Our analysis shows novel and promising results regarding the capability of remote sensing in characterizing suspended sediment in terms of grain size distribution and reveals that erratic stages favour delivering of non-cohesive sandy sediment more than the persistent stages. This conclusion is supported by numerical simulations and is consistent with previous studies on suspended sediment rating curves.Several coastal regions on Earth have been increasingly affected by intense, often catastrophic, flash floods that deliver significant amounts of sediment along shorelines. One of the critical questions related to the impact of these impulsive runoffs is “are flash floods more efficient in delivering non-cohesive sandy sediment along the coasts?” Here we relate flow stages  to the grain size distribution of the suspended load, by performing a synergic analysis of  The Tiber River is the second largest river in Italy, with a length of 409 km and a drainage basin of about 17.156 km2; it is sourced from Central Apennines and debouches in the Tyrrhenian Sea, close to the city of Rome. Mean annual discharge of the Tiber River is about 225 m3/s; maximum annual discharge exceeds 1500 m3/s and the minimum annual discharge reaches 60 m3/s (ref. 3).The hydrologic regime of the Tiber River basin   at the water gauge station of Ripetta Bridge  of the suspended sediment, retrieved from satellite. Grain size plays a fundamental role in settling velocity and dispersal of suspended sediment over shelf areas9. Observations showed that formation of sedimentary bottom layers and their related sandy and/or mud deposits strongly depends on sediment  settling properties13. In particular, the role of sediment cohesion, which is controlled by sediment size, influences morphology of deltas and coastal areas14.Inspired by the 2012 Tiber River flood, here we seek to quantify the impact of stream flow variability, i.e., erratic vs. persistent flow stages15, in particular, off river mouth and estuarine environments, where remote sensing algorithms can be applied to mid-resolution ocean color sensors  that provide near-daily data at ~1 km spatial resolution since two decades. Moreover, remote sensing has the additional advantages of providing a synoptic view of river plumes20. Application of in-situ calibrated MODIS satellite data, for instance, allowed capturing the spatio-temporal variability of the distribution of suspended sediment over the coral reefs21 and assessed the impact of these extreme outflows on wetland sedimentation during the historic 2011 Mississippi River flood18.Although the knowledge of sediment characteristics gives a substantial contribution in understanding and modeling particle dynamics and coastal geomorphology, GSD of suspended particles from river plumes are still seldom quantified and insufficiently documented. Satellite data can be used as an alternative techniquep) and the GSD has been predicted theoretically and verified experimentally by many studies24. However, those findings had no application to remote sensing data since the beam attenuation coefficient is not retrievable from space. Fortunately, after commercial optical backscattering meters became available, research provided evidence that the spectral particle backscattering coefficient (bbp) and the GSD are also related28 in a very similar fashion as cp and the GSD were found to be related. The advantages of this finding are many because bbp can be retrieved from ocean color observations, thus establishing a link between remote sensing measurements and the spatio-temporal variability of particle assemblage  for fluvial geomorphology applications and, in particular, for understanding and monitoring fate and distribution of riverine suspended load characteristics. All this sets the base for our investigation on flow-stream variability and GSD of suspended sediment from a satellite-based analysis off the Tiber River mouth.The relationship between the spectral beam attenuation coefficient (cQw) data at the historic Ripetta flow gauge, matching the period of satellite observations. Ripetta’s station (http://www.idrografico.roma.it), has one of the most complete measurements data set monitoring the river discharge relative at more than 96% of the Tiber River drainage basin. To estimate the streamflow variability we then calculate monthly averages from the daily dataset, as well as the coefficient of variation of streamflow, 18; low (high) CV indicates flow regimes that are weakly (highly) variable around the mean and are termed as persistent (erratic). The use of monthly averages allows us to highlight the link between GSD of suspended sediment off the river mouth and the streamflow variability7. From the comparison between daily water discharge and CV .Light back-scattered by marine particles depends on size distribution, concentration, refractive index, and the detection wavelength see Eq.  below, tbp from remote-sensing reflectances (Rrs) data, by using the quasi-analytical algorithm (QAA) (ref. 31), an approach that was validated in a previous work for Italian coastal and open waters32. In particular, we use observations from the Moderate Resolution Imaging Spectro-radiometer (MODIS) for the period 2002 to 2014, i.e., a data record that includes the November 2012 flood event (see Text S1 in the Supplementary Information). Rrs data were remapped to a ~ 20 x 20 km2 box centered around the Tiber River mouth  ref. 3, an apprη, a parameter that is highly relevant for the GSD estimations. In particular, the default value is given by35:The QAA parameterization was left as originally documented except for where in-situ η vs. rrs(443)/rrs(555) matchups (see below) but the data cloud was highly scattered around the mean regression curve31  and bbp at various stations, covering a wide turbidity range, belonging to two field campaigns, conducted in 2013 and 2015 across Italian seas  at the wavelengths 470, 530 and 660 nm. From surface data, the spectral slope (η) was calculated as the slope of the best linear fit of bbp against the wavelength, both log-transformed. Rrs was measured using the free-profiling technique using OCR-507 radiometers , at the bands 412, 443, 490, 510, 555, 665 and 863 nm using protocols compliant with NASA’s recommendations37.Hence, to provide reliable eas Fig. . bbp wasrs(443)/rrs(555) from our in-situ reflectances respect to our measured η. Data shows high scattering but also high overestimation respect to Lee’s curve (Eq. rs(555)/rrs(665) as predictive variable, which results to be more sensitive to variations in coastal waters; indeed, the fit is largely improved  applied this approach to study the particle abundance and volume in the dimensional ranges corresponding to three main phytoplankton-size classes in the global ocean41. Here, we consider the silt range, between the limits 3.9 µm and 62.5 µm, and the very-fine sand range, between 62.5 µm and 125 µm.The full size range can be divided in several sub-ranges to study the proportion between size classes. For instance, Kostadinov VC) between two given edge sizes  is equal to:By assuming spherical shape, the volume concentration (VC estimation can relate to a size fraction like silt (3.9 µm to 62.5 µm), sand (62.5 µm to 125 µm) or to the global assembly by taking the full size range (3.9 to 125 µm). In this latter case, it is named total volume concentration (VCtot). For these two size classes VCtot is equal to the sum of the particle volume of each class, reported in parts per million28.A useful parameter, related to a GSD, is the median diameter D50), which is the diameter at which the lower and the upper volume fractions of the GSD are equal. It is obtained after the integration of the GSD in Eq. 0, which :6\docume25 and experimental investigation27 showed that the GSD can be associated to bbp by means of η in Eq. (p instead of on bbp (refs. 29). Kostadinov et al. (ref. 25) found that if bbp was predicted using Mie calculations, a relationship between η and ξ also existed, and was similar to that related to cp. Theoretical and empirical studies agree on the fact that ξ increases as a monotonic function of η and thus, as for ξ, lower η is associated to a higher proportion of large particles and vice-versa.Theoretical derivations that used the Mie scattering theoryη in Eq. . Such reet al. (ref. 25). The LUT requires the input of the spectral bbp, which is estimated with the QAA. The LUT returns the Junge slope ξ, and also the GSD value N0 at a reference size D0, which is relevant for VCtot calculations. In this regard, we notice that VCtot is a concentration value of particles within a given size range, but is conceptually and methodologically different than the usual suspended particulate matter concentration which is commonly retrieved by using remote sensing techniques and expressed in mass density units (e.g.44).In this work, we use the look-up table (LUT) provided by Kostadinov rs(667) value within the box for each day  over the same period of the collected water discharge data. From this daily time series, daily time series of η and the GSD were obtained and then monthly averaged.To track the river’s turbid water off the river mouth from satellite-based images, we select the maximum Rday Fig. . In the 46, by using different flood discharge evolution. In particular, we test consistencies between a model of sediment transport based on remote sensing observations, and a generic numerical model of sediment transport, by exploring the relation between GSD and stages (erratic vs. persistent) of river outflows  entering in the domain from the river mouth , in which there is a river flow in inlet on the western side Fig. . The comuth Fig. . We remauth