Orthobunyavirus.Nearly complete genome sequences for three ungrouped viruses, Pacui virus (BEAN27326), Rio Preto da Eva virus (BEAR540870), and Tapirape virus (BEAN767592) isolated in the Amazon region are reported here. All three genomic segments  were recovered and were similar to members of the genus  Bunyaviridae is composed of 350 viruses distributed in five genera  (The family fection)  and somefection) . In addiBunyaviridae are vector-borne viruses , with the exception of hantaviruses, which are basically transmitted by contact with excretes of wild rodents (feces and urine)  . These vd urine) .Oryzomys sp. in the Belém-Brasilia highway, Pará State , Brazil, in 1961. Rio Preto da Eva virus (RPEV) was isolated from a pool of Psychodidae sp. (Diptera: Phlebotominae) in the Rio Preto da Eva municipality , Amazonas state, Brazil, in 1995. Tapirape virus (TPPV) was isolated from an Oxymycterus sp. rodent captured in the Zoobotanic Park area, Parauapebas municipality, Pará State, Brazil .Pacui virus (PACV) was isolated from a rodent Viruses were cultivated into Vero cells, and after 90% cytopathic effect (CPE) they were harvested and their genomes recovered after RNA extraction using the Trizol/Quiagen method as recommended by the manufactures. Nearly complete genomes were obtained using the GS FLX 454  sequencing.de novo strategy , and gene predictions were performed with the software Geneious v. 7. For PACV, the total genome recovered was 12,467 nucleotides (nt) in length ; for RPEV, the genome was 12,329 nt ; and for TTPV, the genome was 12,234 nt in length . All three viruses showed the N, Gn, Gc, NSm, and polymerase genes, but the NSs gene was not found. This is the first report of the complete ORF genome sequences for PACV, RPEV, and TPPV, three ungrouped bunyaviruses isolated in Brazil.The genomes were assembled by use of a strategy  and the KM225256; M, KM225255; L, KM225254); RPEV ; and TPPV .The genome sequences have been deposited in GenBank under the following accession numbers: PACV (S,


Lab-on-a-chip immuno assays utilizing superparamagnetic beads as labels suffer from the fact that the majority of beads pass the sensing area without contacting the sensor surface. Different solutions, employing magnetic forces, ultrasonic standing waves, or hydrodynamic effects have been found over the past decades. The first category uses magnetic forces, created by on-chip conducting lines to attract beads towards the sensor surface. Modifications of the magnetic landscape allow for additional transport and separation of different bead species. The hydrodynamic approach uses changes in the channel geometry to enhance the capture volume. In acoustofluidics, ultrasonic standing waves force µm-sized particles onto a surface through radiation forces. As these approaches have their disadvantages, a new sensor concept that circumvents these problems is suggested. This concept is based on the granular giant magnetoresistance (GMR) effect that can be found in gels containing magnetic nanoparticles. The proposed design could be realized in the shape of paper-based test strips printed with gel-based GMR sensors.  Rec2 ,5,6. TheCommon labels employed in immuno assays are enzymes (ELISA) ,8,9,10, Magnetic beads have several advantages compared to other labels. Unlike fluorescent labels, magnetic beads do not bleach, the MR detector can easily be structured onto the chip surface, there is no background signal from the sample, they are safer to handle than radioactive material and the detection is faster than in ELISA. However, the depositing of solved or dispersed particles such as beads onto a surface is usually diffusion limited. Beads, which generally have a low diffusion coefficient due to their comparably large size , usually settle onto a surface due to gravitational forces only. However, this requires a long time, depending on the size and density of the particles. Polystyrene beads like the Dynabeads M-280 (Invitrogen) with a diameter of 2.8 µm sink at a velocity of 1.7 µm/s when dispersed in water. Thus, sedimentation times in the absence of any flow are on the order of tens of seconds, or even minutes, depending on the system dimensions. Brownian motion, the parabolic flow profile of flows in micro-channels and lift forces  exacerbaet al. [et al. [et al. [There exist a few µTAS that work under static conditions, where no microfluidic flow is applied during the time of the binding so that beads can settle on the surface due to gravitational forces. For such devices, repulsive double layer forces may become a problem. Sharp et al.  demonstret al. ,31. Unbo [et al.  have sho [et al.  showed tOver the decades, different solutions for this problem have been found. These can generally be allocated to one of three categories, one employing magnetic forces, one utilizing hydrodynamic effects and one applying acoustofluidics. In the following paragraphs, these three categories are defined and examples of actual applications are given. However, as these approaches have their disadvantages, a new sensor concept that might solve these problems in the future is presented in the final section.et al. [et al. [et al. [et al. [The trajectory of superparamagnetic beads flowing in microfluidic systems can be controlled with magnetic fields, e.g., produced by on-chip conducting lines ,41,42,43et al.  and Laga [et al.  for spin [et al.  designedet al. [on). However, if the fields are switched off, the beads begin to diffuse apart, resulting in a broader concentration distribution (Coff) after a few seconds of diffusion. When the fields are reactivated, the beads are once again transported to the minima. Due to the asymmetric shape of the potential, a net transport of beads is achieved.However, magnetic fields can be utilized even further. Instead of just assuring contact between bead and sensor surface, they can assist in the transport of beads, rendering microfluidic pumps unnecessary. Lee et al.  developeet al. . In thisAs the diffusion times strongly depend on the hydrodynamic radius of the beads, a separation by size is possible if appropriate time intervals for the switching of the field are chosen. However, the sample size is restricted to objects on the micrometer scale, as magnetic forces are volume dependent and need to overcome the Brownian motion of the particles. On the one hand, objects in the range of a few nanometers show such a strong diffusion that is is not possible to generate strong enough magnetic field gradients within handheld devices. For beads with a diameter above 10 µm, on the other hand, diffusion becomes negligibly small, hindering efficient transport. This problem can be circumvented by modifying the device into a so-called “rocking ratchet” structure . Here, aHowever, utilizing current carrying microstructures to manipulate bead positioning also possesses some disadvantages. First of all, conducting lines have to be structured on-chip and need contacting. Depending on the complexity of the wire structure, this can significantly increase the production costs. To assure that beads contact the sensor surface, conducting lines need to be structured next to the sensors. If they were to be structured right on top of the sensor, an additional layer of insulating material would be required, thus increasing the distance between beads and sensor. This would result in a decreased signal intensity. Thus, the maximum field strength is limited. High field strengths would collect beads right on top of the conducting lines, away from the sensor. Thus, there is a trade-off between field strength (and thus capture range) and resolution. Either the field strength is high and beads in a large liquid volume are attracted but deposit on the conducting lines, or the filtered liquid volume is smaller and the deposition less accurate, leading to more beads being deposited on the actual sensor surface. Additionally, Joule heating caused by the wires might influence sensitive measurements or deteriorate temperature-sensitive analytes.et al. [l the channel’s height drops from h1 to h2 and broadens in width from a1 to a2, forming a ramp-like structure. Instead of employing magnetic fields to draw beads to the sensor surface, hydrodynamic effects caused by variations in the channel geometry can be utilized to support the bead capture process. Weddemann et al. ,47 calcuet al. predicted an increase in the capture rate of beads by more than 100%. However, the overall fraction of captured beads still remains small compared to methods that apply the magnetic approach. In a straight channel, beads only change their altitude through diffusion and buoyancy. In the ramp structure, the fluid gains an additional motion in the z-direction. The fluid profile drags beads at high altitudes faster towards the sensor surface than beads at low altitudes. This way, Weddemann Furthermore, while this approach circumvents the problems of producing magnetic gradients by conducting lines, as mentioned above, it increases the complexity of the channel system, as a three-dimensional design is required. Additionally, the whole concept of collecting beads only works for slow velocities up to a few hundreds of µm/s, depending on the remaining parameters  layers are separated by a third, non-magnetic (nm) layer. The giant magnetoresistance effect (GMR effect) was found and originally studied in magnetic multilayer systems ,57. Its However, GMR sensors require well defined surfaces and well controlled lithography. They need to be integrated into a µTAS structure, including microfluidic pumps for low flow velocities. This increases production costs for the chips, often preventing a successful market introduction. However, newly developed printing methods employing GMR ink could change that . As an aThe possibility of printing gel allows for the realization of granular gel-GMR sensors without employing sputtering or lithography processes. The complexity of the production of such sensors could thus be reduced, leading to a more rapid and less expensive fabrication compared to conventional devices. Besides, biogels such as alginates are abundant in supply and available at low cost. The mechanical flexibility of the matrix might simplify the application in specific geometries or on flexible materials such as paper, thus enabling new lab-on-paper technologies. Finally, the large effect amplitudes will ensure high sensor sensitivity.etc.) with a bead solution containing the marker beads and antibodies bound on the bead surface, the activated strip could be dipped into the mixture. Antigens in the solution would link antibodies on the bead surface and antibodies on the gel, thus resulting in beads that are bound to the gel surface. Subsequently to a washing step, the strip would be inserted into a standardized magnetic field. The developing stray field of the beads would alter the resistance of the GMR gel, thus allowing the direct measurement of the bead density on the gel surface. As the mixing takes place under macroscopic flow conditions (no laminar flow), the usual problem of contact between beads and surface could be circumvented. However, repulsive double-layer forces would have to be prevented by careful choice of the surface materials. Due to the simplified fabrication of these strips, the production costs of this µTAS system could possibly be low enough to allow for mass production. With the help of these gel-based GMR sensors, a new type of paper-based immunoassay µTAS systems could be developed. Instead of structuring sensors on the bottom of a microfluidic channel, spots of GMR gel could be deposited on a small paper strip, similar to a pH test strip. The gel has to contain functional groups that assemble on the surface of the gel and are able to bind antibodies see . Dipping


