. The propagated virus was analyzed by plaque assay and full-genomic sequencing (details to be presented elsewhere).JEV was inoculated into the YS of 8 - 9 day old ECE, and incubated at 35 -37°C for 48 - 72 hrs. The propagated virus was analyzed by plaque assay and full-genomic sequencing (details to be presented elsewhere).MDCK and Vero cell lines were obtained from the ATCC, or from Diagnostic Hybrids, Inc. . The cells were propagated in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with L-Alanyl-L-Glutamine , antibiotics [PSN: penicillin, streptomycin, neomycin . The MDCK and Vero cells tested negative for mycoplasma DNA using a Takara PCR Mycoplasma Detection kit .In-vitro experiments with H5N1 viruses, JEV, and VEEV, and their cultivation in ECE were conducted in an USDA-approved BSL3-enhanced (BSL3+) containment facility.2.Viruses were grown in MDCK cells in serum-free DMEM media supplemented with bicarbonate, antibiotics, and 1.0 μg/mL L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)- treated, mycoplasma- and extraneous virus-free trypsin  at 34 - 37°C (as appropriate for each virus strain) in 5% CO50 values were calculated for influenza viruses and Mousepox virus by the Reed-Muench method [Mousepox virus for 4 days in BSC-1 cells.TCIDh method . For theStandard plaque assays using agarose overlays were used to determine JEV and VEEV titers in Vero cells .The authors declare that they have no competing interests.SBH grew influenza viruses, JEV, and VEEV, interpreted data, helped train technicians, and helped draft the manuscript; DED grew influenza viruses, interpreted data, oversaw the training of technicians, and managed the influenza virus programs; WAS grew JEV, VEEV, and influenza viruses, and interpreted data; ERJ is the MRI biosafety officer and recommended evaluation of methodologies that might reduce biohazards stemming from broken virus-inoculated ECE; JMO grew JEV and VEEV; KSM grew JEV and VEEV and managed alpha- and flavivirus programs; MDH performed molecular genetic studies including sequence analyses and alignments; JOR performed JEV and VEEV studies; JAL conceived of using ethylene breather bags for this application, isolated CDV, participated in molecular genetic studies and sequence analyses, interpreted data, oversaw the training of technicians, and drafted the manuscript. All authors read and approved the final manuscript.


Eidolon helvum (37%), Epomophorus gambianus (3%), and Epomops buettikoferi  from Ghana. These findings confirm the presence of LBV in West Africa.To investigate the presence of Lagos bat virus (LBV)–specific antibodies in megachiroptera from West Africa, we conducted fluorescent antibody virus neutralization tests. Neutralizing antibodies were detected in  Lyssavirus is differentiated into 7 genetically divergent genotypes: classical rabies virus (genotype 1), Lagos bat virus , Mokola virus , Duvenhage virus (genotype 4), European bat lyssavirus (genotypes 5 and 6), and Australian bat lyssavirus (genotype 7) , given a seroprevalence of 5% and assuming random sampling (Bats were sampled in January and May 2007 at 6 sites in Ghana: the center of Accra (urban habitat); the wooded outskirts of Accra (savannah habitat); and forested habitats at Pra, Kibi, Adoagyiri, and Oyibi (a plantation with woodland/forest border). Bats were captured by using 6–18-m mist nets; roosting Epomophorus gambianus and E. helvum, were caught in sufficient numbers  for reasonable inferences to be made about LBV seroprevalence rates  by using a standard fluorescent antibody virus neutralization (FAVN) test  than in E. gambianus . Of 6 Epomops buettikoferi, 2 were seropositive . No sex differences in E. helvum seroprevalence were evident .Levels of specific virus neutralizing antibodies against LBV were higher in E. helvum, we attempted to determine a possible case reproduction rate (R0) for LBV infection in this species by using the equation R0 = 1/x*, where x* = proportion of susceptible hosts in a population (0 = 1.6 (95% CI 1.3–2.0).Because of the high level of seropositivity in E. helvum bats in Ghana. Previous studies have suggested that healthy bats develop antibodies to other lyssavirus infections  were apparently healthy pregnant females. These results support theories that lyssaviruses are endemic within specific bat populations, that they may not cause high mortality rates, that exposure rates of LBV between megachiroptera in Old World African bats are high, and that bats may breed successfully after LBV exposure  (panel A) and migrates annually, compared with E. gambianus, which resides in less dense colonies of tens or hundreds .The underlying cause of the difference in seroprevalence between ousands) , panel At Africa , panel BE. helvum is widely distributed in Africa and a food source in West Africa.No investigations into infections of humans were made during these investigations, but lyssavirus infections in humans in Africa are underdiagnosed (


Ureaplasma urealyticum and Ureaplasma parvum) and mycoplasmas  are potentially pathogenic species playing an etiologic role in both genital infections and male infertility. Reports are, however, controversial regarding the effects of these microorganisms infections in the sperm seminological variables. This study aimed at determining the frequency of genital ureplasmas and mycoplasmas in semen specimens collected from infertile men, and at comparing the seminological variables of semen from infected and non-infected men with these microorganisms.Genital ureaplasmas (t-tests) and nonparametric techniques (Wilcoxon tests) were used for statistical analysis.A total of 120 semen samples collected from infertile men were investigated. Semen specimens were examined by in-house PCR-microtiter plate hybridization assay for the presence of genital ureaplasmas and mycoplasmas DNA. Semen analysis was assessed according to the guidelines of the World Health Organization. Standard parametric techniques  was higher than that of Mycoplasma hominis (10.8%), Ureaplasma parvum (4.2%) and Mycoplasma genitalium (5%). Mixed species of mycoplasmas and ureaplasmas were detected in 6.7% of semen samples.The frequency of genital ureaplasmas and mycoplasmas detected in semen samples of infertile men was respectively 19.2% (23/120) and 15.8% (19/120). The frequency of Mycoplasma hominis DNA in semen samples is associated with low sperm concentration (p = 0.007) and abnormal sperm morphology (p = 0.03) and a negative correlation between sperm concentration and the detection of Mycoplasma genitalium in semen samples of infertile men (p = 0.05). The mean values of seminal volume, pH, vitality, motility and leukocyte count were not significantly related either to the detection of genital mycoplasmas DNA or to the detection of ureaplasmas DNA in semen specimens.Comparison of the parameters of the standard semen analysis between the male partners of the infertile couples with and without genital ureaplasmas and mycoplasmas infection showed that the presence of Our results demonstrate that genital mycoplasmas and ureaplasmas seem to be widespread among the male partners of infertile couples in Tunisia. Genital mycoplasmas infections of the male genital tract could negatively influence semen quality. Our results also indicate that PCR-microtiter plate hybridization assay method provides a rapid and effective technique to detect human genital mycoplasmas and ureaplasmas which is useful for etiological and epidemiological studies of these pathogens. Mycoplasmataceae and Mollicutes class, are widely distributed in humans, mammals, birds, reptiles, fish, and other vertebrates as well as in plants ), necrospermia by a sperm vitality < 50% and teratospermia by a normal morphology <30%.Prior to semen analysis, the men were asked to abstain from sexual intercourse or masturbation for 3–5 days before attending the clinic. All samples for analysis were produced on site and collected into standard containers that had previously been shown not to have any cytotoxic effects on human spermatozoa according to the methods outlined by World Health Organization . Immedia6/mL.Peroxidase staining, a practical and reliable method recommended by WHO for determining leukocytes in the semen, was employed to count and differentiate leukocytes (white blood cells) from immature germ cells . LeukocyU. urealyticum, U. parvum, M. hominis and M. genitalium DNA in these samples according to the following protocol.For each male patient, 200 μL of semen specimens were used for the detection of genital mycoplasmas and ureaplasmas DNA. An amplification-based method was performed to determine the presence or absence of g for 20 minutes. The precipitates were treated with 5 μl of proteinase K (20 mg/ml) at 55°C for 2 h in 600 μl of digestion buffer (30 μl of 10% sodium dodecyl sulphate and 570 μl of TE buffer ).The precipitates from each 200 μL of semen specimens were harvested by centrifugation at 14,000 g in an Eppendorf centrifuge. The aqueous phase was separated, mixed with 750 μl of phenol-chloroform/isoamyl alcohol (25:24:1 [vol/vol/vol]) and centrifuged for 15 minutes at 14,000 g in an Eppendorf centrifuge. The obtained aqueous phase was mixed with an equal volume of isopropanol. The samples were left at -80°C for 1 h and then centrifuged