Fig. . In part4. At the inlet, we impose a constant input of 0.6 kg/m3 of suspended sediment concentration49. All sediment is characterized by specific density of 2,650 kg/m3 and dry bed density of 1,600 kg/m3. On the north, south, and eastern boundary we assigned a water elevation equal to zero . The initial condition of the models consists of a constant bathymetry with 5 m of erodible sediment on the basin bottom and a time step Δt = 9 s.For the simulations we consider the upper limit of very-fine sand size range , according to the fact that the suspended fraction recorded during the Tiber River floods ranges from clay to very fine sand (3.9–125 µm) and that the non-cohesive very fine sand fraction is the most predominant oneero Fig.  to reproQw) and the satellite-based total volume concentration of suspended particles (VCtot) shows a comforting agreement, i.e., r = 0.55  as an indicator of the GSD. By plotting the monthly η versus the coefficient of variation (CV) we find that CV and η correlate negatively, thus suggesting that more erratic river discharges (higher CV) relate to a larger proportion of large particles in suspension. On the contrary, more persistent flow discharges  are related to finer particles in suspension  and the mean grain-size population. Here, we use the monthly particle backscattering spectral slope  and the median of grain-size diameters  within the plume, for the day that records the maximum concentration of suspended sediment of the whole time series off the river mouth, i.e., the flood event of 17 November 2012 , which is a good proxy for turbidity, sharply decreases with the distance from the river mouths  grain-size in suspension, and thus, to be comparable with the satellite-retrieved Junge slope. Numerical results show that, although reaching the same value of water discharge, and despite the fact that the two run show similar along-stream velocity profiles, the erratic event  correlates with the grain size of the suspended sediment. Accordingly, Basso et al. (ref. 7) found that large values of coefficient of variation (CV), are associated to large values of the δ -coefficient of the sediment rating curve, i.e., the power law relation between water and sediment discharges, β are empirical coefficients)50. In turn, the δ-coefficient represents the erosive power of the river, with large values being indicative for rivers with a strong increase in erosive power and in sediment transport capacity when discharge increases38. Moreover, δ-values are also affected by the GSD of the material available for transport, i.e. in streams characterized by sand sized sediments the power of the stream to transport sediment will be more important than in streams that mainly transport silt and clay51 ; this will result in high δ-values52.Analysis of matched remote-sensing and in situ data expands and complements these previous findings from two aspects: i) the satellite-based reconstruction of GSD  time series allows for long-term statistical analyses of both water discharge and suspended sediment load, thus complementing and integrating GSD in situ measurements; ii) the synoptic property of satellite images allows for a spatial view of the sedimentary characteristics of the plume, thus helping the recognition of river plume pattern in terms of GSD. Indeed, by analyzing a ten-year-long time series, we assess the ability of unsteady flow to transport large particle size in suspension in a real field case53. Moreover, by focusing on the 2012 erratic event, we pictured depositional patterns of different grain size off the river mouth, a crucial feature that may largely impact shoreline restoration strategies54.Our results show that the synergy of remote sensing and hydrologic 27. Based on those, we retrieved a GSD long time series within the Tiber River plume off the river mouth. The resulting analysis, which paired river flow discharge data to satellite-based GSD showed i) an encouraging correlation (r = 0.55) between water discharge (Qw) and the total volume concentration (VCtot) of suspended particles and ii) a relationship between flow stage characteristics  and the spectral slope of backscattering (η), in turn, related to the GSD. Finally, by running a numerical model that confirmed what we obtained from remote sensing, therefore, assessing the relationship we find between GSD and flow variability. This, in turn, gives also robustness to our ansatz, that is, the methodology to retrieve GSD from satellite spectral slope of backscattering.Previous researches demonstrated the possibility of using satellite to retrieve GSD26 to coastal geomorphology and sediment transport applications. From the new satellite-based tool, we find an emerging relation between GSD and flow stages (erratic vs. persistent floods), also relating this to seasonality. By setting an ad hoc numerical experiment, we then provide a mechanistic support to our findings, setting novel hypotheses on the role of sharp spatial pressure gradient (rather than shear velocity) in remobilizing sediment during river floods. This demonstrates the geomorphological effectiveness of flash floods, which are able to deliver fine-to-medium suspended load off river mouths.Unsteady flow makes the problem of sediment transport very complex and difficult to describe in general laws. Our work presents a novel, satellite-based tool for retrieving GSD of suspended solids debouching from river mouth, by adapting a semi-analytical techniqueSupplementary Information


The 2014–2016 Ebola virus (EBOV) outbreak in West Africa highlighted the need for improved therapeutic options against this virus. Approaches targeting host factors/pathways essential for the virus are advantageous because they can potentially target a wide range of viruses, including newly emerging ones and because the development of resistance is less likely than when targeting the virus directly. However, systematic approaches for screening host factors important for EBOV have been hampered by the necessity to work with this virus at biosafety level 4 (BSL4).In order to identify host factors involved in the EBOV life cycle, we performed a genome-wide siRNA screen comprising 64,755 individual siRNAs against 21,566 human genes to assess their activity in EBOV genome replication and transcription. As a screening platform, we used reverse genetics-based life cycle modelling systems that recapitulate these processes without the need for a BSL4 laboratory.Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for EBOV genome replication and transcription, and confirmed this using infectious EBOV under BSL4 conditions. An FDA-approved drug targeting this pathway showed antiviral activity against infectious EBOV, as well as other non-segmented negative-sense RNA viruses., and demonstrates the power of life cycle modelling systems for conducting genome-wide host factor screens for BSL4 viruses.This study provides a minable data set for every human gene regarding its role in EBOV genome replication and transcription, shows that an FDA-approved drug targeting one of the identified pathways is highly efficacious in vitroThe online version of this article (10.1186/s13073-018-0570-1) contains supplementary material, which is available to authorized users. Ebolavirus) are members of the filovirus family and cause severe hemorrhagic fever in humans and non-human primates /(μc+ − μc−), with μ being the mean and σ being the standard deviation of the positive control c+ or the negative control c− of the assay) were calculated as previously described [Paired two-tailed escribed . Non-linZ′ factors  to dynamic range ) of 0.28 and a dynamic range of about 3.3 log10 in 96-well format , due to their greatly increased brightness. While using these reporters increases minigenome-plasmid-derived background activity (as seen in the –L control) to an extent similar to the increase in positive signals, it also reduces the impact of two other sources of background signals, i.e., luminometer noise and cellular autoluminescence. Indeed, both nano-luciferases showed an improved dynamic range and an improved separation band, with Z′ factors of 0.48 and 0.41, respectively, which was considered sufficient to proceed with the large-scale siRNA screen in 384-well format, for which the basic nano-luciferase construct was selected.The aim of this study was to identify host factors involved in genome replication and transcription of EBOV. Since work with infectious EBOV is restricted to BSL 4 laboratories, we endeavored to use a minigenome assay, which models EBOV genome replication and transcription under BSL 2 conditions Fig. . As a fimat Fig. . HoweverFor the screen   with a Z-score of at least 1.5 were considered “validated” . Thirty-one of these genes were selected for further testing using infectious EBOV   was perfen siRNA . RoughlyBOV Fig. . For thiResults identified 3 host factors  where the best siRNA resulted in a reduction in reporter activity  comparable to that seen when targeting the viral polymerase L directly, i.e., by about 2 log compared to the negative control siRNA. String analysis of those factors indicated that they are associated with each other, increasing the confidence that these are, indeed, true-positive hits with biological relevance. Two of these three factors, i.e., CAD and NXF1, were also remarkable insofar as all examined siRNAs targeting these factors showed a strong reduction in reporter activity following reporter virus infection Fig. . In the Based on these results, we focused on CAD, and first assessed whether this protein is required only for EBOV, or also for other genetically distinct classes of RNA viruses, such as the New world arenavirus Junín virus (JUNV), which is a negative-sense RNA virus with an ambisense coding strategy that replicates in the cytoplasm in discrete viral inclusion bodies similar to EBOV , 32. To Since CAD is the first of several enzymes in the de novo pyrimidine synthesis pathway Fig. , we reviWhile minigenome assays are ideally suited to assess genome replication and transcription in isolation, they have the disadvantage that they require a number of processes which have no equivalent in the EBOV life cycle. These include the initial transcription of viral minigenome RNA from cDNA plasmids by an exogenously provided T7-RNA polymerase, illegitimate encapsidation of these initially transcribed, naked minigenomes by NP, and dependence on plasmid-driven gene expression for expression of the RNP proteins and T7-RNA polymerase. While we did not observe an effect of teriflunomide on control plasmid-driven gene expression in our minigenome experiments, we could not rule out an off-target effect of teriflunomide on the other artificial processes. To address this issue, we utilized a transcription and replication-competent virus-like-particle (trVLP) assay, which authentically models the complete virus life cycle (with the exception of primary transcription), and which is independent of these artificial processes in target cells . As a co50 concentrations of ~ 10 and 13 μM, respectively, further confirming the importance of de novo pyrimidine synthesis for the life cycle of infectious EBOV and also excluding a cell-type specific action of teriflunomide , for which it has been shown that luciferase activity mirrors infectious titer production . As expeide Fig. . Consistide Fig. .Fig. 6EfFinally, we tested whether teriflunomide might also be active against other RNA viruses, using Newcastle disease virus , Rabies virus , and Influenza A virus   Inhibition of DHODH has been reported to revert mRNA export-blocks induced either by Influenza virus NS1 or Vesicular Stomatitis Virus M, and it has been suggested that this is mediated by an increase in protein levels of the mRNA export factor NXF1 [With respect to the exact role of de novo pyrimidine synthesis in the viral life cycle, and the mechanism by which inhibitors of this pathway exert their antiviral activity, several mechanisms have been proposed for other viruses: (1) De novo pyrimidine synthesis is required for B and T cell proliferation , and thutor NXF1 . This mitor NXF1 , was abstor NXF1 , 60. Surtor NXF1 , 51, 61.tor NXF1 , and alstor NXF1  but alsotor NXF1  and diretor NXF1 –66. Thustor NXF1 , 42. GivGiven that the DHODH inhibitor teriflunomide is an FDA- and EU-approved drug for the treatment of multiple sclerosis, with favorable long-term safety data , and thaIn summary, we have performed a genome-wide siRNA screen for host factors important for genome replication and transcription of EBOV, the primary data from which will allow data mining for novel pro- and antiviral factors against these processes. In our own analysis, de novo pyrimidine synthesis emerged as the top pathway important for genome replication and transcription in our screen. This was further confirmed using an inhibitor of this pathway, which is FDA- and EU-approved, and thus should be further evaluated for its efficacy against EBOV in vivo. Overall, this study highlights the power of reverse genetics-based life cycle modelling systems for understanding the biology of highly pathogenic viruses, and for the development of new approaches for countermeasures.Additional file 1:Supplementary figures and methods. (PDF 884 kb)Additional file 2:Uncorrected and seed-corrected logP values from RSA analysis, excel file format. (XLSX 1121 kb)Additional file 3:Genes selected for follow-up, excel file format. (XLSX 14 kb)Additional file 4:Results of common seed analysis, pdf file format. (PDF 2900 kb)Additional file 5:Results of secondary screen, excel file format. (XLSX 50 kb)Additional file 6:Results of secondary assay, excel file format. (XLSX 18 kb)


Aspergillus thermomutatus (Teleomorph: Neosartorya pseudofischeri) was found to contain ~35 nm isometric virus-like particles associated with four double-stranded (ds) RNA segments, each of which coded for a single open reading frame. The longest dsRNA element (3589 nt) encodes a putative RNA-dependent RNA polymerase (1114 aa), the second longest dsRNA element (2772 nt) encodes a coat protein (825 aa), and the other two dsRNAs  encode hypothetical proteins of 768 aa and 711 aa, respectively. Phylogenetic analysis of the amino acid sequences showed 41–60% similarity to the proteins coded by the dsRNAs of the most closely related virus, Penicillium janczewskii chrysovirus 2, indicating that it is a new species based on the International Committee on Taxonomy of Viruses criteria for the genus Chrysovirus. This is the first virus reported from A. thermomutatus and was tentatively named Aspergillus thermomutatus chrysovirus 1. A virus free line of the fungal isolate, cured by cycloheximide treatment, produced large numbers of conidia but no ascospores at both 20°C and 37°C, whereas the virus infected line produced ten-fold fewer conidia at 20°C and a large number of ascospores at both temperatures. The effects of the virus on fungal sporulation have interesting implications for the spread of the fungus and possible use of the virus as a biological control agent.A clinical isolate of  Aspergillus species have been reported to be infected with mycoviruses, or uncharacterised dsRNA segments [Aspergillus section Nigri [Aspergillus section Flavi [Aspergillus section Circundati, and section Fumigati [Aspergillus section Clavati [A. foetidus [A. niger [A. flavus [A. fumigates [A. ochraceus [The vast majority of the reported mycoviruses contain double-stranded (ds) RNA genomes; a lesser number have single-stranded (ss) RNA genomes, while only a few mycoviruses possess single-stranded DNA . Consequsegments . These ion Nigri , 5, 6, Aon Flavi , AspergiFumigati , Aspergi Clavati , A. foetfoetidus , 11, A. A. niger , A. flav. flavus , 14, 15,umigates , 19, 20 chraceus , 22, witchraceus .Aspergillus thermomutatus (Paden) S. W. Peterson (Teleomorph: Neosartorya pseudofischeri S. W. Peterson) was first described by Paden [Aspergilli [A. thermomutatus. However, A. thermomutatus often produces both anamorph  and teleomorph  stages together in the same culture [by Paden  and re-eby Paden . It is kby Paden , 26. Altby Paden , there aby Paden , 28, 29.pergilli  and ther culture , and a c culture .A. thermomutatus (Ath1) was obtained on 10/7/2012 from the fungal culture collection at Middlemore Hospital, Auckland, New Zealand. While the fungal cultures studied were originally derived from clinical isolates their origin was anonymous and the material analysed consisted of pure fungal cultures that contained no human material. Cultures were maintained on Potato Dextrose Agar  at 37°C in the dark. The Ath1 isolate was studied in a Physical Containment Level 2 laboratory, where all culturing procedures were conducted in a biological safety cabinet under the highest possible level of decontamination.An isolate of DsRNA was extracted from fungal mycelium and purified virus particles, according to the method of Valverde et al.  as modifwww.ronyasoft.com).Virus-infected Ath1 was grown in conical flasks containing 200 mL Yeast Extract Peptone Dextrose broth (YPD) and incubated on a shaking incubator at 180 rpm in the dark. Approximately 10 g of fungal mycelium was harvested on a filter paper using vacuum filtration, ground to a fine powder in liquid nitrogen and transferred to a 50-mL Falcon tube containing 20 mL of sodium phosphate buffer  and 10 mL chloroform. The mixture was incubated for 30 min on ice on an orbital shaker at 230 rpm then centrifuged at 10,000 x g for 30 min at 4°C. The upper aqueous phase was centrifuged at 120,000 x g for 2 h at 4°C and the resultant pellet resuspended in 1 mL SPB (0.02 M pH 7.0) for 4 h at 4°C. The suspension was clarified by centrifugation at 10,000 x g for 20 min at 4°C, the supernatant centrifuged at 120,000 x g for 2 h at 4°C and the pellet resuspended in 0.5 mL SPB  overnight at 4°C. Following centrifugation at 10,000 x g for 20 min a 50-μL drop of the supernatant was negatively stained with 2% uranyl acetate (pH 4.0) and observed for virus particles using a Phillips CM12 TEM. The size of 100 virus particles was determined relative to the scale bar (200 nm) incorporated into the EM image from enlarged photomicrographs (79 x 79 cm) using Poster Printer software v 3.01.43 (12-3`)(5`-CTCACCTTCGGATCCTCC with a unique four nucleotide index at the 5`end was used to generate dsDNA fragments with random lengths (~600–1500 bp), which were sequenced by Macrogen Inc. (South Korea) as described by Roossinck et al. [de novo assembled in Geneious software version 8.0.3 [Initial partial sequence of purified dsRNA, obtained by 454 sequencing (accession: SAMN10160075), was used as the starting point for complete genome sequencing. Preparation of samples was performed as described by Roossinck et al. . A primek et al. . Reads won 8.0.3 .Escherichia coli-competent cells and sequenced using vector-specific primers (M13 forward 5´-GTAAAACGACGGCCAG-3´ and M13 Reverse 5´-CAGGAAACAGCTATGAC-3´). Nucleotide sequences were assembled using Geneious version 8.0.3 [For each of the four dsRNA elements, viral dsRNA was gel purified using an Axyprep DNA gel extraction kit (AXYGEN) and used as a template for cDNA synthesis, dsDNA amplification and determination of 5`and 3`terminal sequences as described by Khalifa and Pearson . PCR proon 8.0.3  as descron 8.0.3 .http://unafold.rna.albany.edu/?q=DINAMelt/Quickfold) with the following parameters: RNA at 37°C, Na+ = 1 M, Mg++ = 0 M, sequence type = linear, distance between paired bases = no limit. The closest published sequences to the consensus sequences (for both the PCR products and 454 sequence data) were determined using BLASTn (blast.ncbi.nlm.nih.gov/Blast.cgi). Where BLASTn failed to find a similar sequence, BLASTx (translated nucleotide to protein) was used to find the closest amino acid pairwise sequence. Deduced amino acid sequences of the RNA-dependent RNA polymerases (RdRPs) of the new A. thermomutatus virus and the closest published sequences were aligned using MUSCLE multiple sequence alignment software [The potential secondary structure of the 5`and 3`termini of the dsRNA1 was predicted and the minimum free energy (ΔG) was estimated using Quickfold software (software  and usedsoftware  for distsoftware .