Southern rice black-streaked dwarf virus (SRBSDV), a new member of the genus Fijivirus, is a double-stranded RNA virus known to lack poly(A) tails. We now showed that some of SRBSDV mRNAs were indeed polyadenylated at the 3' terminus in plant hosts, and investigated the nature of 3' poly(A) tails. The non-abundant presence of SRBSDV mRNAs bearing polyadenylate tails suggested that these viral RNA were subjected to polyadenylation-stimulated degradation. The discovery of poly(A) tails in different families of viruses implies potentially a wide occurrence of the polyadenylation-assisted RNA degradation in viruses. Vaccinia virus (a double-stranded [ds] DNA virus) [Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Odontoglossum ring-spot virus (ORSV), Cucumber green mottle mosaic virus (CGMMV), Tobacco rattle virus (TRV), Turnip crinkle virus (TCV) and Tobacco necrosis virus (TNV) [RNA of many eukaryotic viruses, ranging from DNA to RNA viruses, have 3' poly(A) tails , which aA virus) . Furtherus (TNV) , seven pus (TNV) . The preFijivirus of the family Reoviridae [Rice black-streaked dwarf virus (RBSDV), which is also a member of the Fijivirus genus [18 to prime cDNA synthesis as templates [SRBSDV has been proposed as a new member in the genus oviridae , which coviridae ,12. SRBSus genus ,13. SRBSus genus . P7-1 inus genus . P9-1 isus genus . Howeverus genus . P8 and us genus . SRBSDV emplates , suggestSRBSDV isolate used in the experiment was obtained from rice and maize plants showing typical dwarf symptoms with white waxy galls in 2014 in 8 counties of 4 provinces in China, including Yunnan, Guizhou, Hunan, and Jiangxi provinces. Total RNA from infected rice and maize leaf and stem tissue were extracted following the standard protocol of TRIzol reagent . The isolate was identified as SRBSDV excluding RBSDV by reverse transcription RT-PCR using specific primers for distinguishing the two viruses [To confirm characterization of the polyadenylate tails associated with viral mRNAs, the 3' Rapid Amplification of cDNA End (RACE) PCR was performed using BD SMART™ RACE cDNA Amplification Kit . In this case, reverse transcription reactions were performed using total RNA (respectively from infected rice and maize) as templates and adapter-oligo(dT) primer (P1)  to primeAfter 3' RACE, the 3'-termini sequences of viral mRNAs were obtained, and the results indicated that SRBSDV mRNAs indeed possessed ploy(A) or poly(A)-rich tails in plant hosts. Taking S10-mRNA as an example to analyze the nature of poly(A) and poly(A)-rich tails, a total of 42 polyadenylated viral mRNA molecules were cloned from rice and maize plants. In addition to 10 mRNAs bearing poly(A) tails exclusively comprised of adenosines, a large number of mRNAs possessed poly(A)-rich tails B. NotablTo our knowledge, dsRNA viruses are lack of poly(A) tails at the 3'-ends of the genome segments and their mRNAs. Interestingly, in this paper, we demonstrated that some viral mRNA molecules were polyadenylated at their 3'-terminus in plant cells infected with SRBSDV (a dsRNA virus). Besides their crucial roles for mRNA stability and translation efficiency, the polyadenylate tails were recently described as involved in viral RNA degradation . The PolVaccinia virus, was linked with the conserved RNA degradation mechanism, and non-abundant, fragmented viral mRNAs bearing poly(A) or poly(A)-rich tails were detected in human cells infected with this virus [In this study, the non-abundant presence of SRBSDV mRNAs bearing polyadenylate tails was considered to represent degradation intermediates of an RNA decay pathway, rather than to convey protection to mRNAs. Recently, a dsDNA virus, is virus . Such pois virus . The dis


Salmonella control strategies in Sweden. Current control strategies in Denmark and the Netherlands were used as benchmarks. The true number of human Salmonella cases was estimated by reconstructing the reporting pyramids for the various scenarios. Costs were calculated for expected changes in human morbidity , for differences in the control programmes and for changes in cattle morbidity. The net effects (benefits minus costs) were negative in all scenarios (€ −5 to −105 million), implying that it would not be cost-effective to introduce alternative control strategies in Sweden. This result was mainly due to an expected increase in the incidence of Salmonella in humans (6035–57108 reported and unreported new cases/year), with expected additional costs of € 5–55 million. Other increased costs were due to expected higher incidences of sequelae (€ 3–49 million) and a higher cattle morbidity (€ 4–8 million). Benefits in terms of lower control costs amounted to € 4–7 million.The objective of the study was to analyse the economic effects of introducing alternative  Salmonella. Due to this, EU requirements for Salmonella control in poultry have been met for Swedish poultry long before joining the EU. A severe outbreak of S. Typhimurium in Sweden (SE) in 1953 that involved more than 9000 people prompted the need for a control programme for Salmonella. Since then, the strategy for control has been to prevent Salmonella in any part of the production chain. The current Swedish control programme covers the entire food chain from feed to food Salmonella in animals, animal products or feed is notifiable according to the Swedish law on zoonoses , and measures to eliminate/eradicate Salmonella are taken at any positive finding. Restrictions are put on infected holdings until they can be declared free from Salmonella. The mandatory on-farm eradication is partly funded by the Board of Agriculture (with tax money) and the remaining cost is paid by the affected farmer. The level of co-funding varies with animal species and whether the farm is affiliated to a voluntary control programme. No funding is given for eradication on broiler holdings or beef herds buying animals from more than five sources. For other food-producing animals, 50% of documented costs are covered by the government, unless the farm is affiliated to a voluntary control programme, in which case up to 70% of documented costs may be covered by the government. The cost of HACCP controls in feed mills is paid by the feed producers . The costs for the control in animal products are paid for by the food industry.In addition to the common legislative framework for food safety in the European Union (EU), Sweden has a salmonella control programme that aims to ensure that domestic food of animal origin is entirely free of all Salmonella control was attempted in 1993 Salmonella programme has been in place for many years and a large amount of money is spent on it, an economic evaluation of the programme has been requested on several occasions. On EU-level, a consortium was recently commissioned to perform cost-benefit analyses of reducing the Salmonella prevalence in slaughter pigs and breeding pigs, respectively Salmonella control but also highlight a substantial lack of data. It is important to try to estimate the benefits and costs of reducing the Salmonella prevalence in countries with a high prevalence of Salmonella in food-producing animals. However, in countries where a well functioning and efficient Salmonella control is already in place, analysing a potential relaxation of the current control measures is more relevant. In 2011, based on the above, the SE Salmonella committee  commissioned the authors of this study to perform an evaluation of the costs and benefits of the SE control programme as compared to other less costly options. As it is not known what effect a change in different parts of the SE Salmonella control program would have on public health it was decided to compare the SE programme with other existing, less costly, control programmes. In Salmonella control programmes in Sweden were estimated. The current paper estimates the underdetection and underreporting of Salmonella cases in SE and analyses possible economic effects of introducing these alternative Salmonella control strategies in SE.A general estimation of the costs and the benefits of the SE Salmonella control programmes in Sweden  were calculated. These calculations included costs for 1) an increasing number of domestic human cases of Salmonella and sequelae 2) changes in surveillance and control programmes and 3) production losses in cattle due to S. Dublin. The costs in 1) were based on estimations of expected increases in the number of reported domestic cases in four of the five different scenarios developed in true numbers of cases by a reconstruction of the reporting pyramid in each scenario. The methodologies involved in this reconstruction are discussed in more detail below.The economic effects of a hypothetical implementation of the Danish and Dutch Salmonella cases in SE were estimated in Salmonella incidence in SE would become equal to the incidences in DK and NL, respectively, based on the five different data sources. As discussed by the authors, one of these data sources (based on travel data), yielded unrealistically high estimates and has thus been excluded from the cost analysis in the current paper.Changes in the expected number of reported domestic Salmonella cases only constitutes a minor part of all clinical Salmonella cases. All Salmonella cases that have some degree of clinical symptoms  may lead to direct and indirect costs. In order to estimate the true number of salmonellosis cases, five different stages, at which information regarding a salmonellosis case might get lost, were identified: (1) no medical care is sought, (2) sample is not taken, (3) sample is not analysed for Salmonella, (4) non-perfect test sensitivity and (5) positive test is not reported. In addition, two factors were identified that affects the probabilities of information losses; 1) having bloody diarrhoea was assumed to affect the probability of a) seeking care and b) having stool samples taken, and 2) the type of unit  carrying out the examination was assumed to impact the probability of stool samples a) being taken and b) being analysed for Salmonella. The probabilities of having information losses at any of these five different stages were modelled by betapert distributions with parameter values based on expert opinion . Data regarding reported hospitalized cases (2010) and deaths (1998–2008) were obtained from databases at the National Board of Health and Welfare in Sweden. The proportions of salmonellosis cases allocated to each of these outcome classes were calculated using the model described in Two sequelae of salmonellosis were accounted for in this study: Irritable Bowel Syndrome (IBS) and Reactive Arthritis (ReA). Because of a high background prevalence of IBS, attributable risk (AR) (where risk for controls is subtracted from risk for cases) was used to estimate the expected proportion of salmonellosis cases leading to post-infection (PI) IBS see . The estThe total number of cases of either sequelae was then partitioned into the three different outcome classes . Since neither of these illnesses has been associated with mortality, ‘death’ was excluded from the possible outcome sets. For PI-IBS, the proportions of cases seeking care were based on a SE population study i.e. costs that arise as a direct consequence of an illness) included costs for medication, GP visits, hospitalizations and transports to and from care units. The data sources used to estimate these direct costs are specified in i.e. production losses that arise as a consequence of sick leave) were estimated as the net salaries for all salmonellosis cases (as well as for the parents of child cases) during the sick leave period according to neo-classical wage theory The total costs for a change in the number of salmonellosis cases were calculated as the sum of 1) direct costs, 2) indirect costs and 3) intangible costs. Firstly, direct costs , was used. However, remaining payments (i.e. outstanding claims received within the period 2005–2010) for the feed-borne outbreak of Salmonella Cubana in 2003 The current programme includes surveillance in the entire chain from feed to food, and eradication measures whenever ment see . To estiThe total costs for the current compulsory and voluntary control programmes run by the industry were obtained from each organisation, respectively. Detailed figures were provided by the Swedish Dairy Association, the Swedish Animal Health Services the Swedish Poultry Meat Association and the Swedish Egg and Poultry Association.i.e. those items without certification of Salmonella freedom according to the SE Salmonella guarantees) were obtained from the financial department of the National Food Agency.The current costs for surveillance in slaughterhouses and cutting plants, and for extra import controls of food items , and these additional costs were therefore not included.Surveillance for Salmonella analyses and postage. To assess the sensitivity of the final estimate of the cost for laboratory analyses, the calculations were also performed with assumed prices of € 7 for serology (serum/milk) and € 17 for bacteriology.The costs for a control programme for pigs and cattle according to the current practice in DK and NL, respectively, were re-calculated using the SE animal population. The costs