for 15 minutes at 14,000 g. The DNA pellet was washed up once with 70% ethanol, air dried, and dissolved in a final volume of 100 μl of TE buffer.After homogenisation, the samples were incubated in a solution of CTAB-NaCl (100 μl of 5 M NaCl and 80 μl of 10% CTAB) for 10 minutes at 65°C, and then mixed with 750 μl of chloroform-isoamyl alcohol (24:1 [vol/vol]) and centrifuged for 15 minutes at 14,000 U. urealyticum, U. parvum, M. hominis and M. genitalium. A forward primer, My-ins (5'-GTAATACATAGGTCGCAAGCGTTATC-3'), and two reverse primers, MGSO-2-Bi (5'-CACCATCTGTCACTCTGTTAACCTC-3') and UGSO-Bi (5'-CACCACCTGTCATATTGTTAACCTC-3'), were used to amplify an approximately 520-bp region of the 16S rRNA gene of mycoplasmas and ureaplasmas ; 0.5 μM My-ins; 0.25 μM (each) MGSO-2-Bi and UGSO-Bi; 0.2 mM (each) dATP, dCTP, and dGTP; 0.6 mM dUTP; 1.25 U of Go U. urealyticum, U. parvum, M. hominis and M. genitalium were detected by using molecular hybridization technique in a liquid phase assay  with species-specific internal biotinylated probes [Amplified products of 520-bp of U. urealyticum,Uure-P4-Am (5'-biotin-GGCTCGAACGAGTCGGTGT-3') specific for U. parvum,Upar-P6-Am (5'-biotin-GTCTGCCTGAATGGGTCGGT-3') specific for M. hominis,Mhom-P10-Am (5'-biotin – GACACTAGCAAACTAGAGTTAG-3') specific for M. genitalium.Mgen-P3-Am (5'-biotin-TCGGAGCGATCCCTTCGGT-3') specific for The amplified products were denatured in denaturation solution for 10 minutes and then immobilized to a microtiter plate, DNA-BIND 1 × 8 strip well plates, as described in the manufacturer's instructions. Specific-species capture probe diluted in hybridization solution was added to the wells of each microtiter plate immobilized by the denatured amplicons, and hybridization was carried out at 37°C for 30 minutes. Following hybridization, the wells were washed four times (soaking for 30 seconds) with washing solution. One hundred microliters of ready-to-use conjugate were added per well and incubated for 15 minutes at room temperature. Wells were washed as described above. One hundred microliters of substrate (Tetramethylbenzidine) were added per well and incubated for 30 minutes at room temperature in the darkness. The reaction was stopped by adding 100 μl of stop solution to each well, and the OD at 450 nm was measured.Samples with an OD value of greater than the cut-off value (OD of negative control + 0.15) + 10% of cut-off value were considered positive, as suggested by the manufacturer.Semen characteristics were compared between ureaplasmas semen-positive and ureaplasmas semen-negative groups, and between mycoplasmas semen-positive and mycoplasmas semen-negative groups. Means were calculated for the separate groups for sample volume, pH, sperm concentration, vitality, rapid progressive motility, total progressive motility, normal morphology and leukocyte count for semen samples.t-tests) were used to test significance of factors. Because sperm concentration, vitality, rapid progressive motility, total progressive motility, normal morphology and leukocyte count were not normally distributed and nonparametric techniques (Wilcoxon tests) were used to test the significance of the differences between groups regarding these factors. P < 0.05 was considered statistically significant.All the variables were initially inspected for normally distribution, and the sample volume and pH were all found to be approximately normally distributed. Therefore, standard parametric techniques . Thirteen of them had only U. urealyticum, and the rest consist of mixed species .U. parvum was detected in 5 patients (4.2%). Three of them had only U. parvum and two had mixed species .M. hominis was detected in 13 patients (10.8%). While nine patients had only M. hominis, the rest had mixed species .M. genitalium was detected in 6 patients (5%). All of them had mixture species listed above. The distribution of detected species of genital ureaplasmas and mycoplasmas from the patients is shown in Table Of 120 semen samples, 23 (19.2%) were positive for genital ureaplasmas and 19 (15.8%) were positive for genital mycoplasmas. p > 0.05).The analysis of the semen specimens based on seminological variables according to the WHO criteria  had showThe mean values of seminal volume, pH, sperm concentration, sperm vitality, sperm motility, sperm morphology and leukocyte count were not significantly related to the detection of genital ureaplasmas DNA in semen specimens Table .M. hominis DNA in semen specimens were significantly lower than that of the male partners without M. hominis DNA     Table .We couldn't get semen samples from fertile men, so, we have limited our comparison for seminological variables between semen from infected and non-infected infertile men with genital mycoplasmas and ureaplasmas.U. urealyticum is one of the most common species [U. urealyticum had been differentiated, into biovars 1 and 2. biovar 1 is composed of serovars 1, 3, 6, and 14, and biovar 2 is composed of serovars 2, 4, 5, and 7 to 13 [U. urealyticum biovars 1 and 2 were classified into U. parvum and U. urealyticum, respectively [U. parvum and U. urealyticum [U. urealyticum, U. parvum, M. hominis and M. genitalium in semen specimens. Our results demonstrated that genital mycoplasmas and ureaplasmas seem to be widespread among infertile male patients, as shown respectively by the frequency of 19.2% and 15.8%. These data are comparable with those reported in previous studies [U. urealyticum was the most prevalent species detected (15%) in this study. The frequency of U. urealyticum in the semen samples of male infertile patients in the literature varies from 5 to 42% [It is estimated that 15% of male infertility is related to genital tract infection . Among i species ,22. Sinc species , and esp species  first de 7 to 13 . In 1999ectively . Most ofalyticum . In our  studies ,27. U. u5 to 42% ,27,28. TU. parvum was detected in 4.2% of semen samples. The frequency of this species was lower than that reported by Knox et al. [In our study, x et al.   have been found in 7–14% of semen samples of infertile men [M. genitalium was not separately isolated among patients, it was detected together with U. urealyticum + M. Hominis in one patient, with M. hominis in one patient, with U. parvum in two patients and with U. urealyticum in two patients.U. urealyticum and M. hominis were observed together in two patients. Thus, eight patients (6.7%) had mixed species and our results are similar to those of previously reported studies [In the literature, mixed species  was lower than the normal reference of WHO manual (≥ 20 × 106/ml) in semen of M. hominis-positive infertile men and higher (52.63 × 106/mL) in semen of M. hominis-negative infertile men. The present data show that M. hominis may affect sperm concentration and sperm morphology of infertile men. A negative correlation was also found between sperm concentration and the detection of M. genitalium in semen samples of infertile men. Although, the sperm count with the presence of M. genitalium was within a normal range, a decrease in sperm concentration was significant. However, we have failed to demonstrate a correlation between sperm concentration and sperm morphology and the detection of genital ureaplasmas in semen samples. The comparison of the sperm seminological variables between semen with mixed infections and semen without mixed infections or without infections showed that only the sperm concentration in the male partners of infertile couples with mixed infection in semen specimens were significantly lower than that of the male partners without infections. Our findings show that the mixed infections have no additional effect on seminology.In our study, a positive correlation was found between sperm morphology and the detection of M. hominis presented a higher mean of leukocytes than semen with negative M. hominis; this difference not was statistically significant. The detected mean (1.153 × 106 leukocytes/mL) was higher than the reference value of the WHO manual (≥ 1 × 106 leukocytes/mL). In contrast, the means of leukocyte count of the positive DNA in semen samples for U. urealyticum, U. parvum and M. genitalium were smaller than the reference value of the WHO manual. In addition, no significant difference was detected among our studied patients. These findings indicate that the presence of mycoplasmas and ureaplasmas in semen is not necessary associated with leukocytospermia, and thus, in spite of potentially pathogenic species. Our results are consistent with previous reports [Semen with  reports . The unr reports ,44.M. hominis and M. genitalium and sperm morphology in the infection of M. hominis. Our results also indicate that PCR-microtiter plate hybridization assay method provides a rapid and effective measure to detect human genital mycoplasmas and ureaplasmas which is useful for etiological and epidemiological studies of these pathogens.The results of our study demonstrate that the genital mycoplasmas and ureaplasmas seem to be widespread among male partners of infertile couples in Tunisia. The study of the comparison of the semen parameters of infertile men with and without genital ureaplasmas and mycoplasmas has not shown any significant differences, apart from the sperm concentration in the infection of