(forward primer: 5`-CGAGTGTGAGGCATCAAAGC-3`and a reverse primer: 5`-TGTCGCATGATGCATATAATTGGG-3`) and PrimeScript One-step RT-PCR Kit , according to the manufacturer’s instructions. The RT-PCR protocol was: one cycle at 50°C for 30 min; 2 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 58°C and 1 min at 72°C.To test for the presence of AthCV1, total RNA was extracted from fungal cultures using a Spectrum Plant Total RNA Kit (Sigma-Aldrich), as described by the manufacturer, and used as a template for one-step RT-PCR using a virus-specific primer pair To obtain a virus-free line of Ath1 for comparison with the virus-infected line, plugs of mycelium from the edge of an Ath1 virus-infected culture (grown on PDA plates at 37°C in the dark) were transferred to PDA plates containing 21 mg/mL cycloheximide (Sigma) and incubated in the dark for 1 week at 37°C. Single conidiospore isolates were grown on unamended PDA media for 3 days at 37°C in the dark, followed by a second round of cycloheximide treatment (as described above). Single spore isolates were then grown on unamended PDA media in the dark for 1 week at 37°C, sub-cultured twice and then tested for the presence of virus.ITS1F forward primer 5`-CTTGGTCATTTAGAGGAAGTAA-3`and ITS4 reverse primer 5`-TCCTCCGCTTATTGATATGC-3`[Internal Transcribed Spacer (ITS) regions of the naturally virus infected and the generated virus free cultures were PCR amplified using ATATGC-3` and sequSporulation rate comparisons were conducted at 37°C, to represent human body temperature, and 20°C to represent an environmental temperature. Five virus-free cultures (obtained from virus-infected cultures using cycloheximide treatment) and five virus-infected cultures were grown from single spores and inoculated at the edge of PDA plates (9 cm Ø) and incubated in the dark until the mycelium reached the opposite side of the plate. Spores were harvested from the plates by washing twice with 20 mL 0.05% Tween 80 and filtering through cheesecloth into a 50-mL Falcon tube. The spores were collected by centrifugation at 8000 x g for 10 min and resuspended in 10 mL distilled water before being counted in a Neubauer chamber. For data analysis, an independent samples t-test was performed using SPSS version 21 (IBM SPSS statistics).To separate ascospores from possible contamination by conidia, prior to testing for virus infection, a preliminary heat-treatment experiment was extrapolated from O'Gorman et al.  and GiraOne hundred ascospores were germinated individually on PDA plates for one day at 37°C in the dark and individual germlings transferred to 250-mL flasks containing 100 mL YPD and incubated at 37°C for 2 days on an orbital shaker at 180 rpm. AthCV1-specific one-step RT-PCR was conducted on the resultant mycelium to test for virus presence, as described above.Virus-free and virus-infected cultures were grown for 7 days on PDA in the dark. To compare linear growth, five replicate virus-free and virus-infected single spore isolates were individually inoculated at the edge of PDA plates and grown at 37°C for 6 days and at 20°C for 15 days in the dark. The growth was measured every 24 h and at the completion of the experiment cultures were tested for the presence of the virus using RT-PCR as described above. To compare biomass production, five virus-free and virus-infected single spore isolates were grown at 37°C for 4 days and at 20°C for 15 days in the dark, and five replicate plugs of the resultant mycelium individually transferred to conical flasks containing 200 mL YPD and incubated on a shaking incubator at 180 rpm in the dark. Mycelium was collected by vacuum filtration; 100 mg of each sample being retained for virus testing (using one-step RT-PCR as described above) and the remainder dried at 90°C for 72 h before weighing. An independent samples t-test was performed using SPSS version 21 (IBM SPSS statistics) for data analysis.2) from each sector were tested for the presence of the virus by RT-PCR using virus-specific primer pairs as described above.Formation of sectors with different growth phenotypes was frequently observed in the virus-infected Ath1 cultures. These sectors were either smooth white cottony, or with rough surface texture. Three plugs of mycelium . The mycelium was then coarsely cut with a sterile blade in a glass beaker and transferred into a 150-mL flask containing 17 mL protoplast buffer. Then 0.2 g of Lysing Enzymes from Trichoderma harzianum (Sigma-Aldrich) was dissolved in 3 mL Novozyme buffer , filter sterilised through a 0.40-μm strainer and added to the mycelial suspension, followed by incubation in a shaking incubator at 85 rpm for 4 h at 28°C. Protoplast formation was checked every 20 min, starting from 60 min, and after 4 h the protoplasts were passed through a 75-μm strainer and collected in a 50-mL tube. A 30-mL volume of KC buffer (0.6 M KCl and 50 mM CaCl2) was immediately added to the protoplast suspension and the mixture centrifuged at 4000 x g for 10 min. The protoplast pellet was then washed twice with 10 mL STC  at 4000 x g for 10 min before being resuspended in 0.5 mL STC and kept on ice.Since the virus-free culture of Ath1 did not reproduce sexually, it was important to determine whether the virus-free culture would produce sexual spores when it was transfected with the virus. Protoplasts were prepared according to the method of Kohn et al.  with mod2) and added to the spermidine and virus particles suspension. A 200-μL aliquot of the protoplast suspension was then added to the virus suspension and mixed by twirling the tube for 10 s before it was incubated on ice for 30 min. Following incubation, 200 μL of PEG 4000 was mixed with 100 μL KTC in a separate tube, added to the previous suspension and gently twirled again and incubated at room temperature for 20 min. Following incubation, 40-μL aliquots of the mixture were added to 5 mL warm agar medium  containing 0.7% agar), gently mixed and immediately spread on SMM plates which were sealed with Parafilm and incubated at 37°C [Transfection was conducted as described by Rollins  with mod at 37°C . IndividdsRNA1, forward 5`-AGCTCAGTCATCGAGATAGC-3`, reverse 5`-TCTATGTCGGGTCCCTCTAT-3`; dsRNA2, forward 5`-CGAGTGTGAGGCATCAAAGC-3`, reverse 5`-TGTCGCATGATGCATATAATTGGG-3`; dsRNA3, forward 5`-TGCTTTCTTCAACCACTTCC-3`, reverse 5`-TGAGTACTCGCACACGTC-3`; dsRNA4, forward 5`-TTGGCTGTACGTTGGCGATG -3`, reverse 5`-TGAATGGGTCCAGCCACTC -3`. Electrophoresis and DNA visualization was conducted as described previously.When grown on solid media, the naturally infected Ath1 culture usually exhibited a rough texture from which white cottony ascospore-free sectors repeatedly emerged. When subcultured, the white cottony ascospore-free sectors retained this phenotype and did not produce any sectors. Similarly, virus-transfected lines showed only the white cottony ascospore-free morphology. To try to explain these differences, the different phenotypes were tested for the virus over time to determine the presence and persistence of virus infection. Ten serial sub-cultures (each grown for one week) were made from each phenotype and total RNA extraction, and virus testing performed by RT-PCR, as previously described, using virus-specific primer pairs as follows: Initial electrophoresis of dsRNAs from Ath1 (both mycelium and virus particle) in 1% agarose gel at 90 V for 45 min showed the presence of two dsRNAs (~3600 bp and ~2700 bp), but subsequent electrophoresis in 1.5% agarose gel for 19 h at 80 V separated the lower band into three bands of 2.5, 2.68 and 2.7 kb . HomogenChrysovirus does not exist in any of the four dsRNA elements of AthCV1. The closest sequences in GenBank were the four dsRNAs of Penicillium janczewskii chrysovirus 2 , dsRNA2 2772 nt (MF045842), dsRNA3 2676 nt (MF045843) and dsRNA4 2514 nt (MF045844). Each dsRNA segment contains a single open reading frame and 5`and 3`-UTRs . DsRNA1 ovirus 2 .A. thermomutatus virus in the same monophylogenetic clade with Botryosphaeria dothidea chrysovirus 1 [Alternaria alternata chrysovirus 1 [Penicillium janczewskii chrysovirus 2 [Colletotrichum fructicola chrysovirus 1 [A phylogenetic comparison of the translated amino acid sequences from the RdRP, encoded by dsRNA1, with those of 26 other chrysoviruses  placed tovirus 1 , Alternaovirus 1  and Peniovirus 2  with theovirus 1 . SequencNeosartorya pseudofischeri isolate NRRL (teleomorph of Aspergillus thermomutatus). Sequences of ITS 1 and 2 regions have been reported to identify Aspergillus species [PCR screening and agarose gel electrophoresis of dsRNA revealed that a virus-free culture was successfully obtained  followin species , 53.p<0.05) in the virus-infected culture compared with that in cultures at 37°C where conidial production was ten-fold greater than in the virus-free culture. There was no significant difference (p<0.05) in the rate of mycelial growth or mycelial dry weight