for the DK feed control were also included in the DK scenarios. The costs for laboratory analyses were calculated based on the current prices in the national laboratories, and a discount was only assumed in case of a very large number of samples. Labour costs for sampling and sending samples were not included in the calculations, due to the difficulty of estimating these costs. Costs of € 12 for each serological analysis of serum or milk samples and € 23 for each culture sample were assumed in the DK scenarios. In the NL scenarios, where the total number of samples were fewer and thus no discount on laboratory prices or postage was assumed, € 14 for a serum/milk sample and € 35 for a culture sample were used in the calculations. These costs match the current SE prices for Information about the NL surveillance for the relevant time period was provided by GD Animal Health, Deventer, NL. Information about the DK surveillance was obtained from reports publicly available Sampling in the food chain without the current control programme would be based on the microbiological criteria as stated in the EU legislation. The National Food Agency provided estimates for sampling necessary to fulfil the EU requirements. Detailed cost estimates are provided in S. Dublin if the current control was to be discontinued have been previously estimated S. Dublin prevalence in this report, 10%, was used in the current study. This level was regarded as realistic as it corresponds to the situation in the region with the highest prevalence in Sweden. Detailed cost estimates are provided in Production losses due to In true number of salmonellosis cases. Using this procedure, the mean true number of salmonellosis cases in SE, i.e. after corrections for underdiagnosis and underreporting, was estimated to increase with between 6035 and 15782 for the DK scenarios and between 42888 and 57108 for the NL scenarios  are all positive. This combined effect thus represents a benefit of introducing alternative control strategies  production losses accrue as long as the sick leave persists (until retirement). The alternative approach, called the friction cost approach, assumes that wages are not completely flexible and, as a consequence, a limited pool of involuntary unemployment does exist Salmonella prevalence in one domestic species will affect the prevalence in other species and the environment. For this reason, this study aims to assess the overall situation and how it affects the prevalence and consequent health costs in humans. Domestic cases in humans may stem from sources other than domestic animals and the effect of control in animals may thus be overestimated. However, controlling Salmonella in domestic animals is expected to affect other sources of the infection that often have their origin in domestic animals.The costs for surveillance and control in different animal species must be assessed together. The current Swedish control includes all animal species and it is reasonable to assess the scenarios with this approach as well. The Salmonella in feed, animal production and food only include direct costs. Indirect costs would include e.g. those arising in the dairy or meat industry due to supply shortages caused by restrictions on infected farms or hygiene measures on restricted farms required for milk collection trucks etc. Moreover, only documented costs for on-farm eradication could be included. Thus, costs for extra working hours that could not be documented, or losses due to the need to replace old but functional buildings that could not be properly cleaned as well as other non-refundable costs were not included. This may lead to an underestimation of the costs for the current Salmonella control. However, trying to include indirect costs may lead to an overestimation of the control costs, as some of those costs are associated with a future improvement in the production or other economic benefits.The calculated costs for the current control of Salmonella surveillance in the DK and NL scenarios were also based on direct costs. Any indirect costs in these scenarios were impossible to predict. However, the DK and NL scenarios only include surveillance costs and not heat treatment of poultry feed. Administrative costs for the industry's surveillance programmes were not included either, as they were hard to predict. Thus some of the expenses in the scenarios are underestimated (heat treatment of poultry feed is mandatory in the EU and the surveillance programmes in the scenarios would entail some administrative costs) and consequently the net benefit is overestimated. The surveillance figures are based on calculations of the number of samples that would be taken in the SE animal population according to the strategies in DK and NL, respectively. Substantial changes in the SE animal population would make these estimates less reliable, but it would also affect the cost for the current programme . Thus the comparison between the current control and the DK and NL scenarios would still be useful and the estimated benefits would most likely not be substantially affected. Cost for measures taken in positive herds were not included in the scenarios. These costs are difficult to estimate as they depend on the number of positive farms and on the measures taken by each farm. Losses due to restrictions in dairy herds were included in the estimate for indirect costs for S. Dublin in cattle herds. In pig herds, losses due to restrictions would perhaps be larger.The estimated benefits of introducing alternative strategies for Salmonella Dublin and by existing EU requirements according to the microbiologic criteria. There is therefore no reason to expect that retailers' supply curves would shift for these types of meat more than marginally as a result of exchanging the control programme. For pig farmers it is possible that marginal costs would decrease, at least in the NL scenario where surveillance costs are expected to be quite low. Although rather small , some losses due to this infection are already present. Again, this is difficult to assess and thus it was decided to use figures that were published and based on official calculations by the Swedish Board of Agriculture, but to include a reduced estimate for these losses as well. The laboratory costs in all scenarios were estimated based on current prices (that include postage) and some discounts for large volumes. These prices may have been slightly over- or underestimated but there is already a market in Sweden for large numbers of Salmonella analyses, paid for with private as well as government money, and the current prices are thought to reflect this situation. Reducing the laboratory prices only had a large effect on the estimate for surveillance in pigs in the DK scenarios. However, these reduced estimates may not be realistic as they  do not include sampling costs. The calculations of the current costs include costs for sampling and for sending the samples to the laboratory.As the aim of this study was not to design an optimized alternative control strategy for SE, different scenarios for control of The calculations of the costs of ceasing the current control of cattle include indirect costs and are therefore not directly comparable with the costs for the control (see above). The calculations are, however, highly relevant for the SE situation and the comparison with health benefits in humans is considered valid. Thus it was regarded as more valuable to keep the indirect costs in the estimates than to exclude them. However, halving this estimate had the largest effect on the final cost for the alternative surveillance in both the DK and NL scenario.Salmonella control in animal production may have been underestimated, the size of the increased costs for human illness is by far greater than the expected size of this underestimation. A higher estimate for the cost of the current control would not be expected to change the main result that it is favourable to keep the current control.Although the estimated benefits of ceasing the current Appendix S1Calculation of costs for human cases of salmonellosis.(DOCX)Click here for additional data file.Appendix S2Variables used in the Monte Carlo simulations to estimate costs for the true number of human domestic salmonellosis cases.(DOCX)Click here for additional data file.Appendix S3Variables used in the Monte Carlo simulations to calculate the number of Salmonella-related Reactive Arthritis (ReA) cases.(DOCX)Click here for additional data file.Appendix S4Variables used in the Monte Carlo simulations to calculate the number of Salmonella-related IBS cases.(DOCX)Click here for additional data file.Appendix S5Data sources used to fit a beta distribution for the proportion of salmonellosis cases with bloody diarrhea.(DOCX)Click here for additional data file.


Chrysochloris asiatica). The identification of CaEFV reveals the presence of foamy virus in the placental mammal superorder Afrotheria. Phylogenetic analyses place CaEFV basal to other foamy viruses of Eutherian origin, suggesting an ancient codivergence between foamy virus and placental mammals. These findings have implications for understanding the long-term evolution, diversity, and biology of retroviruses.Endogenous retroviruses provide important insights into the deep history of this viral lineage. Endogenous foamy viruses are thought to be very rare and only a few cases have been identified to date. Here we report a novel endogenous foamy virus (CaEFV) within the genome of the Cape golden mole ( Foamy viruses are complex retroviruses, which are typically nonpathogenic and infect a variety of placental mammals, including primates, cats, cows, bats, and horses Chrysochloris asiatica), which we designate ‘Chrysochloris asiatica endogenous foamy virus’ (CaEFV). This finding provides strong evidence that foamy viruses were already present in the most recent common ancestor of all placental mammals ∼100 million years ago.The steady accumulation of additional animal genome sequences currently offers a great opportunity to discover novel endogenous foamy virus-like elements, which could provide important insights into the evolutionary history and biology of foamy viruses. Here we report the discovery of an endogenous foamy virus within the genome of a small, insectivorous mammal native to southwestern South Africa, the Cape golden mole (Rhinolophus affinis foamy virus (JQ814855), spider monkey simian foamy virus (EU010385), gorilla simian foamy virus (HM245790), chimpanzee simian foamy virus (NC_001364), macaque simian foamy virus (NC_010819), and African green monkey simian foamy virus (NC_010820).All whole-genome shotgun sequences from animals available from NCBI were screened for endogenous foamy viruses using the TBLASTN algorithm and the protein sequences of representative foamy viruses. The following representative foamy viruses were used: bovine foamy virus (NC_001831), equine foamy virus (NC_002201), feline foamy virus (NC_001871), Protein sequences were aligned using MUSCLE pol and env genes) insertion (Chrysochloris asiatica endogenous foamy virus), along with various other retroviruses shows that CaEFV groups with foamy viruses with robust support , we cannot estimate the insertion time of CaEFV into the Cape golden mole genome. However, the presence of multiple premature stop codons suggests the invasion occurred long time ago (see ref 6 for discussion of a similar case) .To further determine the relationship between CaEFV and other endogenous and exogenous foamy viruses, we reconstructed phylogenetic trees using conserved regions of the Env protein. Our phylogenetic analysis shows that CaEFV is basal to other exogenous and endogenous foamy viruses of Eutherian origin . PlacentThe foamy virus phylogeny does not exactly match the species phylogeny . NeverthExogenous foamy viruses have been found exclusively in the superoder Laurasiatheria  and Euarchontoglires (such as primates) Table S1BLASTP results using CaEFV Env protein as a query .(DOCX)Click here for additional data file.Table S2PSI-BLAST results using CaEFV Env protein as a query .(DOCX)Click here for additional data file.Dataset S1The conserved region of Pol proteins of CaEFV and other retroviruses.(FAS)Click here for additional data file.Dataset S2The conserved region of representative endogenous and exogenous foamy virus Env protein.(FAS)Click here for additional data file.Dataset S3Amino acid sequences of CaEFV Pol and Env proteins.(FAS)Click here for additional data file.