Little information was, however, available regarding the effect of mycoplasmas and ureaplasmas on the sperm quality, as well as their relationship with the leukocyte count. Therefore, it can be concluded that the screening of mycoplasmas and ureaplasmas species in routine semen analysis is not clinically relevant in our specific population. It should be restricted for men undergoing complete evaluation of infertility, genital infection and male partners from couples undergoing IVF.The author(s) declare that they have no competing interests.Authors R.G. carried out the PCR experiments, the analyses and interpretation of data, and drafted the manuscript. Author W.K. participated in the collection of data, PCR experiments and analysis of data. Author C.C. participated in the analysis and interpretation of sperm sminological variables. Author A.Z. participated in design and coordination of the study. Authors L.K., T.R and A.H. participated in design, data analyses, coordination and study of the manuscript.All authors readed and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Diarrhea is the third leading infectious cause of death worldwide and is estimated to be responsible for approximately 2 million deaths a year. While many infectious causes of diarrhea have been established, approximately 40% of all diarrhea cases are of unknown etiology. In an effort to identify novel viruses that may be causal agents of diarrhea, we used high throughput mass sequencing to analyze stool samples collected from patients with acute diarrhea.Kobuvirus. We have tentatively named this novel virus klassevirus 1. We also detected klassevirus 1 by RT-PCR in a diarrhea specimen collected from a patient in St. Louis, United States as well as in untreated sewage collected in Barcelona, Spain.Sequences with limited similarity to known picornaviruses were detected in a stool sample collected in Australia from a child with acute diarrhea. Using a combination of mass sequencing, RT-PCR, 5' RACE and 3' RACE, a 6383 bp fragment of the viral genome was sequenced. Phylogenetic analysis demonstrated that this virus was highly divergent from, but most closely related to, members of the genus Klassevirus 1 is a previously undescribed picornavirus that is globally widespread and present on at least three continents. Further investigations to determine whether klassevirus 1 is a human pathogen are needed. The impact of diarrhea is primarily felt in the developing world, where approximately 2 million deaths result from diarrhea annually -3. In de associated with a diverse range of diseases. Viruses in six of these genera potentially infect humans . With the advent of culture independent molecular methods, many diverse new members of the picornavirus family have been identified in recent years. These include novel cardioviruses  and Thermoscript [Invitrogen]).The klassevirus 1 contig had a genomic organization similar to other picornaviruses Figure . ConservKobuvirus, which includes Aichi virus, bovine kobuvirus and porcine kobuvirus.Phylogenetic analysis of the VP3/VP1, P2, and P3 regions of the genome demonstrated that this virus sequence is highly divergent from all previously described picornaviruses Figure . The cloIn order to determine the prevalence of klassevirus 1, two patient cohorts were examined by RT-PCR. In the first cohort, 340 pediatric stool specimens sent to the clinical microbiology lab for bacterial culture at the St. Louis Children's Hospital in St. Louis MO, USA were tested. One sample from this cohort was positive by RT-PCR. The amplicon from this patient's virus (designated klasse-stl1) was found to have 92% identity at the amino acid level and 88% nucleotide identity to the original klasse-mel1 contig. Screening of 143 stool samples from children with acute diarrhea collected from the Royal Children's Hospital  did not yield any additional positive samples. In addition, RT-PCR was performed on concentrated raw sewage collected in Barcelona, Spain. A PCR product of the expected size was obtained and directly sequenced. This fragment (klasse-bar1) shared 85% amino acid identity and 84% nucleotide identity to the original klasse-mel1 sequence  of the genome and the VP0/VP3 region. Approximately one kb of additional sequence was generated from klasse-bar1 and klasse-stl1 in both of these regions (see methods). Pair-wise amino acid identities ranged from 85%–97%, with the greatest degree of sequence conservation in the 3D region . To begin addressing this question, epidemiologic studies including case-control and seroprevalence analyses are needed.This stool was collected in 1984 from a 38 month old child presenting to the emergency department of the Royal Children's Hospital, Melbourne, Australia with acute diarrhea and stored at -80°C. Previous testing of this diarrhea specimen for known enteric pathogens using routine enzyme immunoassays (EIA) and culture assays for rotaviruses, adenoviruses, and common bacterial and parasitic pathogens was negative . Additio120 mg of frozen stool was chipped and then resuspended in 6 volumes of PBS . The samFor sequencing experiments, the stool filtrate was proteinase K treated prior to RNA extraction. RNA was isolated from primary stool filtrate using RNA-Bee  according to manufacturer's instructions. RT-PCR and 3'RACE reactions were performed using SuperScript III and Platinum Taq (Invitrogen One-Step RT-PCR). For 5'RACE reactions cDNA was generated with Themoscript (Invitrogen) and amplified with Accuprime Taq (Invitrogen). Amplicons were either cloned into pCR4 (Invitrogen) or sequenced directly.NP_653075.1), Foot-and-mouth-type-O (NP_658990.1), Equine Rhinitis B virus (NP_653077.1), Theiler murine encephalomyelitis (AAA47929.1), Mengo virus (AAA46547.1), Encephalomyocarditis virus (CAA60776.1), Seneca valley virus (DQ641257), Aichi virus (NP_047200.1), Porcine teschovirus (NP_653143.1), Human Cosavirus E-1 (FJ555055.1), Hepatitis A Virus (M14707), Bovine kobuvirus (NP_740257.1), Porcine kobuvirus (YP_002456506.1), Human coxsackievirus A2 (AAR38840.1), Porcine enterovirus A (NC_003987), Human poliovirus 2 (M12197), Avian encephalomyelitis virus (NC_003990), Duck hepatitis virus 1 (ABI23434), Seal picornavirus type 1 (NC_009891), Human Cosavirus A1 (FJ438902), and Human parechovirus 1 (AAA72291.1). Multiple sequence alignments were performed using ClustalX (1.83). The amino acid alignments generated by ClustalX were input into PAUP [Protein sequences associated with the following reference virus genomes were obtained from GenBank: Equine Rhinitis A virus , RNA was extracted in the same manner as the primary sample. For the remaining specimens (73), chips of frozen fecal specimens (~30–150 mg) were resuspended in 6 volumes of PBS. Total nucleic acid was extracted from 200 μL of each stool suspension using a MagnaPure LC instrument (Roche). 200 μL of water was used to elute the total nucleic acid from each sample.Leftover material from 340 stool specimens that were routinely submitted to the St. Louis Children's Hospital Lab for bacterial culture were collected from January 2008–July 2008. For these specimens total nucleic acid was extracted as described above. This study was approved by the Human Research Protection Office of Washington University.One 10 L-sample of raw sewage was collected in an urban wastewater treatment plant in the area of Barcelona, Spain. The sample was collected in a sterile container and stored for up to 2 hours at 4°C before being processed. The viruses present in the sample were concentrated in 30 mL of phosphate buffer by organic flocculation based on the procedure previously described by Calgua et al., 2008 . A seconGQ253930-GQ253936).Primers expected to generate a 345 bp product were designed to the 2C region of klassevirus 1 (LG0098: 5'-CGTCAGGGTGTTCGTGATTA-3' and LG0093: 5'-AGAGAGAGCTGTGGAGTAATTAGTA-3'). RT-PCR reactions were performed using Qiagen one-step kit under the following conditions: 30 min RT step, 94°C hold for 10 min, followed by 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s. In order to further compare strain divergence, primers expected to produce amplicons of 1001 bp and 1025 bp based on the klasse-mel1 sequence were designed targeting the 3D and VP0/VP3 regions, respectively: (LG0118: 5'-ATGGCAACCCTGTCCCTGAG-3' and LG0117 5'-GGAAACCCAACCACGCTGTA-3') and (LG0119: 5'-GCTAACTCTAATGCTGCCACC-3' and LG0136: 5'-GCTAGGTCAGTGGAAGGATCA-3'). These RT-PCR reactions were performed using the Invitrogen One-Step RT-PCR kit with the following conditions: 30 min RT step at 60°C, 94°C hold for 2 min, followed by 40 cycles of 94°C for 15 s, 56°C for 30 s, 68°C for 90 s. Whenever possible, amplicons were cloned into pCR4 (Invitrogen) and sequenced using standard Sanger sequencing technology. In some instances, PCR products were directly sequenced and only high quality sequence from those samples were included in analysis. All klassevirus 1 sequences have been deposited in Genbank (The authors declare that they have no competing interests.LH performed the experiments, sequence analysis and wrote the paper. SF and GZ performed sequence analysis; CK, RG and JP provided stool and sewage for analysis, DW conceived the project and helped write the paper. All authors have read and approved the final manuscript.