between virus-negative and virus-positive Ath1 cultures at either 20°C or 37°C.The effects of viral infection and temperature on the growth and sporulation of Ath1 are summarized in RT-PCR screening detected AthCV1 in both the creamy rough ascospore-rich and the white cottony ascospore-free sectors of both the initial cultures of naturally infected Ath1 and after four successive subcultures. However, after ten successive weekly subcultures the virus was detected only in the cultures’ originating from and still expressing the creamy rough ascospore-rich phenotype. Similarly, cultures transfected with AthCV1, which showed the white cottony ascospore-free phenotype, tested positive for AthCV1 after four subcultures but negative after ten subcultures.RT-PCR screening for AthCV1 detected the virus in 37% of 100 single ascospore cultures. Although the ascospore isolation method included a heat treatment of 65°C for 15 min, this is unlikely to have inactivated a dsRNA virus.Aspergillus species in culture, a clinical isolate of Ath1 was observed to produce few conidia and large numbers of cleistothecia. In addition, the cultures produced sectors exhibiting two different phenotypes; a creamy rough granulated sector with conidia and ascospores, and a white cottony sector producing conidia only. These cultures were found to be infected with a chrysovirus, closely related to Penicillium janczewskii chrysovirus 2 [Botryosphaeria dothidea chrysovirus 1, the latter being reported to have a hypovirulent impact on its fungal host and to cause sector formation in the infected cultures [Unusually for ovirus 2  and Botrcultures .Chrysovirus is Penicillium chrysogenum virus (PcV) in which dsRNA1 codes for the RdRP (1117 aa), dsRNA2 codes for the CP (982 aa), while dsRNA3 and dsRNA4 code for hypothetical proteins  [Aspergillus spp., including A. foetidus [A. niger [A. flavus [A. fumigatus [Chrysoviruses are non-enveloped isometric particles, with a multipartite genome of four unrelated monocistronic linear dsRNA segments. The type member of the genus ctively) . The 5`-the RdRP 17 aa, ds. flavus  and mostumigatus , 17.A. thermomutatus. It exhibits all the common characteristics of chrysoviruses and is provisionally named A. thermomutatus chrysovirus 1 (AthCV1). The ~35 nm isometric particles encapsidate four dsRNA segments , with amino acid sequence similarity to known chrysoviruses (Penicillium janczewskii chrysovirus 2 (PjCV2) [Chrysovirus [The virus isolated from Ath1 is the first virus described from oviruses , Table 3oviruses . In comm (PjCV2) , the mosysovirus , 56, 57.Aspergillus spp. [A. fumigatus were found to be infected with a chrysovirus, a partitivirus [A. niger cultures [Sector formation in cultures of Ath1 appeared to be associated with the presence of AthCV1. Sector formation in virus-infected fungal cultures, has previously been observed in a range of fungal species, including lus spp. , 46. Conitivirus , a tetraitivirus , and acoNeosartorya hiratsukae (Anamorph: Aspergillus hiratsukae) was very efficient (percentage not given). Transmission of AthCV1 through the ascospores of Ath1 was relatively low (37%), thus providing a means for the fungus to escape from virus infection. The apparent ability of virus-infected cultures spontaneously to produce ascospore-free sectors that after multiple subcultures result in virus-free cultures, may also provide a means for the fungus to escape virus infection in vivo, and is worthy of further investigation.The vertical transmission of mycoviruses through conidia is often highly efficient and can be close to 100% in some cases . In contAspergillus species [The isolate Ath1 was the only isolate of its species being studied, within a biosafety cabinet, and using rigorous decontamination procedures to avoid contamination resulting in an extremely low chance of contamination. Regardless, it was considered important to double confirm the identity of the virus-free culture by sequencing the ITS 1 and ITS 2 regions as it has been previously used to identify  species , 52, 53.The variable production of both asexual and sexual reproductive structures in virus-infected Ath1 cultures has implications for virus spread within the fungal population. The effect of AthCV1 on conidia production was dependent on temperature, with production of conidia in virus-infected cultures being significantly lower at 20°C and significantly higher at 37°C than in the virus-free cultures. It is uncertain whether this is unusual, as the majority of published studies report only the effects of mycoviruses on their host at a single temperature. However, these findings do highlight the importance of the virus-host-environment interaction.Chrysovirus, such as Botryosphaeria dothidea chrysovirus 1 [Magnaporthe oryzae chrysovirus 1-A [Magnaporthe oryzae chrysovirus 1-B [Agaricus bisporus virus 1 [Aspergillus mycovirus 1816 was reported as a probable suppressor of RNA silencing [Use of mycoviruses as biological control agents for fungi is a topic of wide interest, but an effective biological control agent requires consideration of multiple factors , as is well documented for chestnut blight control . The firovirus 1 , Magnapoirus 1-A , Magnapoirus 1-B , Agaricu virus 1 , are knoilencing . While AAspergillus species and whether it could have significant effects on their biological properties, especially on sporulation. Even if it is impractical to use AthCV1 directly as a biocontrol agent, an understanding of the molecular nature of the effects caused by the virus may enable the development of approaches such as targeting gene expression by pharmaceuticals.A major constraint on the use of mycoviruses as biocontrol agents is that natural horizontal spread typically requires hyphal anastomosis, which is limited by genetically controlled hyphal incompatibility , therebyS1 FigSecondary structure proposed for the 5`-UTR (left) and 3`-UTR  of the plus strand of AthCV1 dsRNA1 (putative RdRP). Minimum free energy is -53 and -35.7 kcal/mol for 5`and 3`termini respectively. Jop parameters: RNA at 37°C, Na+ = 1 M, Mg++ = 0 M, sequence type (linear), distance between paired bases (no limit).(TIF)Click here for additional data file.


New reagents have emerged allowing researchers to assess a growing number of vaccine-associated immune parameters. Multiplex immunoassay(s) are emerging as efficient high-throughput assays in malaria serology. Currently, commercial vendors market several bead reagents for cytometric bead assays (CBA) but relative performances are not well published. We have compared two types of bead-based multiplex assays to measure relative antibody levels to malarial antigens.Plasmodium falciparum vaccine candidates using non-magnetic and magnetic fluorescent microspheres were compared for their performances with a Bio-Plex200 instrument. Mean fluorescence intensity (MFI) was determined from individuals from western Kenya and compared to known positive and negative control plasma samples.Assays for the measurement of antibodies to five P. falciparum recombinant antigens were successfully coupled to both non-magnetic and magnetic beads in multiplex assays. MFIs between the two bead types were comparable for all antigens tested. Bead recovery was superior with magnetic beads for all antigens. MFI values of stored non-magnetic coupled beads did not differ from freshly coupled beads, though they showed higher levels of bead aggregation.P. falciparum antibody assays. Magnetic beads were more expensive, but had higher bead recovery, were more convenient to use, and provided rapid and easy protocol manipulation. Magnetic beads are a suitable alternative to non-magnetic beads in malarial antibody serology.Magnetic and non-magnetic beads performed similarly in  Plasmodium falciparum antigens have been correlated in multiple immuno-epidemiological studies with protection from infection . The capacity to measure relative levels of antibodies to multiple antigens could ultimately increase uniformity for comparative reasons. On this front, there has been a surge in the development of multiplex methodologies as reliable replacements of ELISAs .The introduction of magnetic beads demands validation with a Bio-Plex7 beads mL−1 and were stored at 2–4 °C. Both beads and sheath fluid were obtained from Luminex .The carboxylated SeroMAP™ microspheres (non-magnetic) product numbers: S005, S015, S025, S030, S040, S045, S050, S055, and S060; and carboxylated MagPlex™ microspheres (magnetic) beads, product numbers: MC10029-01, MC10036-01, MC10045-01, MC10054-01 and MC10065-01 were used. Bead stock suspensions were at 1. 