Culicoides spp. specimens collected at the China-Laos-Myanmar border in southern Yunnan Province. This virus had a cytopathic effect (CPE) on both insect cells (C6/36) and mammalian cells (BHK-21). Electron microscopy revealed the structure of the virions to be spherical with a diameter of 75 nm. Polyacrylamide gel analysis demonstrated that the viral genome consisted of 10 segments of double-stranded RNA (dsRNA), with a distribution pattern of 3-3-3-1. The coding sequences of 9 genome segments of YN12246  were obtained by high-throughput sequencing and Sanger sequencing. Comparisons of conserved genome segments 1 and 3 (Seg1 and Seg3), encoding the polymerase-VP1 and sub-core T2 protein, respectively, showed that YN12246 groups with the Culicoides-borne orbiviruses. The highest levels of sequence identity were detected between YN12246 and Tibet orbivirus (TIBOV), indicating that they belong to the same virus species . The data presented here confirm that YN12246 is a member of the TIBOV species, which was first isolated from mosquitoes in 2009. This is the first report of the isolation of TIBOV from Culicoides.We isolated a novel virus strain (YN12246) from  Orbivirus is the largest of 15 genera within the family Reoviridae, currently containing 22 recognized virus species [Culicoides, mosquitoes and sand flies), and have a wide host-range that collectively includes domestic and wild ruminants, equines, marsupials, sloths, bats, birds and humans [Culicoides-borne, according to evolutionary data [Bluetongue virus (BTV), African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV) are transmitted by adult Culicoides and are regarded to be the most important orbiviruses with respect to economics [Broadhaven virus  [St. Croix River virus  [The genus d humans –5. Previconomics , 6. Althspecies)  and St. species) . Comparispecies) .Tibet orbivirus (TIBOV) was first isolated by our laboratory from Anopheles maculatus mosquitoes collected in 2009 from Motuo County in Nyingchi Prefecture, Tibet, China. Molecular genetic analysis revealed that this virus was a new species within the genus Orbivirus [Tibet orbivirus. Although this virus was first isolated from mosquito specimens, molecular evolutionary genetics analysis revealed that it was not closely related to the mosquito-borne orbiviruses, and was instead a better fit for the Culicoides-borne group. Because only one strain (XZ0906) was available for analysis, it was difficult to determine the propagation characteristics of TIBOV.The Culicoides spp. specimens, and identified it as a TIBOV strain. Here, we describe the sequence analysis of polymerase (Pol) and T2-subcore-protein of TIBOV. Phylogenetic comparisons with different orbiviruses indicate that TIBOV belongs to the Culicoides-borne orbivirus group. This is the first report of TIBOV isolated from Culicoides.During a survey of arboviruses in Yunnan Province in 2012, our laboratory isolated a virus strain (YN12246) from a pool of Aedes albopictus cells and BHK-21 baby hamster kidney cells were used in this study [3 and 100 U/mL of penicillin and streptomycin. BHK-21 cells were maintained at 37°C in the presence of 5% CO2.C6/36 is study . Both ceCulicoides samples were collected in the summer of 2012 in Daluo, Xishuangbanna Dai Autonomous Prefecture, Yunnan province, China. The specimens were frozen to death in a -20°C freezer, then classified and identified on ice, dispensed into cell-freezing tubes, recorded and labeled, stored in liquid nitrogen and finally transported to our laboratory. Specimens were then homogenized, inoculated and blind passaged three times (7 days per cycle) on monolayers of C6/36 and BHK-21 cells in 24-well plates, as previously described [escribed . The celThe culture supernatant from infected BHK-21 cells showing signs of CPE was centrifuged at 8000 rpm for 20 min at 4°C. The resulting supernatant was further centrifuged at 30,000 rpm for 2 h to remove cellular debris. The final supernatant was fixed in 2% glutaraldehyde and processed for negative-stain electron microscopy with 2% phosphotungstic acid.Viral RNA was isolated as described previously, and analyzed by polyacrylamide gel electrophoresis (PAGE) .Tibet orbivirus (TIBOV) [Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen), and cDNA was synthesized using Ready-To-Go You-Prime First Strand Beads , according to the manufacturer’s procedure. Samples were subjected to Reverse-transcription polymerase chain reaction (RT-PCR) with specific primers designed to detect the 12th segment of Banna virus (BAV) , the 12t (TIBOV)  . Viral RNA was extracted from 140-μL aliquots of virus-infected BHK-21 cell culture supernatant using a QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA was made with a Ready-To-Go kit  using random hexanucleotide primers. Samples were then amplified as described previously . AmplifiRT-PCR was performed to fill in gaps between viral gene sequences obtained with Ion Torrent sequencing using contig-specific primers. Total viral RNA was extracted as described above, cDNA was generated by reverse transcription, and used as a template for complete genome amplification. Next, a set of specific primers was designed to amplify each segment of the viral genome and the amplification products were sequenced using the Sanger method .Reoviridae and Orbivirus sequences were used for phylogenetic analysis. The background information of all virus strains used in this study is shown in Multiple sequence alignments were performed using the Clustal X 1.8 software. Phylogenetic and molecular evolutionary analyses were conducted using the Mega 5.05 software. The phylogenetic tree was generated using the neighbor-joining (NJ) method, distance-p model and 1000 replications of bootstrap values. The sequences obtained in this study were compared with those available in public databases, and representative Culicoides spp. homogenized supernatants was inoculated onto monolayers of C6/36 and BHK-21 cells, yielding a virus isolate designated YN12246. The supernatant of YN12246 resulted in CPE in C6/36 and BHK-21 cells in successive cell passages. By 48 h post-infection, shrinkage, aggregation and shedding were observed in C6/36 cultures, and rounding, lysis and shedding were observed in BHK-21 cells  and Seg3 (713 bp long) of the TIBOV genome, generated cDNA of the expected sizes (885 bp for Seg1 and 706 bp for Seg3) from the YN12246 template. However, reactions using primers designed to amplify BAV, LNV, KDV and YUOV all failed to generate PCR amplicons. Sequence analysis showed that Seg1 and Seg3 from YN12246 has a high level of nucleotide sequence identity , and a high level of aa identity , with TIBOV. Sequence analyses of Seg1 and Seg3 and comparisons with published data, demonstrated that the YN12246 virus from Yunnan is a strain of TIBOV.Culicoides-isolated strain (YN12246) and mosquito-isolated strain (XZ0906) is among 9–135 bp, the difference between Seg1 and Seg8 is above 100 bp, the difference between Seg3 and Seg10 is within 100 bp. It means that there is a big difference in the genome length among TIBOV isolated from different vectors. Besides, there is a larger difference (63.6–99.6%) in the homologous aa with each segment between Culicoides-isolated strain (YN12246) and mosquito-isolated strain (XZ0906). Seg1, 3, 4, 5, 7 and 9 of YN12246 exhibited a relatively high aa identity (95.1–99.6%) to their homologues in the XZ0906. Seg8 and 10 are slightly lower than that aa sequence identity respectively for 81.1% and 74%, and the identity between YN12246 and XZ0906 is 63.6% in Seg 6 .The whole coding sequences of 8 segments of YN12246  and partial sequences of Seg6 were obtained by high-throughput sequencing and Sanger sequencing. Differences were observed in both the nucleotide and amino acid sequences of virus YN12246 relative to XZ0906 . The resThe sequences of Seg2 and partial sequences of Seg6 of YN12246 were not obtained by high throughput sequencing and gene specific amplification see the  section.Orbivirus, including BTV, EHDV, YUOV, Pata virus (PATV), Palyam virus (PALV), and SCRV, was obtained from NCBI GenBank. The Seg1 and Seg3 sequences were downloaded and compared with those of YN12246.Whole genome sequence information of representative members of genus Amino acid sequence analysis of VP1 protein , encoded by Seg1, revealed 98.8% sequence similarity between YN12246 and the TIBOV strain XZ0906, compared to between 35.4% (SCRV) and 73% (PATAV) similarity with the six representative orbiviruses. In the case of T2 protein, encoded by Seg3, sequence similarity between YN12246 and the TIBOV strain XZ0906 was of 96.4%, compared to between 23.3% (SCRV) and 78.3% (PATAV) similarity with the six representative orbiviruses .Reoviridae were obtained from GenBank, and used to construct a phylogenetic tree. These 37 virus strains (including different species and different serotypes of one species) readily clustered into 14 evolutionary branches, with YN12246 clustering within the genus Orbivirus branch  is the most conserved of the orbivirus genes. The T2 protein of he genus . T2 aminOrbivirus species include the reassortment of genome segments, genome segment migration patterns during 1% agarose gel electrophoresis (AGE), conserved terminal nucleotide sequences, serological cross-reactions, comparison of homologous genome segments or proteins by sequence analysis or cross-hybridization, host and vector range and the nature of clinical signs induced [The parameters stipulated by the International Committee on Taxonomy of Viruses (ICTV) for the ‘polythetic definition’ of individual  induced . Recent  induced , 19.Orbivirus. The RdRp protein sequence, encoded by genome segment 1 (VP1), is an important criterion for the identification of members of genus Orbivirus [Orbivirus members. These data indicate that YN12246 belongs to genus Orbivirus. In addition, the sub-core protein (T2) of Orbivirus is used to classify serotypes within the genus. It has also been demonstrated that orbiviruses within a single species group have more than 91% identity with respect to the T2 protein aa sequence [Orbivirus species. These data indicate that YN12246 and XZ0906 belong to a single serotype. From the phylogenetic analysis of family Reoviridae and genus Orbivirus, based on the VP1 and T2 protein aa sequences, it is possible to conclude that YN12246 is a member of the TIBOV species.The genetic sequences of VP1 and T2 can be used to define new species within the genus rbivirus , 8. PrevOrbivirus by bioinformatics analysis. RT-PCR amplification was used to close the gaps between contigs for each segment. Ultimately, the whole coding sequences of 8 segments of YN12246  and partial sequences of Seg6 (660–1633) were available. Then Sanger sequencing was employed to confirm the sequences using primers newly designed for each of the 9 RNA segments  was obtained relative to the genus segments . HoweverCulicoides–isolated strain (YN12246) and mosquito-isolated strain (XZ0906) were 98.8% for VP1 and 96.4% for VP3 (T2 protein) but only 63.6% within the VP6 gene , exhibited the lowest homology. On the other hand, the failure to obtain the sequence of Seg2 by high-throughput sequencing indicates a very low homology in the VP2 gene between Culicoides-isolated strain and known orbivirus. Accordingly, Seg2 may be the hypervariable region in TIBOV, further research will be carried out. We will continue to carry out the sequencing work of Seg2 and Seg6, in order to obtain the whole genome sequence of the virus YN12246.Previous studies have shown that the VP1 and VP3 proteins (encoded by Seg1 and 3 respectively) of BTV are highly conserved, while the components of the BTV outer capsid, proteins VP2 and VP5 (encoded by Seg2 and 6 respectively), are the most variable of the viral proteins. VP2, in particular, contains neutralising epitopes and controls the interactions between the virus particle and neutralising antibodies, exhibits a relatively low amino acid identity among the different BTV serotypes . Amino aOrbivirus can be divided into three groups, namely Culicoides-borne orbiviruses (such as BTV), mosquito-borne orbiviruses (such as YUOV) and tick-borne orbiviruses  [Culicoides clusters onto the same branch as the TIBOV strain XZ0906 isolated from mosquitoes, in addition to BTV and EHDV, which are transmitted primarily by Culicoides, but is phylogenetically distinct from YUOV, UMAV (Umatilla virus) and SLOV (Stretch Lagoon virus), isolated from mosquitoes, as well as GIV, BRDV and LIPV (Lipovnik virus) isolated from ticks , Tilligery virus (TILV), and PATAV were initially isolated from mosquitoes, EUBV has also been isolated from Culicoides, and the evolutionary relationship based on the analysis of the T2 subcore shell protein indicates that these three viruses group more closely with the Culicoide-borne orbiviruses, suggesting that Culicoides may act as their biological vector [The cumulative genetic information provided by many studies has confirmed that us; GIV) , 19. Theom ticks . YN12246l vector . There iOrbivirus species/serogroup usually have similar dsRNA migration profiles in 1% agarose gel electrophoresis (AGE) [Orbivirus [Culicoides, has a 3-3-3-1 RNA migration pattern almost identical to that of XZ0906, isolated from mosquitoes [Viruses within a single is (AGE) . The virrbivirus . YN12246squitoes , indicatCulicoides was infectious to pigs, although the results of IgG antibody positive need to be verified by neutralization test.The indirect immunofluorescence assay (IFA) was applied to detect the IgG antibodies of swine serum samples collected in the virus isolation region against YN12246 strain. The results showed that a special fluorescent reaction appeared between YN12246 virus and swine serum, the antibody positive rate was 14.0% (8/57), while no positive for the serum samples collected from other regions was detected (data not shown). The results suggest that the TIBOV isolated from Culicoides and collected in Yunnan, could replicate in insect cells (C6/36) and mammalian cells (BHK-21). While TIBOV XZ0906 only caused CPE in BHK-21 cells, and not in C6/36 cells, TIBOV YN12246 caused CPE in both cell types. This result may reflect phenotypic differences between strains of one virus species isolated from different vectors. Further study is required to investigate this phenomenon further. In addition, although CPE progressed quickly after viral inoculation of cell cultures, with up to 75% of BHK-21 cells showing CPE after 3–4 days of YN12246 infection, multiple plaque formation revealed a relatively low virus titer. The titer of the TIBOV strain YN12246 was approximately 1.0 × 104–1.5 × 104 PFU/mL, similar to the XZ0906 strain (data not shown). This low virus titer might represent a defining characteristic of the TIBOV orbivirus.Both TIBOV XZ0906, isolated from mosquitoes, and TIBOV YN12246, isolated from S1 Table(DOCX)Click here for additional data file.


Here we report that acute CO2 exposure leads to a rapid cessation in the contraction of the pharynx muscles in Caenorhabditis elegans. To uncover the molecular mechanisms underlying this response, we performed a forward genetic screen and found that hid-1, a key component in neuropeptide signaling, regulates this inhibition in muscle contraction. Surprisingly, we found that this hid-1-mediated pathway is independent of any previously known pathways controlling CO2 avoidance and oxygen sensing. In addition, animals with mutations in unc-31 and egl-21 (neuropeptide secretion and maturation components) show impaired inhibition of muscle contraction following acute exposure to high CO2 levels, in further support of our findings. Interestingly, the observed response in the pharynx muscle requires the BAG neurons, which also mediate CO2 avoidance. This novel hid-1-mediated pathway sheds new light on the physiological effects of high CO2 levels on animals at the organism-wide level.Carbon dioxide (CO 2) is a key molecule in many biological processes. High levels of CO2 in patients with pulmonary diseases are associated with worse outcomes. However, mechanisms by which organisms sense and respond to high CO2 levels remain largely unknown. Using Caenorhabditis elegans as a model system, we found that exposure to high CO2 levels leads to a very rapid cessation in the contraction of the pharynx muscles. Further analysis revealed that the pharynx muscle response is controlled by dense core vesicle secretion from the BAG neurons in a hid-1-mediated pathway. This novel hid-1 pathway sheds new light on the physiological effects of high CO2 levels on animals at the organism-wide level.Carbon dioxide (CO Of particular importance is the ability of many animals to sense and respond to high levels of CO2 by either attraction or aversion 2 (hypercapnia) impair alveolar epithelial function of the lungs by activating the stress sensor AMPK, which leads to Na,K-ATPase endocytosis, impaired cell proliferation, and loss of distal lung epithelial function Drosophila and mice, which increases mortality in a model of pneumonia and leads to changes in gene expression through the NF-kB pathway 2 levels One of the fundamental features shared by most, if not all, living organisms is the ability to maintain levels of carbon dioxide –sensing BAG neurons, in which the guanylyl cyclase receptor, gcy-9, controls the response to CO2gcy-9 in the BAG neurons and plays a role in BAG neuron differentiation 2 sensing and avoidance 2; whereas BAG neurons reach maximal activation within 30 s, ASE neurons reach maximal activation only after 2 min, and AFD neurons show intricate dynamics in which Ca2+ levels first drop and then increase to maximal levels after 2 min C. elegans do not avoid high CO2 levels, nor do animals with defects in the daf-2 signaling pathway, which is an important regulator of the starvation response High levels of COC. elegans exposed to high CO2 levels show specific phenotypes independent of pH In addition to avoidance, C. elegans animals to short (10 s) hypercapnia-inducing levels of CO2 (≥5%) leads to a significant reduction in the rate of pharyngeal muscle contraction (pumping). Strikingly, this effect is independent of any currently known molecular pathways that regulate CO2 avoidance or O2 sensing. Specifically, through a forward genetic screen, we identified a novel participant in the response to CO2, hid-1, that plays a role in continued pumping in the presence of high CO2 levels. Moreover, we show that dense core vesicle secretion pathways in the BAG neurons contribute to the reduced pumping rate in response to high CO2 levels.Here we report that exposure of C. elegans (N2) to high levels of CO2, we exposed 1-day-old adult animals grown on standard NGM plates in a small chamber to gas mixtures containing 21% O2 and 5%, 10%, or 20% CO2 at 22°C. In normal air (0.0391% CO2), the rate of muscle contraction of the pharynx was ∼200 pumps/min. Within 10 s of exposure to 5% CO2 balanced with 21% O2 and 74% N2, the pumping rate of the pharynx was reduced from ∼200 to ∼60 pumps/min , which catalyze the conversion of CO2 into bicarbonate, had no effect on the pumping rate  or TAX-4 (tax-4[p678]) do not avoid high CO2. In addition, the insulin-IGF pathway mediates CO2 avoidance, as daf-2 mutants show reduced CO2 avoidance osm-3 and che-10 have abnormal cilia as well as defective CO2 avoidance. In C. elegans strains carrying mutations in daf-2, osm-3, or che-10 the CO2 avoidance response is either reduced or absent 2 levels on the C. elegans pharynx is quick and robust, similar to the avoidance response. However, rather than CO2 avoidance, tax-4(p678), daf-2(e1370), osm-3(n1540), and che-10(e1809) mutants show a significant reduction in pumping following exposure to 10% CO2, similar to the reduction observed in wild-type (N2) animals  animals to 10% CO2 resulted in a response similar to that of wild-type animals , ets-5(tm1734), and ets-5(tm1755) mutants stopped pumping, similar to wild-type animals  that bind O2 and sense decreases (gcy-31 and gcy-33) or increases (gcy-35 and gcy-36) in O2 levels C. elegans strains gcy-31(ok296), gcy-33(ok232), gcy-35(ok769), or gcy-36(db42) to 10% CO2 resulted in pumping inhibition similar to that observed in wild-type animals  mutagenesis. Specifically, we screened for mutant animals that do not stop pumping in response to 10% CO2 (Movie S2). We screened the progeny of ∼1200 F1 animals and found three strains that continued pumping when exposed to 10% CO2. One of these strains was further crossed to the Hawaiian strain, and deep sequencing was performed on DNA from recombinant F2 progeny. The region containing the mutant gene that enabled continuous pumping in 10% CO2 was identified by searching for a low number of Hawaiian single-nucleotide polymorphisms (SNPs), as described elsewhere hid-1. In 5% CO2, unlike in wild-type animals, the pumping rate of animals with the isolated hid-1(yg316) allele was similar to the pumping rate in normal air conditions, and significant pumping continued after exposure to 10% CO2, whereas in 20% CO2 pumping was abolished  or hid-1(yg316) strains , an important component of DCV exocytosis egl-21 encodes the C. elegans ortholog of carboxypeptidase E, an important component in neuropeptide maturation unc-31(e928) or egl-21(n476) deletion strains to 10% CO2, the pumping rate of the pharynx was significantly higher compared with that in wild-type animals exposed to the same concentration of CO2 (2. The unc-13 gene is involved in synaptic vesicle secretion of neurotransmitters rab-3 gene is a Rab GTPase that affects the distribution of synaptic vesicle populations unc-13(e1091) or rab-3(js49) mutant strains to 10% CO2 showed pumping behavior similar to that of wild-type strains (2 levels and that compromising DCV secretion probably impairs the pumping response to high CO2 levels.Dense core vesicles (DCVs) secrete neuropeptides in peptidergic neurons n of CO2 . We also strains . These dC. elegans2 requires