Lyssavirus infection was determined by use of direct fluorescent antibody assays on bat brains and virus neutralization assays on bat sera. Although no bats were found to have active infection with a Lyssavirus, 22 had evidence of neutralizing antibody against the Australian bat lyssavirus (ABLV). Seropositivity was statistically associated with one species of bat, Miniopterus schreibersi. Results from the virus neutralization assays are consistent with the presence in the Philippines of a naturally occurring Lyssavirus related to ABLV.Active surveillance for lyssaviruses was conducted among populations of bats in the Philippines. The presence of past or current  Australian bat lyssavirus (ABLV) (Rabies virus (RABV) are members of the genus Lyssavirus. These viruses are genetically similar and cause indistinguishable clinical syndromes in infected mammals. In the United States, where endemic canine rabies has been eliminated through vaccination and animal control, bat-associated variants of RABV have accounted for 24 (75%) of the 32 cases of human rabies reported since 1990  was originally obtained from an insectivorous bat in Australia and was amplified by passage in BHK and MNA cells at CDC.At the Centers for Disease Control and Prevention (CDC), the presence of virus-neutralizing antibodies was determined by a modification of the rapid fluorescent focus inhibition test (RFFIT) 2 incubator. MNA cells  were added to each serum-virus mixture, and the incubation was continued. After 48 hours, culture medium was removed, and the slides were fixed in acetone, air-dried, and stained for residual virus with FITC-conjugated anti-rabies monoclonal antibodies. A sample was defined as positive for neutralizing antibody if at least a 90% reduction in infectious centers as observed relative to the positive control. All positive samples were retested at increasing dilutions to estimate endpoint antibody titers. Standard human rabies immune globulin (HRIG) diluted to contain 2 IU/mL antibodies was used as a positive serum control for all tests. The titer of HRIG ranged from 1:125 to 1:625 against both ABLV and CVS-11.All bat serum samples were thawed and placed in a 56°C water bath for 30 minutes to inactivate complement. Serum samples were then diluted to 1:10 if possible. Samples with insufficient volume were screened at a higher dilution. The RFFIT was conducted by using Lab-Tek 8-well glass slides with covers . Sera were screened for antibody by incubating 100 μL of diluted serum with 100 μL of ABLV or CVS-11 that had been diluted to contain approximately 100 infectious units when incubated for 90 minutes at 37°C in a COResults from the serologic testing were used to detect patterns in seropositivity by location or type of bat, by using the Chi-square test.Of the 821 bats collected, all but three were identified to species . The colLyssavirus.Brains from all 821 bats were tested for the presence of RABV antigen by DFA. None of the bats had detectable antigen consistent with an active infection with rabies or a related Of the 821 bats collected, 231 had sufficient volume and quality of serum to be diluted to 1:10 and successfully screened at 1:20, after being combined with the challenge virus . An addiOf the 231 bat sera tested, 22 (9.5%) were positive for neutralizing antibodies against ABLV. Antibody titration studies demonstrated decreasing percent neutralization at progressively higher serum dilutions. Of those 22 bat sera, 8 demonstrated no virus neutralization at the next highest dilution tested; 8 demonstrated some neutralization as dilute as 1:40; 3 had some at 1:80; 2 had some at 1:160; and 1 had evidence of some neutralization at a dilution of 1:320. When the strict definition of 90% to be considered positive was used, only two bat sera remained positive at the 1:40 dilution. This dilution is the equivalent of 0.6 IU/mL antibody. Five of those 22 samples were also positive when tested against CVS-11. Only 1 of the 209 bat sera that was negative when tested against ABLV was positive when tested against CVS-11.Mineopterus schreibersi (Schreiber’s long-fingered bat) tested positive (p=0.01).The 22 bats with neutralizing antibodies against ABLV included six different species collected from four islands . No locaM. schreibersi remained the only species with a statistically significantly greater proportion of serum samples positive for neutralizing antibody.The data analysis was repeated with a less strict case definition of 75% reduction in infectious centers relative to the positive control and including the 43 additional samples that could only be screened at higher dilutions. When these criteria were used, 53 (19%) of 274 bat sera tested were positive. The two samples with the highest positive endpoint titers in the initial analysis remained highest, but now at a 1:80 dilution. Although additional species would have been identified as having neutralizing antibodies, Lyssavirus related to ABLV in the Philippine bat populations studied.This study presents evidence of neutralization of ABLV by serum from Philippine bats. This neutralizing activity correlated with the ability to neutralize RABV (CVS-11) and titrated steadily with serial dilutions of the serum. These findings are consistent with the presence of naturally occurring antibodies against a Duvenhage virus (DUVV) (genotype 4), European bat lyssavirus (EBLV) 1 (genotype 5), EBLV-2 (genotype 6), and ABLV (genotype 7). Phylogroup II includes Lagos bat virus (LBV) (genotype 2) and Mokola virus (MOKV) (genotype 3). Antibodies to viruses within one phylogroup should cross-neutralize viruses of that same phylogroup. The ability to cross-neutralize is directly proportional to the relative nucleotide and amino acid homogeneity between the two viruses being compared Lyssavirus infection were discovered from which nucleotide and amino acid sequences could be determined and subsequently compared with ABLV and CVS-11. More samples neutralized ABLV than CVS-11, suggesting that the Lyssavirus responsible for the induction of antibodies in these bats might be more similar to ABLV than CVS-11, while still being a member of phylogroup I. Repeating the RFFIT assays with a challenge virus from phylogroup II, such as MOKV or LBV, could have tested this hypothesis further. Although it is possible that we might have been able to demonstrate some cross-reactivity, a finding of greater neutralization activity against MOKV (compared with what was found against ABLV) would not be expected since all known phylogroup II viruses have a rather limited geographic distribution in Africa. In addition to the more widespread distribution of phylogroup I lyssaviruses, the small quantities of available bat sera precluded repeated RFFIT testing with an additional challenge virus such as MOKV.Lyssaviruses are classified into groups on the basis of their relative pathogenicity, their binding affinity to specific monoclonal antibodies, and their nucleic acid sequences. There are seven putative genotypes that have been aggregated into two basic groups on the basis of their overall phylogenetic relatedness Lyssavirus antibody in these bat populations. Reduction in infectious centers by 90% compared with the positive control provided a conservative estimate of the prevalence of anti-Lyssavirus neutralizing antibodies. Previous studies have used a cutoff as low as 50% neutralization for the interpretation of data The strict case definition used in the interpretation of the RFFIT assays resulted in the identification of 22 positive bat sera. As noted in the results of the second analysis, for which a lower threshold for positivity was used, additional bat sera that had 75% to 89% neutralization also had progressively decreasing neutralization at increasing dilutions, a finding similar to that for the 22 positive samples that met the strict case definition. Thus, we may have slightly underestimated the actual prevalence of anti-M. schreibersi and in locations with the highest prevalence of neutralizing antibodies. Ideally, if a large stable colony of such bats could be identified, surveillance among sick and dying bats could be conducted. Such a study would increase the likelihood of obtaining a virus isolate and would minimize the potential adverse impact on the bat populations from oversampling large numbers of otherwise healthy bats. Similarly, the routine virus variant typing of human and domestic animal rabies cases in the Philippines and throughout Southeast Asia will provide basic epidemiologic information on the prevalence of different RABV isolates and enhance the likelihood of discovery of any new lyssaviruses affecting the populations in these regions.On the basis of the 9.5% prevalence of neutralizing antibodies, it is not surprising that all brain samples studied showed no evidence of RABV antigen by DFA in these clinically normal bats. Most studies of healthy bats have found a low prevalence of active infection, usually <1%


