We present a systematic theoretical analysis of how this location-dependence affects the sensor response. The analysis is done for beads magnetized by a homogeneous in-plane magnetic field. We determine the expected value and standard deviation of the sensor response for a given coverage, as well as the accuracy and precision with which the coverage can be determined from a single sensor measurement. We show that statistical fluctuations between samples may reduce the sensitivity and dynamic range of a sensor significantly when the functionalized area is larger than the sensor area. Hence, the statistics of sampling is essential to sensor design. For illustration, we analyze three important published cases for which statistical fluctuations are dominant, significant, and insignificant, respectively.Magnetic biosensors detect magnetic beads that, mediated by a target, have bound to a functionalized area. This area is often larger than the area of the sensor. Both the sign and magnitude of the average magnetic field experienced by the sensor from a magnetic bead depends on the location of the bead relative to the sensor. Consequently, the signal from multiple beads also depends on their locations. Thus, a given  In medicine and biology it is often of great interest to quantify the presence of biomolecules accurately. For example, in molecular oncology the ability to detect cancer biomarkers determines how early the disease can be discovered .μm wide stripe or an array of such stripes. Consequently, magnetic beads will be randomly distributed both on top of and outside of the sensor structure. However, the signal depends on the bead locations relative to the sensor. For randomly distributed beads, this can cause problems, as the signal from some beads may cancel the signal from others, depending on their relative location [bead coverage) defined as the projected area of the beads relative to the total area over which the beads are distributed. For a given bead coverage, the sensor signal changes from sample to sample because bead locations differ between samples. These fluctuations in the signal have not been considered in the literature on magnetic biosensors.Magnetic labels used with magnetoresistive sensors offer an alternative to fluorescence detection that may provide higher sensitivity, lower detection thresholds, and wash-free assay protocols –4. Magnelocation . The senHere, we derive the expected value and standard deviation of the signal from a single sensor at fixed coverage and with fluctuations due to statistical sample-to-sample variations in bead locations. The presented approach is generally applicable. The specific condition chosen in this study is magnetic field sensors showing a linear in-plane field response and where the signal is due to magnetic beads magnetized by a homogeneous in-plane magnetic field. These may include, for example, spin-valve giant magnetoresistance sensors –9, tunneR. Further, we assume that the beads are superparamagnetic with constant magnetic susceptibility χ and no magnetic remanence. Moreover, we assume all beads to be magnetized by a homogeneous external field, Hext, which is applied in the y-direction. This field creates a constant magnetic dipole moment, We assume the magnetic beads to be spherical with radius A = ℓw × w, where ℓw and w respectively denote the length and the width of the sensor, and ℓ is the sensor aspect ratio. We choose a coordinate system with origin at the center of the sensor  is proportional to the average A0. We will refer to A0 as the functionalized area and assume that beads are independently distributed, each bead with the same uniform probability density within this area. Unless otherwise specified, we assume that the functionalized area is quadratic and symmetrically placed with respect to the sensor stripe such that A0 = a × a. Because of the random distribution of beads, the sensor signal will vary between experiments even if the same number of beads is detected in each experiment. We refer to these random sample-to-sample variations as configurational fluctuations or statistical sampling fluctuations. In the best case scenario where electrical noise can be neglected, these still produce fluctuations of the sensor output and thereby limit the minimum coverage of magnetic beads that can be assessed reliably from measurements on a single sensor.The magnetic beads may attach via specific biological interactions to biomolecules within a functionalized surface area, w, and areas in units of w2. The resulting dimensionless variables are denoted by tilde, e.g., In the following analysis, it is convenient to express lengths in units of the sensor width, y-componentA single homogeneously magnetized bead with center located at The average over the sensor area, r0 relative to the sensor. This is illustrated for a square sensor in The average magnetic field from a single magnetic bead, sor area .Thus, depending on the sensor geometry and the functionalized area, both positive and negative signals are possible. When beads are located both inside and outside the sensor area, we have found that the sensor signal is often dominated by the signal from beads outside the sensor area as these are typically placed at a lower height . Here, wAssuming that a single magnetic bead is located with equal probability at any point within the functionalized area, it is convenient to define the configurational expected value of a function S1, of the normalized magnetic field and its variance, A0 areUsing this notation, the expected value, S1 and σ1 depend on S1 and σ1, with their dependence on A0 = a2 is assumed.We note that iid) and the single bead analysis applies to each of these beads. Later, we will revisit and discuss the validity of these simplifying assumptions. Then, the expected value and variance of the summed bead response from N beads areWhen multiple beads are present, the sensor signal is the sum of their individual contributions. We assume that steric interactions (excluded volume effects) between these beads are negligible. Further, we assume