expression of hid-1 in the gut, neurons, or both, we used transgenic lines that express HID-1 fused to GFP driven by either the pan-neuronal promoter rab-3 or the gut-specific promoter ges-1. Expression of HID-1 under the rab-3 promoter in neurons of hid-1(sa722) background was sufficient to restore inhibition of the pharynx pumping almost to the levels shown by wild-type animals (ges-1 promoter in the gut of hid-1(sa722) had no significant effect on the response of the pharynx to 10% CO2.HID-1 is expressed in all neuron and gut cells of  animals . In cont2 levels on the pharynx. The nlp-3 gene is expressed in sensory neurons  as well as in pharyngeal neurons  (nlp-3 promoter in hid-1(sa722) background was sufficient to restore pharynx pumping inhibition after exposure to 10% CO2 (2 activate the AFD neurons gcy-8 promoter in the thermosensory AFD of hid-1(sa722) background did not restore the CO2-mediated pumping inhibition , did not restore the CO2-mediated pumping inhibition (2-dependent pumping inhibition of the pharynx. Transgenic lines expressing HID-1::GFP under the promoter of flp-17 showed expression in the BAG neurons (2-dependent pumping inhibition (gcy-33 promoter in hid-1(sa722) background was specific to the BAG neurons (2-dependent pumping inhibition (flp-17 and gcy-33 promoters in hid-1(sa722) background. We found that following the removal of the HID-1::GFP-expressing BAG neurons, the pumping in 10% CO2 was similar to that of hid-1(sa722) animals  and gcy-9(tm2816) mutations. The pumping rate was similar to that of hid-1(sa722) animals   [38]. Tr 10% CO2 . High lehibition , which shibition . We next neurons . This exhibition . Similar neurons  [21]. Thhibition . Next, w animals . These r animals . These r animals . MoreoveO2 level , further2 levels have diverse effects on the lung epithelium, immunity, and muscle function. However, the effects of acute exposure of muscle cells to high CO2 levels were unknown. In addition, recent studies suggest that mammals, like C. elegans, are able to sense elevated CO2 levels, which is of broad physiologic significance.In humans, high COC. elegans animals to high CO2 levels quickly reduces the pumping rate of the pharynx. This effect depends in part on the nutritional status of the animal, since starved animals exposed to 10% CO2 in air continue to pump, albeit at a significantly slower rate. Our genetic data suggest that the effect of acute exposure to high CO2 levels on the pumping rate is independent of the avoidance responses of C. elegans to high CO2 levels. First, cGMP signaling is required for mediating the avoidance response, as mutations in the cGMP gated ion channel encoded by tax-2 and tax-4 completely disrupt the avoidance response tax-4 does not completely rescue the immediate response of the pumping rate to high CO2 levels. Second, mutation in the insulin-like receptor encoded by daf-2 also disrupts the avoidance response daf-2 mutants under exposure to 10% CO2 is dramatically reduced, like in the wild-type animals. The limited recovery of the pumping rate in daf-2 mutants at 10% CO2 could be due to the effect of daf-2 on starvation regulating pathways osm-3 and che-10 also significantly changes the avoidance response. Again, in the pumping assay, similar mutations in these genes did not change the response of C. elegans to high CO2 levels. In addition, mutations in ets-5 and gcy-9, which were previously shown to be required for the calcium response of the BAG neurons to CO2, did not change the response of the pharynx to high CO2 levels. Finally, the rescue of pumping by hid-1 in the BAG neurons was not affected by gcy-9 mutations.Acute exposure of well-fed adult C. elegans animals presumably interpret high CO2 levels as a harmful cue that leads to avoidance and pumping inhibition. The ability of the animal to stop eating for several minutes probably allows it to avoid undesirable food. Surprisingly, although both the avoidance and the pumping responses to the same stressful cue are immediate, our genetic data suggest that different molecular pathways mediate the two responses to high CO2.hid-1 as a regulator of the pumping response to high CO2 levels, as mutations in the hid-1 gene blunted the response of the pharynx to high CO2 levels. HID-1 is required for the neuropeptide secretion pathway unc-31 and egl-21, could also partially suppress the pharyngeal pumping suppression upon exposure to 10% CO2. Neuropeptides are important signaling molecules in many physiological responses both in C. elegans and in other organisms. In C. elegans there are more than 250 neuropeptides that play a role in feeding and metabolism, and most neurons in C. elegans secrete neuropeptides hid-1, an important peptidergic signaling gene, is expressed both in the nervous system and in the intestine unc-31 mutants demonstrate continuous pumping in the absence of food, unlike wild-type animals hid-1 also partially suppresses the inhibition of pumping in the absence of food (data not shown), we cannot completely rule out the possibility that hid-1 generally inhibits pumping and acts in parallel with CO2.Our genetic screen identified 2 is probably mediated by several different neuropeptides, since pumping inhibition could not be inhibited by deletion of individual neuropeptide genes known to be overexpressed in the BAG neurons, including flp-10, flp-16, flp-27, nlp-1, flp-17, and nlp-14  exhibited the pumping rate seen at normal air levels (unc-31 and egl-21 are due to the other pathway(s) that must be acting in parallel with hid-1. This implies the existence of other, HID-1-independent mechanisms that must regulate the response of the pharynx to CO2 levels. For example, it is possible that high CO2 levels trigger other presynaptic inputs that mediate the effect on the pharynx in parallel with the peptidergic signaling, or that CO2 has also a direct postsynaptic effect on the pharyngeal muscles that inhibits their normal function. Interestingly, such parallel pathways depend on the CO2 levels, since hid-1 completely rescues the pumping inhibition at 5% CO2 and fails to rescue the pumping at 20% CO2 ; CB3329, che-10(e1809); CX2948, tax-4(p678); PR802, osm-3(n1540); DA609, npr-1(ad609); CZ3714, gcy-31(ok296); CZ3715, gcy-33(ok232); CX6448, gcy-35(ok769); AX1296, gcy-36(db42); JT722, hid-1(sa722); JT1058, hid-1(sa1058); YG316, hid-1(yg316); YG2310, hid-1(yg316); jsEx896 [hid-1p::HID-1::GFP]; NM3017, hid-1(sa722) and lin-15(n765); jsEx896[hid-1p::HID-1::GFP]; NM3053, hid-1(sa722) and lin-15(n765); jsEx897[rab-3p::HID-1::GFP]; NM3139, hid-1(sa722) and lin-15(n765); jsEx909[ges-1p::HID-1::GFP]; YG2313, hid-1(sa722); ygEx317 [gcy-8p::HID-1::GFP]; YG2318, hid-1(sa722); ygEx318 [nlp-3p::HID-1::GFP]; YG2319, hid-1(sa722); ygEx319 [flp-17-p::HID-1::GFP]; YG2340, hid-1(sa722); ygEx320 [gcy-33-p::HID-1::GFP]; DA509, unc-31(e928); KP2018, egl-21(n476); YG2302, unc-13(e1091); YG2320, ets-5(tm1734); YG2321, ets-5 (tm1755); YG2322, gcy-9(tm2816); YG2323, gcy-9(tm2816) and hid-1(sa722); YG2324, gcy-9(tm2816) and hid-1(sa722); ygEx321 [flp-17-p::HID-1::GFP]; RB1340, nlp-1(ok1469); RB2575, flp-19(ok3587); VC2012, flp-27(gk3331); VC1108, nlp-14(ok1517)/szT1 X; RB1989, flp-10(ok2624); RB2275, flp-16(ok3085). All strains were obtained from the C. elegans Genome Center (CGC) or the National BioResourse Project (NBRP), except for CX2948, which was kindly provided by the De-Bono laboratory, and NM3017, NM3053, and NM3139, which were kindly provided by the Nonet laboratory Worms were handled as described elsewhere 2 in air. The chamber was connected to a mechanical valve that controlled the humidified gas mixture entering the chamber. For all pumping assays, NGM plates were seeded with 20 µL of OP50 5 h before the experiment to allow normal feeding and to keep worms in a restricted area. A single 1-day-old adult worm was seeded on a plate just before the start of the experiment. Initially, normal air mixture  flowed into the chamber and worms were allowed to adjust for 1 min. The number of pharynx muscle contractions was subsequently measured for 1 min under normal air conditions. Then the airflow was switched to a high-CO2 gas mixture, and after 10 s the pharynx muscle contraction rate was measured again. To measure pumping rate after starvation, well-fed wild-type 1-day-old adult worms were collected using M9 buffer and washed four or five times in M9 buffer. Worms were then seeded on either NGM plates with no bacteria or NGM plates seeded with OP50, for 4 h prior to measurements. All pumping assays were performed at 22°C.A standard NGM plate covered with a lid-shaped chamber with inlet and outlet holes to allow gas flow was used to measure the pumping rate under different concentrations of CO0) for recovery. After recovery, five P0 animals were transferred again to an NGM plate and allowed to lay F1 progeny. Adult F1 animals were cloned onto individual NGM plates and their L4-adult F2 progeny where exposed to 10% CO2. F2 worms that continued the pumping of the pharynx even after exposure to 10% CO2 were isolated. In total, we scored the progeny of ∼1200 F1 animals. The isolated strains were outcrossed three times. The mutation was mapped as described elsewhere 2 were isolated, and the DNA of their F3 and F4 offspring was extracted using a Gentra Puregene kit . Whole genome sequencing was performed using the Applied Biosystems SOLiD 3 deep sequencing apparatus. The positions of the Hawaiian SNPs were mapped on the DNA of the yg316 strain. A 1.2-MB region in chromosome X that did not contain any Hawaiian SNP was found. Within this region a premature stop codon (W625X) in the coding sequence of hid-1 was found to cause the phenotype as described in the text.The EMS mutagenesis was performed essentially as described elsewhere hid-1 promoter driving the genomic hid-1 coding region fused to eGFP, was a kind gift from the Nonet laboratory hid-1 promoter with various neuron-specific promoters. To drive AFD-specific expression an 800-bp fragment upstream of the gcy-8 start codon was amplified and subsequently digested with KpnI-AatII to generate pKS10. Similarly, to drive sensory and pharyngeal specific expression, a 700-bp fragment upstream of the nlp-3 start codon was amplified and digested with KpnI-AatII to generate pKS20. To drive BAG-specific expression a 3.4-kb fragment upstream of the flp-17 start codon was amplified and fused by PCR to HID-1::GFP from NM1699. In addition, to drive BAG-specific expression a 980-bp fragment upstream of the gcy-33 start codon was amplified and subsequently digested with KpnI-AatII to generate pKS30. All plasmids were verified by sequencing and microinjected to either JT722 or YG316 with an elt-2::GFP marker as described elsewhere The NM1699 construct, which contains the Laser ablation was performed using an Andor Revolution XD confocal spinning disk system with a Nikon TiE inverted microscope equipped with a nitrogen pulsed laser and a 365-nm Micropoint dye cell. The microscope and laser were