The aims of this study were to evaluate the construct validity (known group), concurrent validity (criterion based) and test-retest (intra-rater) reliability of manual goniometers to measure passive hip range of motion (ROM) in femoroacetabular impingement patients and healthy controls.Passive hip flexion, abduction, adduction, internal and external rotation ROMs were simultaneously measured with a conventional goniometer and an electromagnetic tracking system (ETS) on two different testing sessions. A total of 15 patients and 15 sex- and age-matched healthy controls participated in the study.P < 0.001); good concurrent validity was only achieved for hip abduction and internal rotation, with intraclass correlation coefficients (ICC) of 0.94 and 0.88, respectively. Both devices detected lower hip abduction ROM in patients compared to controls (P < 0.01). Test-retest reliability was good with ICCs higher 0.90, except for hip adduction (0.82-0.84). Reliability estimates did not differ between the goniometer and the ETS.The goniometer provided greater hip ROM values compared to the ETS  rather than true hip ROM. It is likely that uncontrolled pelvic rotation and tilt due to difficulties in placing the goniometer properly and in performing the anatomically correct ROM contribute to the overrating of the arc of these motions. Nevertheless, conventional manual goniometers can be used with confidence for longitudinal assessments in the clinic. Hip joint range of motion (ROM) is a basic clinical parameter for diagnosing hip diseases, such as osteoarthritis ,2 or femThree-dimensional measurement tools based on electromagnetic tracking have recently been used to precisely measure shoulder -12 and sThus, the aims of this study were (i) to verify the validity of a conventional manual goniometer  to measure passive hip ROM against a criterion standard instrument (ETS)  and to discriminate between individuals with and without FAI , and (ii) to examine the test-retest (intra-rater) reliability of hip ROM goniometric and ETS assessments.A total of 15 subjects (7 women) with a diagnosis of FAI, verified both clinically and radiologically , were evSubjects were tested on two occasions, one week apart, at the same time of day and at constant room temperature to establish the test-retest reliability of the goniometer and the ETS for hip ROM assessment. Concurrent (criterion-based) validity was examined by simultaneously recording the same hip ROM movement with both devices, the ETS serving as the criterion instrument . Known gPrior to data collection, subjects completed a standardized warm-up consisting of ergometer cycling (5 minutes at 50 W). Then, each hip ROM movement was performed twice for further warm-up and familiarization. All assessments were performed in the supine position according to Clarkson , and setA simple long-arm goniometer  with a 360° scale marked in one degree increments was used. Hip flexion was measured with the goniometer as the deviation from the neutral zero position in which the lower limb and trunk are in the horizontal plane Figure . The staThe ETS , which consists of an electronic unit linked to a host computer, one transmitter and four sensors Figure , sampledOne sensor was attached to the sacrum with double-sided tape and flexible medical adhesive tape (5 × 10 cm), whilst the subject was in a standing position Figure . SubjectAnatomical and global calibration trials were performed with the subject in the supine position. The three-dimensional positions of several anatomical landmarks were located by sequentially placing the tip of the pointer on each landmark. Position and orientation data were subsequently sampled from both the calibration sensor and the sensor attached to the segment. Dynamic calibration trials were performed to enable the calculation of functional hip joint centers. This required the participants to perform two hip circumductions with a ROM of approximately 30° in flexion and 30° in abduction .In order to further standardize hip joint ROM measurements, the force applied by the main investigator was controlled by a modified hand-held load cell system . Two plastic grips allowed the investigator to easily handle the load cell during the measurements. For hip flexion, the pad of the dynamometer was pressed against the shank, 5 cm distal to the tibial tuberosity Figure . For hipData processing was performed using software written in the Matlab programming language . Functional hip joint centers, estimated using a functional approach, and digitized anatomical landmarks  were used to define the local coordinate systems of pelvis and thigh, which were then linked to the segment's individual receiver by means of coordinate transformations. For hip flexion, abduction and adduction, the sensor on the lateral aspect of the thigh was chosen as the relevant sensor to which the thigh coordinate system was related. As this sensor yielded considerably lower ROM for internal and external rotation compared to goniometry (-50% approximately), and also compared to the sensor on the medial aspect of the knee, the latter sensor served as the reference for these two motion patterns . In geneP ≤ 0.01 was accepted as significant (Bonferroni correction).Data were first checked for normality and for homogeneity of variance. Paired Student t-tests were then used to detect any systematic bias between goniometer and ETS  and test sessions (test-retest reliability). The Mann-Whitney U test was used to examine differences in ROM values between FAI patients and healthy controls . For FAI patients, only the involved side was considered. For controls, the mean of the right and left hips was consistently used as no significant side-to-side difference was observed. Concurrent validity between the two systems was analyzed using intraclass correlation coefficients (ICC)  with their 95% confidence intervals, and Bland-Altman plots. This was done by plotting the difference between goniometer and ETS measures against their means and calculating the systematic bias ± random error, i.e., 95% limits of agreement (LOA) . As propP < 0.01; Table For both measurement tools, hip abduction was significantly lower in FAI patients compared to controls P < 0.0; Table 1P < 0.001), therefore indicating systematic bias. The percentage difference between the two systems was 18% for flexion, 6% for abduction, 13% for adduction, 12% for internal rotation and 20% for external rotation. The highest systematic bias and random errors were observed for hip flexion ROM . For the different ROM, ICC ranged between 0.44 and 0.94 Table . The higM Figure , Table 2M Figure , Table 2Test-retest ICCs were above 0.90 for all ROM assessments Table , except This study examined whether manual goniometers (i) are sensitive enough to discriminate between individuals with and without FAI , (ii) measure the anatomical correct hip joint ROM  and (iii) produce consistent results (test-retest reliability). The major findings of this study were that goniometric measurements of passive hip motion provided greater ROM data than the criterion instrument ETS. Interestingly, the agreement between the two devices was high for hip abduction and internal rotation, but low for flexion, adduction, and external rotation. The finding that goniometers are particularly valid for measuring hip abduction was also confirmed by the comparison between FAI patients and healthy controls. Moreover, it was demonstrated that goniometric evaluation of passive hip joint angles was reliable between days, with similar reliability scores compared to the ETS. Manual goniometers can therefore be used with confidence during longitudinal assessments, which rely on repeated measurements over time.The assessment of construct validity was performed by comparing hip ROM between FAI and healthy hips . Considering the advanced number of FAI patients in our clinic and the quickly increasing interest for this pathology worldwide , informaAlthough all hip ROM values measured by the goniometer were significantly greater compared to the ETS, concurrent validity of manual goniometers was particularly good for hip abduction, with high ICC, and low systematic bias and random error. Subjects were positioned supine with the contralateral leg hanging down on the edge of the massage table during hip abduction assessments. Thus, the pelvis was not only stabilized by the belt, but also by the abducted contralateral limb, regardless of any other movements. In this configuration, excessive rotation of the pelvis around a vertical axis was prevented, which was not the case for hip adduction. Considering that the sacrum sensor was assumed to be rigidly attached to the pelvis and therefore representative of the pelvic coordinate system, most of the differences between the goniometer and the ETS can be attributed to this phenomenon. For motion patterns others than hip abduction, it is plausible that the difference between the goniometer and the ETS was largely due to pelvic rotation when the passive limit of motion was reached. Although the co-investigators performing the goniometer assessments tried to minimize this source of error by stabilizing the subject manually, they could not adequately correct for this misalignment. The obtained results for hip flexion are in agreement with Elson and Aspinall , who fouApart from the uncontrolled pelvic tilt or rotation and neutralization of lumbar lordosis (in case of hip flexion) for goniometric hip ROM assessments, there are other possible factors leading to the disagreement between the goniometer and ETS data. It is unlikely that between-device bias was attributable to differences in the physiological mechanisms underlying hip ROM testing, because measurements were performed simultaneously with the two devices. Rather, the observed discrepancies were certainly due to visual estimation of the true anatomical reference lines  and potential alignment of the goniometer to the position of the massage table or to the laboratory arrangements, rather than to true bony orientation. Another reason for the disagreement between the goniometer and ETS is the two-dimensional characteristics of goniometric hip ROM measurements. Cheng and Pearcy  showed tThe results obtained in the present study suggest that the hip ROMs "read" by the goniometer are in fact intersegmental thigh-trunk angles  rather than true hip joint ROM. This study clearly demonstrates the "harmful" effect