that magnetostatic interactions between the beads and between the beads and the sensor are negligible. Then the beads are independently and identically distributed (ϕ. It is the surface area covered by the beads (when projected onto the surface) divided by the total functionalized area,Often, the exact number of beads is not known, and it is more convenient to describe the response in terms of the bead coverage, ϕ has a limited range with maximum less than one, because a close-packed monolayer of spheres leaves surface area uncovered,The bead coverage can be estimated independently by, e.g., microscopy, scanning electron microscopy or fluorescence. We note that ϕ1 for ϕ, corresponding to a single bead being present in the functionalized area,There is also a lowest non-zero value ϕ1 also describes the smallest possible increment in ϕ, since the bead coverage is incremented by addition of whole beads.The value of ϕ are only approximative and would be subject to correction terms due to excluded volume effects in a more accurate model.The assumption of iid is justified when the bead coverage is low. A calculation analogous to that leading to Eq (6) in Ref.  gives thϕ asThe expected value and variance of the normalized magnetic field can be written in terms of σϕ > Sϕ, a single measurement of the bead coverage cannot be trusted. The signal-to-standard deviation ratio (SDR) is defined as the expected value of the signal measured in units of its standard deviation,If N or, equivalently, identical ϕ, under the assumption of negligible sensor noise. Since beads were assumed iid, SDR is proportional to N1/2 or, equivalently, ϕ1/2. Note again that S1 and σ1 depend on Absolute values are used because both positive and negative average values of the signal may occur. ϕstat denote the bead coverage corresponding to SDR = 1. Solving SDR = 1, we obtainFor convenience below, let ϕstat is the minimum bead coverage required to ensure a signal of the same magnitude as the statistical sampling fluctuations for a single sampling of the magnetic bead distribution.ϕnoise, that gives rise to a signal of the same magnitude as the sensor output voltage noise, Vnoise. Using the definition of the normalized variables, Eqs ϕnoise, ϕ1, ϕstat, and ϕnoise may all affect the lower limit of bead coverages that can be resolved. Making the practical simplifying assumption that one of the mechanisms dominates, we can define the resolution asNote, that DR) for the bead coverage readout isHence, the maximum possible dynamic range  signal, Sϕmax. From Eqs , we, weσϕ, The latter factor, The above expressions include the discrete nature of the magnetic beads, the position dependence of the signal from magnetic beads, the statistical sampling of a bead distribution, and the sensor noise in the evaluation of the sensitivity and dynamic range of the bead coverage on an area over the sensor. Thus, this provides a much more complete picture of the biosensing capability of a sensor system than considerations of only the sensor noise.In this section, we first present and discuss the effects of the size of the functionalized area and the bead size. Then we discuss the validity of the simplifying assumptions and finally we apply the developed theory to analyze three important cases found in the literature.a. Sϕ/ϕ, (b) SDR) vs. In this subsection, we study the impact of the size of the functionalized area relative to the sensor area. For simplicity, we consider a square sensor (ℓ = 1) and a square functionalized area of side length A. Moreover, the average bead field, S1 is approximately constant. This, combined with Sϕ is proportional to the area covered by magnetic beads, σ1 shows a quadratic dependence on σϕ is proportional to σϕ grows faster than Sϕ for increasing SDR-value is proportional to If the functionalized area is small compared to the sensor size 1avg(r0) , is wellS1 and σ1 are proportional to Sϕ is proportional to σϕ approaches a constant, i.e., it becomes independent of σϕ is constant, the SDR is proportional to Sϕ and thus the SDR decreases as Sϕ. However, it has a strong impact on σϕ, which increases significantly with If the functionalized area is larger than the sensor size . We assume that the sensor area is given a protective coating of thickness h, such that the height of a magnetic bead over the sensor is given by z0 = h + R. We will consider square bead functionalized areas with a = w  = 3 × 10−4. ϕ.In this section, we study the impact of the bead size on the sensor signal and on the range of values of ϕ = ϕmax, red lines) in ϕ = ϕmax is significantly steeper than that observed for a single magnetic bead (ϕ = ϕ1). For The normalized sensor signal for a close-packed monolayer  or to have the largest dynamic range. The lowest LOD is obtained when ϕ and hence the dynamic range for the biosensor. This is obtained for the value of ϕres obtained as the value of ϕ where ϕnoise = max{ϕ1, ϕstat}. For the parameters used in ϕnoise also depends on the sensor operation conditions, i.e., on the excitation field, Hext. Also note that for real sensors, there may be a height difference between the magnetic beads on top of the sensor area and outside the sensor area, which will modify the behavior compared to the results above [The considerations also show that for given sensor parameters, it is possible to identify an operating condition that maximizes the dynamic range in ts above . FinallyThe results presented above were based on a number of simplifying assumptions. Here, we discuss the validity of these assumptions and the anticipated consequences of relaxing them.We assumed that the beads have a constant magnetic susceptibility. Generally, the magnetic field sensors are operated at low magnetic fields (< 5 mT) to ensure that the sensors operate in their linear regime and to reduce constraints on the electromagnets producing the magnetic excitation field. Generally, magnetic beads and nanoparticles show very small deviations from linear behavior for such small magnetic fields , and thH. Following the analysis of [rbb is the center-to-center