controlled by means of IQ software and the Micropoint Mosaic I System 85-75, respectively. The region of interest was set according to the size of the neuron cell body as revealed by the GFP marker. We used a frequency of 15 Hz, an energy range of 80%–90%, and 3–5 repeats in order to completely ablate the GFP marker in the neuron cell body.Figure S12, pH of 5.0 or 7.0, or mutations in the carbonic anhydrase genes. (A) One-day-old wild-type (N2) adult C. elegans were continuously exposed to 10% CO2 for 30 min and pumping rate was measured at different time points. (B) One-day-old wild-type (N2) adult C. elegans were transferred to NGM plates buffered at pH of 5.0, 6.0, or 7.0 followed by exposure to 10% CO2 and measurements of the pharyngeal pumping. (C) One-day-old adult worms with mutations in cah-2, cah-5, or cah-6 genes exposed to 10% CO2 showed pharyngeal pumping rate similar to that of wild-type animals.Pumping inhibition is not rescued by either 30 min of exposure to 10% CO(PDF)Click here for additional data file.Figure S2hid-1(yg316) animals exposed to 10% CO2 is similar to that of wild-type animals. Gravid animals were exposed to either normal air conditions or air containing 19% or 10% CO2 for 6 h. The number of embryos laid during this period was measured.The egg-laying rate of (PDF)Click here for additional data file.Figure S3yg316 or under gcy-8, nlp-3, osm-6, flp-17, or gcy-33 promoters in the background of sa722. Arrows indicate the AFD neurons (gcy-8p) and BAG neurons (flp-17p and gcy-33p).The expression of hid-1p, nlp-3p and osm-6p was detected in several neurons. Scale bar, 10 µm.Transgenic expression of HID-1::GFP. HID-1 fused to eGFP was expressed under its own promoter in the background of (PDF)Click here for additional data file.Figure S42-mediated pumping inhibition. One-day-old animals with mutations in neuropeptide genes, which are known to be overexpressed in the BAG neurons, were exposed to 10% CO2 and pumping rate was measured. The pumping rate of nlp-1, nlp-14, and flp-16 mutants in 10% CO2  was significantly different from that of the wild-type (N2) animals and showed small but significant rescue. *P<.01. Error bars indicate SEM.Animals with deletions in neuropeptide genes expressed in the BAG neurons still show strong CO(PDF)Click here for additional data file.Movie S1C. elegans exposed to 10% CO2. Pumping rate of wild-type animal under dissecting microscope is presented. Worms are first exposed to normal air and then exposed to 10% CO2.Pumping of wild-type (AVI)Click here for additional data file.Movie S2hid-1(yg316) mutant exposed to 10% CO2. Pumping rate of hid-1(yg316) mutant animal under dissecting microscope is presented. Worms are first exposed to normal air and then exposed to 10% CO2.Pumping of (AVI)Click here for additional data file.


Heterocapsa circularisquama. HcRNAV strains are grouped into 2 types (UA and CY), based on intra-species host specificity and the amino acid sequence of the major capsid protein (MCP). In the present study, we report the isolation of novel HcRNAV clones (n=51) lytic to the H. circularisquama strains, HU9433-P, HCLG-1, 05HC05 and 05HC06. HcRNAV34, HcRNAV109, HcRNAV641, and HcRNAV659, which displayed lytic activity against the strains, HU9433-P, HCLG-1, 05HC05, and 05HC06, respectively, were selected as typical virus clones and were intensively examined. The infection intensity of each host-virus combination was analyzed by examining the algicidal activity, detecting the intracellular replication of the viral RNA as well as the appearance of host cells with a morphologically abnormal nucleus post-infection. Interestingly, the strains, 05HC05 and 05HC06, were markedly sensitive to HcRNAV641 and HcRNAV659, respectively. Tertiary structural modeling predicted 4 unique amino acid (aa) substitutions in HcRNAV659-MCP to be exposed to an ambient water environment, which contributed towards determining its infection specificity. Neighbor-joining analysis of MCP aa sequences from HcRNAV clones revealed 3 clades, namely, the CY type and the UA1 and UA2 subtypes. The HcRNAV clones lytic to HCLG-1 (ex. HcRNAV109), HU9433-P and 05HC05 (ex. HcRNAV34), and 05HC06 (ex. HcRNAV659) were categorized into CY type, UA1 and UA2 subtypes, respectively. The present study highlights the complexity of the H. circularisquama-HcRNAV host-virus system, i.e., clonal variation, microbial control, and ecology in a natural algal population.HcRNAV is a single-stranded RNA (ssRNA) virus that specifically infects the bivalve-killing dinoflagellate,  Heterocapsa circularisquama Horiguchi, is one of the most harmful red-tide bloom-forming microalgae, which specifically kills shellfish  genome of approximately 4.4 kb in length  that exhibit complementary intra-species host specificity , which did not show any apparent virus-induced cell lysis when inoculated with either HcRNAV34 or HcRNAV109 , HCLG-1 , 05HC05, and 05HC06  were used in this study (2SeO3 (−2 s−1) was provided by cool white fluorescent illumination at 20°C. The HcRNAV clones obtained in this fashion were then screened for lysis, according to the method described by Tomaru et al.  was inoculated with 1 mL of virus suspension at a multiplicity of infection of ca. 100–500, and incubated under the conditions described in the previous section. Host cultures inoculated with sterilized SWM3 medium served as controls. An aliquot of each culture was sampled every 24 h for 6 days.Sixteen combinations of cross-reactivity tests were performed between the 4 H. circularisquama cells was measured immediately after sampling, without fixation, using an Epics XL-MCL flow cytometer  equipped with a 488-nm air-cooled argon ion laser (15 mW) and standard filters. Fluorescent microspheres  were added as an internal reference. Readings were collected for 1 min in logarithmic mode and were analyzed using EXPO32ADC software (Beckman Coulter).The abundance of g for 3 min, and the resultant pellets were stored at −80°C until RNA extraction. The frozen cells were homogenized with plastic pestles in 100 μL RNase-free water and 40 μL RNA extraction buffer  and the samples were further analyzed by northern hybridization. An aliquot of cell suspension was removed from the culture centrifuged at 13,000×n buffer . The aqun buffer , 10. Brin buffer , and theH. circularisquama cells sampled every 24 h for 6 days were fixed with 1% glutaraldehyde  and maintained at 4°C. The cells were then stained with SYBR-GOLD  according to the methods described by Tomaru et al.  was completely sequenced according to the method described by Nagasaki et al.  (http://pd-fams.com/index.html). The results were manually modified, and the HcRNAV659 model was compared against those of HcRNAV34 and HcRNAV109, as previously reported by Nagasaki et al. .The structural data were viewed using the RasMol 2.6 Beta-2a program , 2 μL of 2 mM dNTPs, 2 μL of 10× buffer, 0.2 μL of Blend Taq , and 2 μL of cDNA as a template. PCR conditions were as follows: initial denaturation at 94°C for 4 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 5 s, extension at 72°C for 1 min, and final extension at 72°C for 5 min. The resultant products were electrophoresed in 2% (w/v) agarose S gels (Nippon Gene). The nucleic acids were visualized by ethidium bromide staining.The PCR products were purified using an ExoSAP-IT kit , and directly sequenced in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and the primers ORF2-A1 and ORF2-B1. Each nucleotide sequence was translated into an amino acid sequence using the universal genetic code. The deduced amino acid sequences were then aligned, and a phylogenetic tree was constructed with the Clustal W program using the neighbor-joining method (NJ)  and NJ-pn) lytic to H. circularisquama strains, HU9433-P (n=21), HCLG-1 (n=9), 05HC05 (n=9), and 05HC06 (n=12) were successfully isolated (n=51) were in close agreement with the ORF-2 genome size (1.08-kb) for HcRNAV34 and HcRNAV109, predicted using denaturing gel electrophoresis. HcRNAV34 (lytic to HU9433-P), HcRNAV109 (lytic to HCLG-1), HcRNAV641 (lytic to 05HC05), and HcRNAV659 (lytic to 05HC06) were selected as typical virus clones and were intensively examined in the subsequent experiments.By employing seawater and sediment samples collected from the western coast of Japan, HcRNAV clones  and 05HC05, but not in the other 2 host strains . In contrast, no amino acid substitution was found on the reverse (interior) of the capsid protein  and HcRNAV109 (CY-type), the tertiary structure of its MCP was predicted n silico . By align silico . Upon an protein .Phylogenetic analysis was performed to investigate the relationship between the novel HcRNAV isolates. A phylo-genetic tree was constructed based on the deduced amino acid sequences of HcRNAV MCP encoded by ORF-2, which also included the previously reported sequence data , Fig. 5 i.e., Gly119, Phe141, Gly160, and Thr248), which significantly differed amongst the other 2 typical HcRNAV clones HcRNAV 34 and HcRNAV 109 , 20H. ci  RNA. Although the factors determining the replication efficiency have not been determined yet, it is likely that the intensity of the negative-stranded RNA signals might correspond to the efficiency of viral replication in the host cell. Further lending support to this hypothesis, our results showed the presence of strong signals in host-virus combinations where, the virus clone was isolated using the corresponding host strain. In theory, in every host-virus combination there exists a high binding affinity of the virus to the specific host and host-virus infection efficiency . Nanostructural differences of the MCP, in addition to the replication-related proteins and the 3′-stem-loop structure, might contribute to determining the infection efficiency of the virus; however, future studies elucidating the detailed molecular mechanism underlying the pathogenesis of HcR-NAV infection are warranted. Nevertheless, our results indicate that every viral clone-host strain combination has its own outcome, and that this diversity might influence the stagnation and succession of H. circularisquama populations in nature.To date, the detailed mechanism underlying the pathogenicity of HcRNAV infection has not been elucidated. The HcRNAV virion has been shown to be composed of 180 ORF-2 products, et al., HcRNAV clones were roughly grouped into 2 types  based on intra-species host specificity and the MCP amino acid sequences  on host-virus combinations with which marked cell lysis is observed, whether intentionally or not, to obtain novel results that are easy to interpret. Consequently, there is the risk of a novel finding being misinterpreted as typical. Hence, the processes that occur in natural waters may be completely different from those observed in intensively studied “special” cases; therefore, it is important to recognize the diversity of host-virus systems in nature and the necessity of realistic as well as objective interpretations of the obtained experimental results.