of flexing hip while evaluating ROM if pelvic tilt or rotation is not adequately controlled. Future research is needed to find hints allowing the goniometer to measure an angle close to the real ROM and to verify if other ways of goniometric estimation  would provide similar results compared to the ETS, all the more because one of the goniometer's arms could be placed on the table, potentially adding accuracy due to standardization. Even if manual goniometers are logically preferred in the clinic to more accurate devices, such as ETS, because of the limited time available for routine medical examinations, clinicians should be aware of this misinterpretation and they should try to minimize pelvic rotation. Apart from other advantages , the use of conventional manual goniometers for longitudinal evaluations is supported by the present test-retest reliability results, which were good and comparable to those obtained with the ETS.The excellent absolute and relative reliability estimates for hip flexion are in agreement to those reported on persons with hip osteoarthritis ,2,42-44 The current study has a few limitations. Firstly, human movement analysis based on electromagnetic tracking technology is affected by instrument errors, anatomical landmark uncertainty and skin movement artifacts . These sThis study was designed to determine the validity and reliability of manual goniometers for measuring passive hip ROM. This study is unique to the literature, and thus offers new information of clinical importance. The current findings suggest that goniometer-based assessments conventionally used in orthopedic clinical practice overestimate the majority of passive hip motion patterns by measuring intersegmental angles  rather than true hip ROM; it is indeed difficult to reproduce true hip ROM by placing the goniometer properly and performing the anatomically correct ROM. It is likely that uncontrolled pelvic rotation and tilt contribute to the overrating of the arc of these motions. Future work is needed to find hints to perform hip ROM assessments more accurately. It is concluded that, as manual goniometers yielded good test-retest reliability estimates, they would remain the first choice tool for the assessment of hip ROM in the clinic, especially when longitudinal monitoring of hip function is aimed.The authors declare that they have no competing interests.SN, NAM, ML and HG were responsible for the conception and design of the study. SN, JFG and SS coordinated the practical work and performed data acquisition. SN prepared the data for analysis and performed together with NAM the statistical analyses, data interpretation and manuscript drafting. ML, HG, JFG and SS assisted with some of the statistical analyses and with the interpretation of the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/11/194/prepub


Molecular characterization of wild-type measles viruses in China during 1995-2004 demonstrated that genotype H1 was endemic and widely distributed throughout the country. H1-associated cases and outbreaks caused a resurgence of measles beginning in 2005. A total of 210,094 measles cases and 101 deaths were reported by National Notifiable Diseases Reporting System (NNDRS) and Chinese Measles Laboratory Network (LabNet) from 2006 to 2007, and the incidences of measles were 6.8/100,000 population and 7.2/100,000 population in 2006 and 2007, respectively. Five hundred and sixty-five wild-type measles viruses were isolated from 24 of 31 provinces in mainland China during 2006 and 2007, and all of the wild type virus isolates belonged to cluster 1 of genotype H1. These results indicated that H1-cluster 1 viruses were the predominant viruses circulating in China from 2006 to 2007. This study contributes to previous efforts to generate critical baseline data about circulating wild-type measles viruses in China that will allow molecular epidemiologic studies to help measure the progress made toward China's goal of measles elimination by 2012. Morbillivirus, family Paramyxoviridae, order Mononegavirales. Measles is a vaccine-preventable disease, but is still a major killer of infants worldwide. During 2000-2008, global measles mortality declined by 78%, from an estimated 733,000 deaths in 2000 to 164,000 in 2008 [Measles virus (MeV) is a single-stranded, negative-sense RNA virus, belonging to the genus  in 2008 ,6. MeaslThe WHO Regional Committee of the Western Pacific Region (WPR) formally declared a measles elimination goal in 2005, and established a target date of 2012 for regional measles elimination ,10. In CThis study describes the genetic characterization of wild-type measles viruses reported from 24 of 31 provinces in China from 2006-2007. Five hundred and sixty-five wild-type measles viruses were isolated from throat swab or urine specimens collected from outbreak and sporadic cases. The results documented the continued circulation of genotype H1 viruses throughout China.In 2006 and 2007, the incidence of measles was 6.8/100,000 and 7.2/100,000, respectively, with a total of 100,267 and 109,827 cases and 35 and 66 fatalities reported by NNDRS. In 2004, measles incidence was > 10/100,000 in 6 of 31 provinces  which were located in the developing areas of China with the exception of Zhejiang province. However, in 2005, the incidence of measles was > 10/100,000 in 14 of 31 provinces, which included 8 developed  and 6 developing  provinces . Population denominators for calculation of incidence and mortality rates were determined on the basis of data reported by the National Bureau of Statistics. Epidemiologic data were analyzed by using Microsoft Excel and Epi Map.Urine, throat swab and blood samples were collected from patients who had acute, febrile maculopapular rash from 24 provinces in China. All clinical samples were collected within five days of rash onset and transported in accordance with standard protocols ,21. IsolRNA was extracted from 250 μl of infected cell lysate using a Trizol reagent, following the manufacturer's instructions. For all virus isolates, RT-PCR amplification was performed using previously described primers to amplify a 600bp fragment in the N gene which included the 450bp fragment recommended for genotyping . PCR proGU237175-GU237481Sequences of the PCR products were derived by automated sequencing and the BigDye terminator v3.0 chemistry according to the manufacturer's protocol in both sense and antisense strands by an automated ABI PRISM™ 3100 DNA Sequencer (Perkin Elmer). Sequence proof reading and editing was conducted with Sequencer™ (Gene Codes Corporation). Sequence data were analyzed using version 7.0 of Bioedit and phylogenetic analyses were performed using Bioedit and Mega4 . The robNNDRS: National Notifiable Diseases Reporting System; MeV: Measles virus; RT-PCR: reverse transcriptase polymerase chain reaction; CPE: cytopathic effect; N: Nucleoprotein; WHO: World Health Organization; WPR: Western Pacific Region;The authors declare that they have no competing interests.YXJ carried out most of the studies and drafted the manuscript. WBX designed the study and organized the coordination. YXJ, STX, YZ, ZZ and NYM performed parts of the studies and sequence analysis. XHJ, JHZ, PSL, CYW, YL, HYZ, collected specimens and performed virus isolation, viral identification. PAR and DAF provided consultation and editing of the manuscript. All authors read and approved the final manuscript.


After its introduction, laparoscopic cholecystectomy rapidly expanded around the world and was accepted the procedure of choice by consensus. However, analysis of evidence shows no difference regarding primary outcome measures between laparoscopic and small-incision cholecystectomy. In absence of clear clinical benefit it may be interesting to focus on the resource use associated with the available techniques, a secondary outcome measure. This study focuses on a difference in costs between laparoscopic and small-incision cholecystectomy from a societal perspective with emphasis on internal validity and generalisabilityA blinded randomized single-centre trial was conducted in a general teaching hospital in The Netherlands. Patients with reasonable to good health diagnosed with symptomatic cholecystolithiasis scheduled for cholecystectomy were included. Patients were randomized between laparoscopic and small-incision cholecystectomy. Total costs were analyzed from a societal perspective.Operative costs were higher in the laparoscopic group using reusable laparoscopic instruments . There were no significant differences in the other direct cost categories (outpatient clinic and admittance related costs), indirect costs, and total costs. More than 60% of costs in employed patients were caused by sick leave.Based on differences in costs, small-incision cholecystectomy seems to be the preferred operative technique over the laparoscopic technique both from a hospital and societal cost perspective. Sick leave associated with convalescence after cholecystectomy in employed patients results in considerable costs to society.ISRCTN Register, number ISRCTN67485658. Langenbuch's classical cholecystectomy has been the gold standard for over a century . Since tAnalysis of evidence in Cochrane reviews shows no difference regarding primary outcome measures  between the three operative techniques of cholecystectomy  -8. In abThe costs of LC and small-incision cholecystectomy (SIC) have been compared in six randomized trials. -15 TheseThe research question is whether there is a difference in costs from a societal perspective between small-incision and laparoscopic cholecystectomy using a blind randomized approach. In a detailed cost analysis attention has to be paid to both direct and