25 ×10200 . The Bio-Plex200 consists of a Luminex 200 suspension array reader, high-throughput fluidics system, a microplate platform, monitor and computer operated with BPM 5.1 software (Bio-Rad). All washing steps of magnetic beads were carried out on a HydroFlex™ microplate washer  using a magnetic plate carrier, while washing of non-magnetic beads was performed using a millipore vacuum manifold. Plates were incubated on an IKA micro-titer shaker ®MTS . During the coupling procedures, the magnetic microspheres were captured using a DynaMag magnetic separator . All centrifugation steps were carried out in an Eppendorf centrifuge.The antibody measurements were performed using a Bio-PlexFor all multiplex bead assays, a plasma pool made of 30 plasma samples from adults living in a Ugandan area of seasonal malaria transmission ; erythrocyte-binding antigen ; glutamate rich protein ; liver stage antigen ; merozoite surface protein-1 ; merozoite surface protein-3  and recombinant thrombospondin-related adhesion protein . Recombinant AMA-1, LSA-NRC and TRAP were expressed in Escherichia coli and provided by David E. Lanar, Walter Reed Army Institute for Research. Recombinant MSP-142 and MSP-3 were expressed in E. coli, and EBA-175 was expressed in Pichia pastoris, and provided by David Narum, National Institutes of Health. Recombinant GLURP-R0 and GLURP-R2 were expressed in E. coli and provided by Michael Theisen, Statens Seruminstitut, Copenhagen, Denmark. Recombinant MSP-119 expressed in Saccharomyces cerevisiae, was provided by the Malaria Research and Reference Reagent Resource Center , and originally deposited there by David Kaslow.The recombinant . P. falciparum antigens were directly coupled to non-magnetic beads and each to a unique bead set, using a previously described procedure  at 600 rpm for 30 s, followed by 300 rpm for 30 min at room temperature (RT). Plates were aspirated using a millipore vacuum manifold, washed twice with 100 µl/well of PBNT, and beads resuspended in 50 µl PBNT. Fifty µl of goat anti-human IgG 2 fragment-R-phycoerythrin  was added to each well, and incubated in the dark with shaking at 600 rpm for 30 s, followed by 300 rpm for 30 min. Plates were aspirated using a millipore vacuum manifold and washed twice with 100 µl/well PBNT. The beads were resuspended in 100 µl PBNT and finally analyzed on a Bio-Plex200  equipped with Bio-Plex Manager 5.0 software (Bio-Rad). Samples were analyzed with the following Bio-Plex200 settings: 100 beads per region, timeout time was set at none, 50 µL sample volume, and DD gate 5,000–2,5000, default settings and the RP1 target were turned on.All reagents and samples were brought to room temperature before use for both types of beads and plasma samples were diluted before use. Non-magnetic beads were diluted with PBNT  and used at a working concentration of 1,000 beads/region/well. Bead stocks were then combined in a 15 ml amber conical tube. 96 well-millipore microtiter plates  were pre-wetted with 100 µl of PBNT/well and aspirated using a millipore vacuum manifold and 50 µl of working bead solution was transferred to each well. Plasma samples were thawed at room temperature, mixed and centrifuged at 16,000 g for 3 min then diluted to 1:100 in buffer . Fifty µl of diluted plasma was added into each well of a microtiter plate in duplicate. The plasma was mixed with the beads three times by pipetting up and down and the plates incubated in the dark on a shaking micro- plate shaker  equipped with Bio-Plex Manager 5.0 software (Bio-Rad) for median fluorescence intensity (MFI). Analyses were performed with a minimum count of 100 beads per region as per the manufacturer’s instructions.The magnetic-based assays were carried out according to the protocol as provided by the manufacturer. Briefly, 50 µl of working bead solution  was transferred to each Greiner 96-well microtiter black, flat bottom plate . The beads were incubated in the dark on a microplate shaker at 600 rpm for 30 s, followed by 300 rpm for 30 min with 1: 100 diluted plasma samples added in duplicate at room temperature. Wells were washed twice with an automated HydroFlex™ microplate washer  using 100 µl of PBNT, and beads resuspended in 50 µl PBNT. Fifty µl goat anti-human IgG 2 fragment-R-phycoerythrin was added to each well. Wells were washed twice with 100 µl PBNT with an automated microplate washer and beads resuspended in 100 µl PBNT and finally analyzed on a Bio-PlexP-value of <0.05 was considered to be statistically significant. Statistical analysis was performed using GraphPad Prism version 5.0 .After subtraction of the background blank, the mean of results from the duplicate wells was reported as the median fluorescence intensity minus background (MFI–BG). To obtain the cut off value for positivity, three standard deviations (SD) were added to the average MFI of the negative controls. Mann Whitney test was used to compare between MFI values from non-magnetic and magnetic beads. In addition, Spearman correlation coefficients (Rho) was used to compare MFI values obtained from stored coupled beads for 18 months and freshly coupled beads. A P. falciparum recombinant antigens to the two bead types (P > 0.05) (To assess the performance of the multiplex assays the range of fluorescence generated was used as an indicator of the effectiveness of antigen coupling to the beads. The mean MFIs (SD) ranged from low values (for blanks) to high values (for positive plasma controls) for both non-magnetic and magnetic beads. These ranges of values suggested successful coupling reactions for each of the five ad types . A compa > 0.05) .The instrument detection limit (IDL) was defined as the analyte concentration that would be required to produce a signal greater than three times the standard deviation of the noise level (negative controls) by both non-magnetic and magnetic beads. IDL provided the lowest quantity of an analyte that could be distinguished above the negative control samples. This experiment provided a proxy measure of accuracy of the two sets of beads from the raw MFI values generated. Both non-magnetic and magnetic beads exhibited a wide dynamic range and had the ability to read concentrations down to the minimum reported concentration for each of the analytes. Most importantly, the two bead types provided similar detection limits at low plasma concentrations for all analytes .P > 0.05). In addition the number of positive responders for AMA-1, EBA-175, MSP-119, MSP-142 and MSP-3 IgG antibodies were almost identical (data not shown).A comparison of ranges of MFI values obtained in a range of samples from individuals from western Kenya and North Americans showed that the two bead types had equivalent MFI values  –76%, MSP-1(42)–63% and MSP-3–66%) for non- magnetic beads. Bead recovery in magnetic beads, ranged from 83–92% –86%, MSP-1 (42)–89% and MSP-3–92%). Our findings indicate that magnetic beads had higher yields microsphere recoveries post-coupling for each of the five coupled P.  falciparum antigens were measured in plasma samples from individuals from malaria endemic area of western Kenya, using freshly coupled beads and beads coupled 18 months ago and stored at 4 °C during this time. Percentage bead aggregation between the types was higher in beads that had been stored for 18 months (data not shown). Freshly coupled beads had similar MFI values to beads that had been stored for 18 months for each of the antibody measured, and all antibody measurements were highly correlated with Spearman correlation rho values, r for all >0.9  To determine if antigens decoupled from the beads during 18 months storage at 4 °C, antibodies to nine P. falciparum antigens. Since the two bead assays are comparable in this respect, other parameters such as ease of use, quick magnetic bead separation and availability should determine suitability of the assay. For both non-magnetic and magnetic beads antigen coupling was successful, the non-specific background was low for blank samples and, the specific signal intensity was high for positive control samples. Also, they were able to discriminate different concentrations of antibodies on serially diluted test samples. Above all, the antigen coupled magnetic microspheres had higher efficiency of bead recoveries. The findings suggest that magnetic bead assays would provide a powerful tool for detection and quantification of P. falciparum antibodies.This paper shows that non-magnetic and magnetic beads perform similarly when used to measure antibodies to 7 beads/ml, $726.00 vs. $438.00 (Bio-Rad Corporation) as at the time of this manuscript submission). In addition, coupling magnetic beads to proteins of interest does not require centrifugation and sedimentation processes. Elimination of centrifugation increases bead recoveries and ease of the assay, making it simple and convenient to the user. Further, use of magnetic beads may overcome limitations associated with non-magnetic beads such as leaking, clogging of micro-plates after washing steps and the need for expensive filter plates. Further, development of automated plate washers for use with magnetic beads further facilitates rapid and accurate testing and overcomes limitations in effectively washing samples using non-magnetic beads. In summary, the process of coupling magnetic beads to proteins of interest is more straightforward and efficient, reduces bead loss, and does not require the use of disposable filter plates. Although magnetic beads are more expensive than non-magnetic beads, the price is comparable when considering the efficiency and use of standard microtiter plates.The results show that both types of beads were comparable . MFI valP. falciparum (In a prior study, storage of coupled non-magnetic beads for seven months at 4 °C in the dark resulted in a decrease in MFI, and it was recommended that lyophilization  could belciparum , suggestP. falciparum antibody patterns in humans plasma samples, which will help advance our understanding of malaria biology and associated host immunological responses. However, this study had a limited sample size and a few analytes were tested because of design aspects based on the amount of stored reagents available for use. Future work should assess the effect of storage of coupled magnetic beads on antibody detection.Overall, the magnetic based Bio-Plex assays offer a novel multiplexed approach to assess P. falciparum antibody assays and provide a suitable alternative to non-magnetic beads for measurement of antibodies to P. falciparum antigens. In addition, use of magnetic beads provide the capacity to evaluate a higher number of P. falciparum analytes due to improved bead recoveries.The magnetic beads performed similarly in 10.7717/peerj.6120/supp-1Supplemental Information 1Click here for additional data file.