We developed viral integration site detection tool called Virus-Clip, which makes use of information extracted from soft-clipped sequencing reads to identify exact positions of human and virus breakpoints of integration events. With initial read alignment to virus reference genome and streamlined procedures, Virus-Clip delivers a simple, fast and memory-efficient solution to viral integration site detection. Moreover, it can also automatically annotate the integration events with the corresponding affected human genes. Virus-Clip has been verified using whole-transcriptome sequencing data and its detection was validated to have satisfactory sensitivity and specificity. Marked advancement in performance was detected, compared to existing tools. It is applicable to versatile types of data including whole-genome sequencing, whole-transcriptome sequencing, and targeted sequencing. Virus-Clip is available at  Viral infection is a common risk factor for various human malignancies . Particuhttp://web.hku.hk/~dwhho/Virus-Clip.zip.Here, we present our viral integration detection tool, namely Virus-Clip. Virus-Clip makes use of the virus genome as the primary read alignment target. Then, it extracts soft-clipped reads from the alignment and maps the soft-clipped segments  to the human genome. Making use of the mapping information, Virus-Clip can report the human and virus integration breakpoints to single-base resolution. Besides, all the integration sites are automatically annotated with the affected human genes and their corresponding gene regions. With streamlined procedures involving minimal steps and tools, Virus-Clip delivers a simple, fast and memory-efficient solution to viral integration site detection Figure . ExecutiTo evaluate the performance of Virus-Clip, we applied it to whole-transcriptome sequencing (RNA-seq) data of two human HBV-associated hepatocellular carcinoma (HCC) samples. The RNA-seq data was generated by 101bp paired-end Illumina HiSeq 2000 platform. Viral integration site detection was similarly performed by VirusFinder and ViralFusionSeq with default parameters. VirusSeq was not included in the comparison as it cannot report exact breakpoint positions. Raw execution result data is available as Virus-Clip identified 8 and 14 HBV integration sites respectively for the two studied samples while 1 and 3 sites were found by VirusFinder. ViralFusionSeq was failed to execute on our dataset but its execution could finish on its example data, suggesting there was no installation error.TERT gene and inside KMT2B gene were frequently reported on HBV-associated HCC [TERT integration event were 12 and 6, while they were 17 and 8 on the KMT2B integration event, for Virus-Clip and VirusFinder respectively. To further evaluate the sensitivity and specificity of the detection by Virus-Clip, we selected 17 HBV integration events supported by at least 1 soft-clipped sequencing read and designed primers that flank the identified HBV integration junctions  data of extremely large size. With the strategy of initial read alignment to virus reference genome instead of human reference genome and streamlined procedures involving only a few essential tools, these issues lead to the superior performance of Virus-Clip. Due to the relatively small size of virus genome, the alignment to it is significantly more efficient. Moreover, Virus-Clip makes use of BWA-MEM for initial alignment to virus genome, SAMTools for soft-clipped reads extraction, BLASTN for local alignment of human chimeric fragment to human genome, and ANNOVAR for annotation. Such minimal combination of tools and workflow allows streamlined procedures. Virus-Clip substantially shortened the process and time in analyzing viral integration event. It also requires significantly fewer computational resources. The installation of Virus-Clip is also simplified, as a result of the simple overall workflow. Furthermore, the automatic annotation capability of the integration sites can facilitate the practical use of the obtained viral integration information. Therefore, to our best knowledge, Virus-Clip contributes a major advancement in viral integration site identification. It provides a simple, fast and memory-efficient solution to identify viral integration event at single-base resolution that requires minimal computer resources and applicable to versatile types of NGS data including WGS, RNA-seq and targeted sequencing. Apart from the RNA-seq data mentioned above, we have also applied Virus-Clip on targeted DNA sequencing data. Similarly satisfactory performance could be obtained (data not shown). One limitation of Virus-Clip is that it requires the provision of virus reference genome as input and hence it is not applicable to data without virus reference genome available (which is unlikely in most circumstances).Virus-Clip is implemented in shell script that executes third-party tools and our own Perl program Figure . The virVirus-Clip consists of a shell script (Virus_Clip.sh) that executes third-party tools and our own Perl program (virus_clip.pl). The actual procedure involves 3 major steps. First, it maps sequencing reads to virus reference genome by Burrows-Wheeler Aligner (BWA-MEM) , which iThen, with the use of SAMTools , it examftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) with default parameters. Top match (if any) is reported as the virus integrated location. Using ANNOVAR [Finally, Perl program virus_clip.pl reads the temporary file and obtains the soft-clipped portions of the reads . It subsequently maps them to the human reference genome by the BLASTN stand-alone version (available at  ANNOVAR , annotatWe selected 17 HBV integration events supported by at least 1 soft-clipped sequencing read and designed primers that flank the identified HBV integration junctions Table . To conf


Hepatitis E virus is a common cause of acute hepatitis in humans. Related viruses have been isolated from multiple animal species, including rats, but their impact on human health is unclear. We present the first full-length genome sequence of a rat hepatitis E virus strain isolated in the United States (LA-B350). Hepeviridae family that also comprises several related viruses isolated from different animals, including rats, ferrets, bats, rabbits, chickens, and teleost fish (–Hepatitis E virus (HEV) is a positive sense, single-stranded RNA virus and is a major cause of acute hepatitis worldwide . Severe ost fish . The epiost fish . Rat HEVost fish –8, but nHere we present the first full-length rat HEV sequence isolated in the United States. The virus was originally isolated from rats trapped in urban Los Angeles, and then passaged twice in Sprague-Dawley rats and once in an athymic nude hooded rat B350), as reported 0, as rep.–3, and may thus be slightly higher than expected for errors introduced by reverse transcription (range of 10–4 to 10–5) (–4) (http://www.qiagen.com/media/ebooks/Maximising_PCR_and_RT_PCR/index.htm), suggesting that LA-B350 may exist as a diverse viral quasispecies, as was described for human HEV as well  in length [excluding the poly(A)-tail] and encodes the expected open reading frame 1 (ORF1) , ORF2 (644 aa), ORF3 102 aa), and also the ORF4 (183 aa) that seems to be conserved in rat and ferret HEV (2 aa, and as well .In conclusion, the availability of the first U.S. rat HEV genome may help in further elucidating rat HEV epidemiology and its possible impact on human health.KM516906.The complete genome of rat HEV strain LA-B350 can be accessed under the GenBank accession no.


By genetically engineering pigs to degrade a crucial viral protein, livestock can be made less susceptible to foot and mouth disease virus. Related research article Hu S, Qiao J, Fu Q, Chen C, Ni W, Wujiafu S, Ma S, Zhang H, Sheng J, Wang P, Wang D, Huang J, Cao L, Ouyang H. 2015. Transgenic shRNA pigs reduce susceptibility to foot and mouth disease virus infection. eLife4:e06951. doi: 10.7554/eLife.06951Image Pig cells showing signs of foot and mouth disease infection (top panels); genetic techniques can be used to make cells less susceptible to the infection (bottom panels)In 2001, an epidemic of foot and mouth disease in the UK resulted in the destruction of over six million livestock, restrictions on international trade, and economic costs in excess of £3 billion . Foot anThe virus that causes foot and mouth disease (FMD) comes in seven types, each of which requires a specific vaccine for immunization . The ChiThe Chinese team - which includes Shengwei Hu, Jun Qiao and Qiang Fu as joint first authors, and Chuangfu Chen, Wei Ni and Hongsheng Ouyang as corresponding authors - looked for the small RNA that was best able to stop the replication of the FMD virus. They then integrated the gene coding for this small RNA into the genome of single pig cells; transgenic embryos were then generated from these cells by the same technique that produced Dolly the sheep. The embryos were subsequently implanted into donor sows that gave birth to transgenic piglets.Hu et al. observed that the FMD virus was almost completely inhibited from growing and dividing inside the cells derived from transgenic pigs. The antiviral activity of the small RNA was confirmed when genetically modified pigs were directly injected with one of the FMD virus types common in Asia. Whereas control groups displayed the classical clinical signs of FMD - high fever, mouth blisters, foot sores and lameness - all transgenic pigs were protected from disease for up to seven days after infection, and only later developed milder clinical signs associated with FMD. It remains to be shown if protection extends to other types of FMD virus.The FMD virus replicates quickly and mutations accumulate when it duplicates its genome. If mutations appear in the region of the genome targeted by the small RNAs, variants that are resistant to treatment can emerge: such ‘escape mutants’ have been observed for other viruses that are similar to the FMD virus . Hu et aCurrent FMD vaccines require at least seven days to induce a protective immune response , and durThe work of Hu et al. is a proof of concept for the use of RNA interference technology to produce transgenic livestock with increased resistance to viral infection. Although this technology could potentially increase food security, it also faces major challenges since, to date, neither the Food and Drug Administration in the United States nor the European Food Safety Authority have approved genetically engineered animals for human consumption. The processes used by these regulators to ensure the safety of genetically modified animals as food resources are strenuous and expensive, and have prevented other transgenic animals, such as salmon, from entering the market in the US . A globa