indirect costs as well as the perspective of the analysis. Furthermore, cost prices, budget prices and tariffs have to be distinguished.In meta-analyses we found no major differences in clinical outcome measures  between LC and SIC for patients with symptomatic cholecystolithiasis. We also Medical Ethics Committee approval for this single-centre trial was obtained in September 2000. Between January 2001 and January 2004, all patients referred to our surgical outpatient's clinic with symptomatic cholecystolithiasis (confirmed by ultrasonography) were considered for inclusion in this study.Inclusion criteria were male or female patients with symptomatic cholecystolithiasis, aged 18 years or older at recruitment, reasonable to good health (ASA I or II), no known relevant allergies, and a signed informed consent letter.Exclusion criteria were age younger than 18 years, choledocholithiasis , cholangitis, known pregnancy, moderate to severe systemic disease (ASA III and higher), known cirrhosis of the liver, history of abdominal malignancy, previous upper abdominal surgery (precluding a laparoscopic approach), psychiatric disease, or a reasons (e.g. lack of knowledge of the Dutch language) that might make follow-up or completion of questionnaires unreliable.Obesity was not an exclusion criterion. Recovery after successful endoscopic treatment of choledocholithiasis was not a contra-indication. Acute cholecystitis was excluded.A random number table was used for generation of the allocation sequence  and the nd year on) performed most of the operations. In case of technical difficulties either trial technique could be converted to open cholecystectomy (OC). Wounds were covered with standard wound dressings.[All patients had a standard anesthesia regime. Premedication, medications for induction and continuation of anesthesia, as well as respiration during surgery were standardized. Residents  were accepted. Hospital stay was defined as the number of postoperative nights in hospital. For logistic reasons, we were not able to blind the surgeon during follow-up. Follow-up was standardized after 2 weeks, 6 weeks and 3 months. Patients were encouraged to resume work as soon as possible.In meta-analyses no differences in patient-relevant outcomes appear to be present between these two techniques. Assuming no differences in primary outcome measures, sample size calculation was based on anticipated differences of costs. The direct costs of the first 50 patients in the trial were calculated so as to estimate the likely range of differences in costs and their standard deviations. On this basis, we estimated that 120 patients per group would be needed to detect a difference of 10% in direct costs using an α of 0.05 and a β of 0.9.Analyzing differences in costs due to complications between both techniques would require another sample size including thousands of patients in order to possibly find significant results. Consequently, differences in complication costs were therefore not statistically tested in our study. However, these costs were reported to illustrate their impact on total costs.®) based on a patient-linked trial registration number. A double data entry was performed. Comparisons were made on an intention to treat basis. Calculations were made using SPSS 11.0®.All data were stored in a case record form . All direct medical costs were summarized in different categories including costs due to complications .As there are no relevant or significant differences in clinical outcome ,16 or quIn evaluating costs, it is important to be complete in accounting for all items. Thereforcosts resulting from outpatient clinic, hospital admittance, surgery, complications, etcetera.costs arising from outside health care immediately related to treatment (e.g. traveling costs from patients). The problem is that it is very difficult to estimate these costs accurately in all patients. We assume that these costs are equal for both groups. Moreover, since the vast majority of patients have an uncomplicated recovery, these costs are assumed not to contribute importantly to total costs.costs due to loss of productivity related to employment status of patients. These costs arise from loss of productivity caused by sick leave, disabled for work or mortality. We decided to use the friction cost method. -36In cost assessments tariffs, cost prices and budget prices have to be distinguished. Tariffs are costs that are calculated for insurance companies. Tariffs are nearly always different from real costs. Budget prices on the other hand are figures used for internal  calculations, and do not reflect real costs either. Cost prices are real costs for procedures and are usually not used in hospital administrations. These cost prices can be calculated but are sometimes difficult to retrieve.Apart from these differences there are costs for depreciation and interest. Different methods are used and no uniform guidelines exist how to estimate these overhead costs, neither do arguments exist that one method is superior over the other. In our hospital a percentage incremental value is added to every cost price. We applied this method in our study.In this cost-minimization analysis, all resources were prospectively recorded. A visit to the general practitioner, diagnostic examinations, and costs due to preoperative outpatient clinic visits were counted. Hospital stay was counted as the number of overnights stay. Medication use is included in admittance cost prices. Operative costs and the costs of anesthesiologists were calculated according to operating room occupancy. Standard materials and equipment used in the operating room including costs associated with cleaning and sterilisation are included in hospital costs for surgery. The costs of the surgeon and resident were calculated from the time of incision to last suture. In laparoscopic instruments extra laparoscopy-specific materials like clips and endobags were calculated, but monitor, gass-insufflator, and camera were not calculated as these were considered present. In the small-incision procedure no extra equipment other than standard instruments is needed. In follow-up the time of the surgeon was calculated. Finally, if complications occurred, all extra costs were included. Costs of consultants were calculated using the national agreed honorarium (140 euro per hour) , and American Society of Anesthesiologists (ASA) classification table . None ofThe numbers of converted procedures, hospital stay, the number of residents performing the procedure, and the number of intra-operative and postoperative complications were not significantly different. Operative time was significantly shorter in the small-incision group  6 to 17 minutes; p < 0.001) table . The totThere is an important difference in direct costs. The difference is caused by differences in costs due to complications and rendering total costs in favor of the laparoscopic procedure.The difference in operation theatre cost is in favor of the SIC group however. Operation theatre costs are over 23% more expensive in the LC group compared to the SIC group . Indirect costs are higher in the laparoscopic group.Data of costs are non-Gaussian distributed, importantly influenced by one extreme outlier. Therefore, although intention-to-treat is violated, the results excluding one outlier in each group are also shown Table . ResultsIn order to be able to compare uncomplicated LC and SIC procedures, costs were also calculated excluding all complicated cases. There still are differences present in the operation theatre related costs  and in total direct costs . No significant differences were observed in other direct cost categories, indirect costs, and total costs.In order to estimate the influence of indirect costs on total costs we performed calculations only including employed patients Table . IndirecWe performed a sensitivity analysis assuming the use of disposable instead of reusable laparoscopic instruments. Calculations show increase of operation costs resulting in differences of approximately 960 euro in favor of the SIC group .A sensitivity analysis assuming a decrease in work leave by 1 week for the employed patients (n = 101) in this trial results in savings of 82790 euro. In another sensitivity analysis assuming a decrease in hospital stay by 1 night for the employed patients (n = 101) in this trial results in savings of 22980 euro.When no differences in primary outcomes are found, consequently, several secondary outcome measures like operative time, hospital stay, and time to recovery as well as costs can be chosen as focus for a trial. Most of these secondary outcome measures are incorporated in a total cost assessment. Our cost analyses show that SIC is more expensive compared to LC when all patients are included . Thus SIC is the preferred operative technique over LC both from a hospital and societal cost perspective.Sick leave associated with convalescence after surgery results in considerable costs to society especially in the employed patient.The authors declare that they have no competing interests.FK and CJHML had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. FK, CJHML, EB and HGG participated in study concept and design. FK, TJ and CJHML participated in the acquisition of data. FK, HGG, CJHML participated in the analysis and interpretation of data. KF and TJ drafted the manuscript. HGG, EB and CJHML were responsible for critical revision of the manuscript for important intellectual content. FK, EB and CJHML participated in statistical analysis. FK and CJHML obtained funding. FK, TJ and CJHML were responsible for administrative, technical, and material support. HGG and CJHML supervised the study.