Shaanvirus. Through sequence analyses and in vitro testing, the isolated virus was also found to have haemagglutinin-neuraminidase (HN) protein as one of the structural proteins. When mouse antiserum was generated against the isolated virus and tested, it was cross-reactive to human parainfluenza virus 1 in an indirect immunofluorescence assay but could not cross-neutralize human parainfluenza virus 1. In addition, the bat paramyxovirus B16-40 was not infectious in the mouse model. Collectively, this study provided basic information on further classification of the bat paramyxovirus B16-40 and related viruses in the proposed genus Shaanvirus.The bat paramyxovirus B16-40 was first isolated in Korea in this study. Using the isolated virus, we could obtain not only genomic information, but also several biological characteristics of the virus. In the phylogenetic analysis, the virus was found to belong to the recently proposed genus  Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Nipah virus, Hendra virus, and Ebola virus are all thought to be bat-borne virusesParamyxoviridae, order Monone-gavirales4. While Henipaviruses (Nipah and Hendra viruses) in South East Asia and Australia are associated with fruit bats5, other paramyxoviruses have been detected not only in fruit bats but in insectivorous bats worldwide9. A potential pathway for Nipah virus transmission from bats to humans was found to be associated with a human-bat interface, specifically date palm sap shared by bats and humans10. In addition, serological evidence of possible human infection with a bat-originated paramyxovirus, Tioman virus11, reinforces the epidemiological role of bats in the emergence of pathogens such as paramyxoviruses in humans.Notably, bats also host major mammalian paramyxoviruses from the family Paramyxoviridae12. Recent bat-associated paramyxoviruses were proposed to be grouped in a separate phylogenetic clade within a potentially separate genus such as Shaanvirus13 which was distantly related to Jeilongvirus14. In addition, novel strains of bat paramyxoviruses in diverse genera have been reported continuously17. Based on the recent papers, bat paramyxoviruses found worldwide to date have belonged to the genera Rubulavirus, Morbillivirus, Henipavirus and the unclassified proposed genera Shaanvirus. Expanded classifications for grouping newly identified viruses in bats can be accomplished by further studying the biological characteristics of novel paramyxoviruses as well as genome characterization18.In addition to these bat paramyxoviruses with zoonotic potential, other new paramyxoviruses have been reported. These include several new mammalian paramyxoviruses such as Beilong virus and J virus, which remain unassigned under the family In this study, active surveillance was performed to reveal paramyxoviruses circulating in Korean bats. A total of 232 bat samples were collected at 48 sites in natural bat habitats and tested for the possible existence of paramyxoviruses.19, five samples were positive and confirmed to be bat paramyxoviruses by sequencing. B16-6 was from a urine sample of Miniopterus schreibersii at BT cave in Hapcheon in March 2016, and B16-40 was from a feces sample of Miniopterus schreibersii at G cave in Danyang in April 2016. B16-148 and B16-154 were from feces samples of Murina ussuriensis at OJ cave in Youngwol and at S cave in Pyungchang in July 2016, respectively. B16-230 was from a urine sample of Miniopterus schreibersii at BLR cave in Seogwipo in December 2016. Among these, one paramyxovirus (B16-40) was successfully isolated.In this study, 232 bat samples were collected at 48 sites from natural bat habitats and tested for the possible existence of paramyxoviruses  method 50/mL or 102.5 TCID50/mL of bat paramyxovirus B16-40, we found no evidence of infection, i.e. no viral shedding, histopathological findings, or seroconversion.When 6-week-old female C57BL/6 mice were inoculated via the intranasal and intragastric routes with 10de novo sequencing platform), 23,336 contigs totaling 8,010,635 base pairs (bps) with an average length of 343 bps were obtained. In the taxonomy annotation with MG-RAST, 12,522 contigs were annotated. While most of the annotated sequences associated with eukaryotes, 13 sequences were associated with family Paramyxoviridae. The 17,771 bases of viral genomic sequences were obtained by RT-PCR using primers designed based on the Paramyxoviridae-associated contigs and RACE sequencing.Using high throughput sequencing  and BtMI-ParaV/QH2013 (KJ641657) strains rather than Beilong virus, J-virus, and Tailam virus  Fig. , which irus Figs  and S1. Shaanvirus, with 68.8–79.0% nucleotide similarities  gene, the bat paramyxovirus isolates and other Korean bat paramyxoviruses belong to the genus ies Fig. . This suConsistent with the genetic findings, the bat paramyxovirus B16-40 was found to have haemagglutinin and neuraminidase activities in the haemadsorption assay (0.5% chicken red blood cells in PBS (pH 7.4)) and neuraminidase assay, respectively Fig. .Figure 3In the preliminary studies of mouse antisera (BALB/cA mice) against human respiratory syncytial virus A (KBPV-VR-41), human mumps virus (KBPV-VR-51) and human parainfluenza virus 1 (KBPV-VR-44), the antisera against human parainfluenza virus 1 (KBPV-VR-44) was only cross-reactive with the bat paramyxovirus B16-40 as determined using the immunofluorescence assay. Therefore, further cross-reactivity and cross-neutralizing tests between human parainfluenza virus 1 (KBPV-VR-44) and the bat paramyxovirus B16-40 were performed.As shown in Table While the mouse antisera neutralized the homologous virus with 20–160 serum neutralizing titer, they did not neutralize the heterologous virus . The proposed genera Shaanvirus and Jeilongvirus have two novel genes putatively encoding SH  and TM (transmembrane) proteins13. Two viral genes were also located between the fusion and haemagglutinin-neuraminidase genes of bat paramyxovirus B16-40. Further, their nucleotide and amino acids sequences were similar to the SH and TM genes identified from bat paramyxovirus strain Ms-ParaV/Anhui 2011. Therefore, the isolated bat paramyxovirus B16-40 might belong to the proposed genus Shaanvirus rather than Jeilongvirus.The bat paramyxovirus B16-40 in this study was similar to viruses in the recently proposed genera Shaanvirus, it was also related to that of Sendai virus and human parainfluenza virus 1, which belong to a different genus, Respirovirus , two of these (E at the third site and Y) were located within the conserved amino acid sequences. These bioinformatics findings might indicate that the HN proteins of bat paramyxovirus B16-40 and human parainfluenza virus 1 (KBPV-VR-44) are genetically related, even though they are in different genera. Further study on their antigenic and evolutionary relationship should be followed up.The bat paramyxovirus B16-40 in this study was predicted to have a haemagglutinin-neuraminidase (HN) protein as an attachment glycoprotein in the sequence analysis. Consistent with the genetic findings, the bat paramyxovirus B16-40 was found to have haemagglutinin and neuraminidase activities in the haemadsorption assay (0.5% chicken red blood cells in PBS (pH 7.4)) and neuraminidase assay, respectively Fig. . NotablyMicropteropus pusillus was reactive with human parainfluenza virus 1 in an indirect immunofluorescence assay3. There may be two possibilities to explain this. First, the African bat might have been infected with a human parainfluenza virus 1-related virus in the genus Respirovirus that has not yet been found. However, as there has been no evidence of bat paramyxoviruses related to the genus Respirovirus in recent studies16, the first possibility seems low. Second, the bat might have been infected by other bat paramyxoviruses which were cross-reactive with human parainfluenza virus 1. In fact, in this study, when mouse antisera were made and tested against bat paramyxovirus B16-40 and human parainfluenza virus 1 (KBPV-VR-44), the two viruses were partially cross-reactive to each other in an indirect immunofluorescence assay. This result provided an evidence that there could be cross-reactivity between viruses belonging to different genera. Therefore, the bat paramyxovirus B16-40-related viruses circulating in bats might be one of the reasons why the wild bat sera was reactive with human parainfluenza virus 1 in the previous study.Notably, in a previous study, bat serum (diluted 1/40) from African wild bat, Respirovirus24, and human mumps virus and African bat mumps virus, which both belong to the genus Rubulavirus25. Further, the engaged proteins in the cross-reactivity between bat paramyxovirus B16-40 and human parainfluenza virus 1 had not been revealed. Therefore, although bat paramyxovirus B16-40 in this study was cross-reactive with human parainfluenza virus 1 in indirect immunofluorescence assay, it is difficult to say that the bat paramyxovirus B16-40 was antigenically related with human parainfluenza virus 1.However, we could not find the cross-neutralization between bat paramyxovirus B16-40 and human parainfluenza virus 1. Based on the recent studies, cross-neutralization was found between the viruses in the same genus of paramyxoviruses: Sendai virus and human parainfluenza virus 1, which both belong to the genus 18. Additional input from the biological context may be helpful. In this study, a bat paramyxovirus B16-40 belonging to the proposed genus Shaanvirus was first isolated. Through virus isolation, we could obtain not only nucleotide sequence information, but also several biological characteristics of the virus. Its putative HN protein was demonstrated to have haemagglutinin and neuraminidase activities. Mouse antisera against the bat paramyxovirus B16-40 was also cross-reactive to human parainfluenza virus 1 in the indirect immunofluorescence assay, but could not cross-neutralize human parainfluenza virus 1. Additionally, the bat paramyxovirus B16-40 was not infectious in 6-week-old female C57BL/6 mice. Therefore, this study provided basic information on further classification of the bat paramyxovirus B16-40 and related viruses in the proposed genus Shaanvirus.Since a number of paramyxoviruses have been newly found around world, their classification has been difficult due to the limited criteria, such as sequence aloneAll animal experiments were conducted at the Korea