Orbivirus for which reverse genetics has not been developed yet.Since 1998, several serotypes of Bluetongue virus (BTV) have invaded several southern European countries. In 2006, the unknown BTV serotype 8 (BTV8/net06) unexpectedly invaded North-West Europe and has resulted in the largest BT-outbreak ever recorded. More recently, in 2008 BTV serotype 6 was reported in the Netherlands and Germany. This virus, BTV6/net08, is closely related to modified-live vaccine virus serotype 6, except for genome segment S10. This genome segment is closer related to that of vaccine virus serotype 2, and therefore BTV6/net08 is considered as a result of reassortment. Research on orbiviruses has been hampered by the lack of a genetic modification method. Recently, reverse genetics has been developed for BTV based on ten in vitro synthesized genomic RNAs. Here, we describe a targeted single-gene modification system for BTV based on the uptake of a single in vitro synthesized viral positive-stranded RNA. cDNAs corresponding to BTV8/net06 genome segments S7 and S10 were obtained by gene synthesis and cloned downstream of the T7 RNA-polymerase promoter and upstream of a unique site for a restriction enzyme at the 3'-terminus for run-off transcription. Monolayers of BSR cells were infected by BTV6/net08, and subsequently transfected with purified in vitro synthesized, capped positive-stranded S7 or S10 RNA from BTV8/net06 origin. "Synthetic" reassortants were rescued by endpoint dilutions, and identified by serotype-specific PCR-assays for segment 2, and serogroup-specific PCRs followed by restriction enzyme analysis or sequencing for S7 and S10 segments. The targeted single-gene modification system can also be used to study functions of viral proteins by uptake of mutated genome segments. This method is also useful to generate mutant orbiviruses for other serogroups of the genus  Culicoides. Bluetongue is listed as a 'notifiable disease' by the Office International des Epizooties (OIE) [Reoviridae, genus Orbivirus [Bluetongue (BT) is an arthropod-borne disease; transmission to ruminants, including cattle, sheep, and goats, occurs by bites of species of es (OIE)  causing rbivirus .The genome of BTV consists of ten linear double-stranded RNA genome segments encoding the seven structural proteins VP1 to VP7, and three nonstructural proteins, NS1, NS2 and NS3/NS3a [Since 1998, BTV serotypes 1, 2, 4, 9, and 16 have invaded European countries around the Mediterranean Basin. The outbreak by BTV8/net06 (sample nr. BTV-8 NET2006/04 in the dsRNA virus reference collection (dsRNA-VRC) at IAH Pirbright, ) was repin vitro synthesized viral RNA in an ongoing infection. We have focused on the uptake of genome segments S7 or S10 originating from BTV8/net06 in BTV6/net08, although the method is proposed to be broadly applicable for all genome segments and all orbiviruses.Research on BTV, including research on reassortment, has already a long scientific record . RecentE. coli DH5α, and were purified using QIAfilter Plasmid Midi Kit (Qiagen).Genome segments S7 and S10 were synthesized by Genscript Corporation  based on the identical sequences AM498057.2 and FJ183380.1 for S7, and the identical sequences AM498060.1 and FJ183383.1 for S10 of Genbank. cDNAs were cloned in plasmid pUC57 under control of the T7 RNA-polymerase promoter and a site for a restriction enzyme at the 3'-terminus for defined run-off transcription . In this reaction, a ratio of 4:1 of anti-reverse cap analogue to rGTP was used. Synthesized RNA was cleaned by use of MEGAclear columns (Ambion) according to the manufacturer's instructions, and eluted RNA was stored at -80°C.Plasmid DNA was digested with 5 BSR cells  supplemented with 5% FBS and 1% of Penicillin/Streptomycin/Fungizone. At 40 hpi, supernatants were harvested, and virus was cloned by endpoint dilution in M96-wells on BSR cells. At 3 days post infection (dpi), supernatants were collected from wells with cells developing cytopathogenic effect (CPE).Monolayers of 10R cells  produced by ATCC-CRL-1875 directed against VP7 (data not shown). Typically, viruses in 48 supernatants were multiplied in M24 wells in BSR cells by adding 75 μl supernatant in 1 ml of DMEM supplemented with 5% FBS and 1% of Penicillin/Streptomycin/Fungizone. After development of CPE, 2-3 dpi, supernatants were collected and stored at -80°C. Viral RNA was isolated from 200 μl of supernatant with the High Pure Viral RNA kit (Roche).PstI and BglII and analyzed by agarose gel electrophoresis. For S10, gel-purified amplicons were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit  in a ABI PRISM® 3130 Genetic Analyzer . In order to genetically serotype the cloned viruses, in-house developed serotype-specific PCR-assays for serotypes 6 and 8, based on segment S2 of BTV, were carried out using LightCycler RNA Master Hybridization Probes kit and a LightCycler 2.0 PCR machine . For the BTV6-S2 serotype-specific RT-PCR forward primer 5'-AGGAACAGTCGGCTTATCAC-3', reverse primer 5'-TTCGCTAATGTGCTTCTCCAT-3'  and taqmanprobe 5'-6FAM- TTGTCAGCTTTACGCAAACCCCG-BHQ-3'  were used. For the BTV8-S2 serotype-specific RT-PCR forward primer 5'-CGGAGACAGCGCAGTATGTA-3', reverse primer 5'-CCTCGGTAGTATCCCTCACG-3'  and taqmanprobe 5'-6FAM-ACATACGATGCCYTCGGAGGATTCTG-BHQ-3'  were used. Template RNA (5 μl) was added to a reaction mixture containing 0.25 μM of the forward and reverse primer, 0.25 μM probe, 2.75 mM MnCl2, 7.5 μl LightCycler mix and 0.2 μl RNAsin  in a final volume of 20 μl. Thermocycling conditions of the RT-PCR were: 20 s 98°C, 20 min 61°C, 30 s 95°C  × 45 cycles followed by 30 s 40°C and storage at 4°C. Amplification was monitored real-time by OD530/OD640 using LightCycler software version 4.05 .A serogroup-specific duplex RT-PCR was used for amplification of genome segment S7 . For par8). This finding was based on both positive and negative differentiation; the presence of a PstI site in S7 of BTV8, and the absence of a BglII site in case of S7 of BTV6  based on nucleotide differences on several positions in the amplicon. Again, the presence and absence of S2 of respectively serotype 6 and 8 was confirmed , BTV8/net06(e1/bhk3), BTV6/net08/S78(bsr2) and BTV6/net08/S108(bsr2). After attachment to cells for 1.5 h at 37°C, supernatant was removed and stored at -80°C (t = 0). One ml of fresh DMEM with 5% FBS, 1% Penicillin/Streptomycin/Fungizone was added to the monolayers and incubation was continued. At 21, 27, 45 and 79 hours post infection (hpi), samples of the supernatants were harvested and stored at -80°C. Virus titers were determined by end-point dilution. The observed differences in virus titer at 0 hpi, which was approximately 10-fold higher for BTV6/net08/S78  was minimal. Since no great differences in the slopes of the different growth curves were detected, the observed enrichment of this reassortant by passaging (6 times) of a mixture of BTV6/Net08 and BTV6/net08/S78 could be the result of factors other than replication and remains unclear.In one occasion, we have also observed a mixture of both segments S7 in candidate reassortant viruses Figure , lane 1.findings  in whicht08e1/bhk/bsr2, BTDespite optimizing the uptake of an exogenous genomic RNA-segment, the here described method to generate reassortants of bluetongue virus is not very efficient. The percentage of rescued reassortant virus is approximately 3-5% for genome segments S7 and S10. However, the method is relatively easy to perform, and mass screening of reassortant candidates can be easily performed depending on the targeted gene and available tools, like discriminating Mabs and/or discriminating PCR-assays. Particularly, this method is of interest for research focusing on one genome segment, since a full set of ten cDNAs encoding complete genome segments is not required. Boyce et al  have devin vitro synthesized RNA. On one hand, the method could be significantly improved by reducing this virus background with discriminating specific siRNAs. Very strong reduction of virus growth has been published for African horse sickness virus, another serogroup of the genus Orbivirus [in vitro synthesized RNA in infected cells could be further increased to improve the efficiency to rescue reassortants. This could be achieved by in vivo RNA synthesis by T7 RNA-polymerase expressing BSR cells after transfection of plasmids containing cDNA of a genome segment flanked by the T7 promoter and a functional ribozyme sequence. Alternatively, repeated transfection of in vitro synthesized RNA could increase the presence of RNA in the BTV-infected cell. Using reverse genetics, recently Matsuo et al. have shown that repeated transfection of BTV transcripts strongly improve the recovery of infectious BTV [The major drawback of the here described method is the high percentage of parental virus not reassorting with delivered rbivirus . On the ious BTV . This suLikely, the first event, the uptake of the transfected RNA by the replicating virus is a random process. This makes this method also suitable for rescue of reassortants with other genome segments. For instance to generate reassortant virus with a different serotype by uptake of RNA of genome segment S2. For this special case, neutralizing sera or neutralizing Mabs could be used to further reduce the background of parental virus and to screen for reassortant virus.in vitro with cDNA as template. This opens the opportunity to use this method as genetic modification system for BTV by uptake of mutated genome segments to study viral proteins. However, we realize that rescue of mutant BTVs with a lower fitness will be more difficult. Presumably, significant improvement of the method is necessary for this purpose by either lowering the virus background, increase the chance on uptake of synthesized mutant RNA, or both. However, the opposite was not seen, reassortant BTV6/08/S78 was rescued with a similar efficiency, although this reassortant multiplies to a higher virus titer than the parental virus. Apparently, efficiency of uptake of transfected synthetic RNA and cloning of mutant virus is at least as important as growth characteristics of desired mutant BTVs.The developed method results in the uptake by replicating BTV of RNA that was synthesized Orbivirus for which reverse genetics has not been developed yet, such as Epizootic hemorrhagic disease virus, this targeted single-gene modification system method will also be applicable in order to generate mutant orbiviruses.In conclusion, a targeted single-gene modification system for BTV was successfully developed without use of positive selection for rescued reassortants or desired (mutant) viruses. This method is also applicable for more detailed genetic modification of BTV to study functions of viral proteins. In addition but not proven here, the method could also be successful to incorporate more than one genome segment, like genome segments S2 and S6 encoding together the complete outer shell of BTV. Finally, for other serogroups of the genus The authors declare that they have no competing interests.RGPvG contributed to experimental design, performed experiments, data analysis and manuscript preparation, DV and SGPvdW carried out experiments and data analysis, PAvR initiated this project, contributed to project design, data analysis and manuscript preparation, and supervised the project. All authors read and approved the final manuscript.