Research Institute of Bioscience and Biotechnology , and were approved by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of KRIBB .26. These species were Rhinolophus ferrumequinum, Murina leucogaster, Miniopterus, Murina ussuriensis, Eptesicus serotinus, Myotis bombinus, Myotis macrodactylus, Hypsugo alaschanicus, and Myotis daubentoni  in a 10% suspension and were transported to the lab (BSL2) for further analysis. The major bat species at the collection sites were determined based on morphology and on previous data from bats’ roosting sites19. The nucleotide sequences of the positive samples were determined by Cosmogenetech Co. Ltd. Five of the 232 bat samples were confirmed positive to the bat paramyxoviruses by sequencing. Virus isolation was performed from the RT-semi-nested PCR-positive samples including feces, urine, and oropharyngeal swabs from bats. Each bat sample was diluted 10-fold with fresh Dulbecco Modified Eagle Medium (DMEM) and filtered with a 0.2-μm filter before being inoculated into the MARC 145 cell line, a clone from the MA-104 cell line from kidney epithelial cells of an African green monkey20; blind passaging was performed up to three times. The cytopathic effect (CPE) was confirmed in cells infected with one sample (B16-40) of five PCR-positive samples. Next, a monolayer of MARC 145 was infected with bat paramyxovirus (B16-40) at a multiplicity of infection (MOI) of 0.1. Two days post-infection, the infected cells were harvested and pelleted. The infected cell pellet was fixed with 2.5% glutaraldehyde in PBS (pH 7.4) for 2 hours and sent for transmission electron microscopy (TEM) to the Advanced Analysis Center in the Korean Institute of Science and Technology in Korea. Briefly, after washing with PBS the cells were post-fixed in 2% osmium tetroxide and dehydrated in an ethanol series ). Then, the sample was transited with 100% propylene oxide and embedded in Epoxy resin. The embedded sample was sectioned into 70–80 nm sections with an ultramicrotome , stained with 2% uranyl acetate and lead citrate on a copper grid, and examined using TEM mode on a CryoTecnai F20 .RNA extracted from bat samples was tested by RT-semi-nested PCR using consensus paramyxovirus primers targeting the RdRp region5/mL of MARC-145 cells were seeded to 25 cm2 tissue culture flasks in DMEM (5% FBS) and incubated for 24 hours to create a monolayer. The virus was then inoculated to the monolayer of MARC-145 cells at a MOI of 0.1 and maintained in DMEM (2.5% FBS) for 6 days. Every 24 hours (6 hours on day 0), culture supernatant was collected for viral titration. The remaining cell layer was washed three times with PBS (pH 7.4), and 1 mL of fresh DMEM was added to release the cell-associated viruses and subsequent freezing and thawing. The collected culture supernatant and freeze-thawed cell lysate were centrifuged at 3,000 × g for 10 minutes to remove cell pellets and then used for viral titration.To observe growth kinetics of the isolated bat paramyxovirus B16-40, 1 × 1050) method. Briefly, the prepared samples were 10-fold serially diluted and inoculated to monolayers of MARC-145 cells in 4 wells. The highest dilution showing 50% of viral infection was obtained by observing cytopathic effects with a light inverted microscope . The TCID50 was calculated using the Spearman-Karber method.The infectious viruses were quantified from the prepared samples using the tissue culture infectious dose 50 was filtered through a 0.2 μm filter and ultra-centrifuged at 250,000 × g for 1 hour. Each pellet was suspended in 500 µL of 1x digestion buffer  and treated with Turbo DNase; the suspension was incubated at 37 °C for 30 min. The RNA from the suspension was isolated using the Trizol LS reagent according to the manufacturer’s instructions (Ambion). The extracted RNA was submitted to Macrogen  for high throughput sequencing in a HiSeq. 2000 sequencing system based on the transcriptome 28. The obtained genomic RNA sequences and RT-PCR-positive amplicons were further analysed with related sequences in GenBank using MAFFT29 and MEGA version 630.For human parainfluenza virus 1 (KBPV-VR-44), the 1,992 bases of the HN gene were obtained by RT-nested PCR following the previously published method31. Putative HN protein structures were predicted using the Phyre2 website32 and visualized with the UCSF Chimera 1.11.2 program33.Genomic sequence data generated in this study have been deposited in GenBank under accession number MG230624. The full HN sequence of human parainfluenza virus 1 (KBPV-VR-44) has also been deposited in GenBank under accession number MG255129. The HN amino acid sequences of human parainfluenza virus 1 (KBPV-VR-44) and bat paramyxovirus were further compared with the BioEdit programTo investigate the activity of the HN protein, the haemadsorption assay was used. Monolayer cultures of MARC 145 cells in 96-well plates were inoculated with the bat paramyxovirus B16-40 at an MOI of 0.1. After 48 hours, the cells were gently washed with PBS and incubated for 1 hour with 0.5% chicken red blood cells (RBCs) in PBS (pH 7.4) at room temperature. Then, the cells were washed with PBS twice to remove unbound RBCs and observed under a microscope. In addition, the neuraminidase assay was performed with the viral isolate using the NA-Fluor Influenza Neuraminidase Assay Kit (Thermo) according to the manufacturer’s protocol. The virus was diluted 2-fold with the NA-Fluor 2× assay buffer in a black 96-well plate and incubated for 1 hour at 37 °C with the NA-Fluor substrate. After adding the stop solution, the plate was read at the excitation and emission wavelengths of 355 nm and 460 nm, respectively, using a PerkinElmer VICTOR2 fluorometer.5 TCID50 or 102.5 TCID50 of the bat paramyxovirus B16-40 via intra-gastric administration routes, and 30 µl of 105 TCID50 or 102.5 TCID50 of the bat paramyxovirus B16-40 via the intranasal administration routes. Each inoculation was conducted twice with 1 week in between. Weight loss was monitored daily for up to 2 weeks. The lungs, liver, brain, and intestine were harvested at 7 or 14 days post-challenge, fixed with 4% paraformaldehyde in PBS, pH = 7.2, and submitted to BioLead Inc., Korea, for histopathological examination. Additionally, RNA was extracted from each tissue and from oral swabs and fecal samples collected daily for 7 days post-challenge. The extracted RNA was tested using RT-semi-nested PCR with the consensus paramyxovirus primers19. In addition, real time PCR was performed with newly designed primers and probes targeting regions of the membrane and fusion protein. Briefly, the designed primers were as follows:A total of 44 6-week-old female C57BL/6 mice maintained under specific-pathogen-free (SPF) conditions were divided into two groups of negative controls (n = 4 each) and four inoculation groups (n = 9 each). The inoculation groups were inoculated with 400 µL of 10forward primer: PVM-F5′-CCCAGGAGTATGGTTATCAAGTGAGG-3′; reverse primer: PVM-R 5′-TCCATTGGGCTCTCTTTGTTTGC-3′; Taqman probe: PVM-P 5′-FAM-CCCATCCCAGACCAGCCACCAGACCC-TAMRA-3′Real time RT-PCR was performed as follows: Reverse transcription at 45 °C for 10 min, followed by PCR −95 °C for 5 min, cycling 40 times at 95 °C for 10 sec, and 60 °C for 30 sec. Using the 10-fold diluted virus (bat paramyxovirus B16-40), standard curves were performed for every reaction.Paramyxoviridae and the bat paramyxovirus B16-40. 6-week-old female BALB/cA mice maintained under SPF conditions were intramuscularly inoculated twice, with 2 weeks in between, with a 1:1 ratio mixture of AddaVax adjuvant and live virus stock , human mumps virus (KBPV-VR-51), and human parainfluenza virus 1 (KBPV-VR-44), respectively, from the Korea Bank for Pathogenic Viruses. Two weeks after the last immunization, antisera against the virus were obtained by cardiac puncture for further studies related to cross-reactivity in indirect immunofluorescence assay. Mouse antisera against the bat paramyxovirus B16-40 (105.75 TCID50/mL) and human parainfluenza virus 1  were further compared through serum titration using the indirect immunofluorescence assay and serum neutralization test.We next observed preliminary cross-reactivity in indirect immunofluorescence assay between several human viruses belonging to the family 50 of the bat paramyxovirus B16-40 and human parainfluenza virus 1 (KBPV-VR-44), respectively, or were mock-infected. Mock-infected cells were used as a negative control for non-specific binding of the mouse serum. The plates were incubated at 37 °C for 48 hours. Then the supernatant was discarded and cells were washed with PBS (pH 7.4). The washed cells were fixed with 100% ethanol for 1 hour at −20 °C and rehydrated with PBS (pH 7.4) for 10 min at room temperature. Primary antibodies used in the experiments were the mouse antisera against bat paramyxovirus B16-40 and human parainfluenza virus 1 (KBPV-VR-44). The sera were diluted with 50% culture supernatants from the cell lines in fresh DMEM; the corresponding mock-infected cells were treated in the same way as the target virus culture. Then, the sera were serially diluted 2-fold from an initial dilution of 1:10 and added to the fixed infected or mock-infected cells and incubated for 1 hour at room temperature. The plates were washed three times with PBS containing 0.05% Tween 20 . FITC-conjugated goat anti-mouse IgG secondary antibody  was diluted 1:2000 with the same serum diluent and was added to the plates and incubated for 1 hour at room temperature. After three washes with PBS-T, the plates were viewed with a fluorescence microscope .To evaluate the level of serological cross-reactivity, monolayers of MARC 145 and LLC-MK2 cells in 96-well plates were inoculated with 200 TCID50 of each virus. The plates were then incubated at 37 °C for 1 hour. The virus-serum mixtures for the bat paramyxovirus B16-40 and human parainfluenza virus 1 (KBPV-VR-44) were added to monolayer cultures of MARC 145 and LLC-MK2 cells, respectively, and incubated at 37 °C for 1 hour. The MARC 145 cells and LLC-MK2 cells were then washed once with PBS (pH 7.4) and maintained in DMEM (2.5% FBS) for 5 days. After incubation, the plates were examined for CPE.The mouse serum samples were inactivated at 56 °C for 30 min. They were then diluted 2-fold from an initial dilution of 1:10 in 96-well plates and mixed with 200 TCIDThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplement Information