separation of two beads. Due to demagnetization effects, the upper limit of the magnetic susceptibility for a homogeneously magnetized sphere is χ = 3. Thus, we can reduce the criterion to rbb ≥ 2R, this criterion is seen to be fulfilled for all cases but the most dense packing of magnetic beads. When magnetic interactions are significant, the effective magnetic susceptibility of an ensemble of beads may be larger than that based on non-interacting beads and therefore a dense packing of beads will give a correction to the signal calculated assuming non-interacting beads. In that case our predictions are only estimates, lower bounds on the true results.In the analysis above, we neglected dipole interactions between magnetic beads. These interactions may modify the magnetic response of the beads as well as their distribution. Hence it is relevant to discuss the validity of this assumption in some detail. The magnetic dipole interaction between two magnetic beads can be considered negligible when the magnetic dipole field from a bead is much smaller than the external magnetizing field, lysis of , this asfluctuations in the signal due to the external magnetic field cannot be neglected. Due to the strong position-dependence, these are likely to dominate the statistical fluctuations of the total sensor signal.In the analysis above, we also neglected the influence of the magnetostatic field due to the magnetic layers of the sensor structure. The magnetic field close to the surface of a structure that is magnetized along the external magnetic field, will be parallel to the external field outside the structure and antiparallel to the external field on top of the structure. Consequently, the contribution to the sensor signal from beads magnetized by the magnetostatic field will generally not suffer from the cancellation effects observed for a homogeneous magnetic field . TherefoWe also assumed that the beads are independently and identically distributed. We have already discussed the validity and impact of this assumption in the theory section on the Sensor response to multiple beads. Finally, we assumed that the beads are uniformly distributed. This assumption is plausible and the simplest possible case. This simplicity simplified our presentation and derivations above. It is easily repeated for a more general assumption about the distribution of beads. A non-uniform distribution could result from the protocol of the assay. For example, a magnetic-field-accelerated incubation may enhance the probability of beads binding at certain locations. As long as the beads are independently distributed, our calculation of the statistical variance of the signal remains valid with little change. One just cannot characterize the bead distribution fully using the constant magnetic bead coverage that we introduced for a uniform distribution. However, the signal and its fluctuations are dominated by contributions from areas near the sensor edge at et al. [et al. [et al. [In this section, we use the theoretical framework from the theory to analyze the signal and dynamic range in terms of our simple presented model for three quite different GMR-based magnetic biosensors from the literature, presented by Graham et al. , Martins [et al. , and Gas [et al. . Detailset al. [μm2 sensor stripe to detect the DNA-mediated binding of 250 nm magnetic beads. They employ tapered current lines to attract and focus the magnetic beads onto an area on and near the sensor stripe. For simplicity, we approximate the functionalized area with a square centered with respect to the sensor stripe and with edge length a = 16 μm, corresponding to the length of the tapered part of the current line. The authors investigate experimentally (in singlet) the sensor response to a series of three subsequent exposures to a suspension of DNA-functionalized magnetic beads and correlate the sensor response to the number of magnetic beads on the sensor stripe observed in a microscope. They find a linear correlation between the number of magnetic beads and the sensor response and conclude that concentrations in the 10 fM range, corresponding to the binding of a single magnetic bead, may be within reach.Graham et al.  use a 6 et al. [μm2 and a U-branch length of 40 μm to detect the DNA-mediated binding of 250 nm magnetic beads. The sensor stripe is placed inside a U-shaped current line structure used to attract and focus the magnetic beads to the sensor stripe. The sensor stripe is covered by a patch of gold (43 × 13 μm2), which is selectively coated with capture DNA to define the functionalized area. The authors study the response vs. DNA concentration and conclude that, using field-assisted hybridization, they can detect concentrations from 1 fM to 10 pM corresponding to normalized signals ranging from 0.005 (1 fM) to 0.05 (saturation). Error bars of about 5% (1 fM) to 10% (saturation) of the saturation signal are reported in their dose-response curve. Values were obtained from measurements on at least five sensors.Martins et al.  is a latet al. [μm2 to detect the binding of 50 nm magnetic beads in a sandwich assay for protein detection. The functionalized area, defined by spotting of capture antibodies, is significantly larger than the extent of the meandering sensor. For simplicity, we therefore assume this area to be infinite. The authors study the response vs. antigen concentration and conclude that they can detect and discriminate between concentrations in the range from 1 fM to 5 nM, corresponding to rms signals ranging from 1 μV to 200 μV. Error bars corresponding to relative signal standard deviations of a few percent are reported. Values were obtained from measurements on at least four nominally identical sensors on the same sensor chip.Gaster et al.  use a meϕ1, ϕstat, log10(DR) and Sϕmax calculated from the geometrical parameters given in ϕnoise could not be assessed from the information presented in the case studies.ϕres is limited by the discrete counting of individual beads—i.e., by ϕ1—under the assumption that the sensor noise is negligible. We estimate values of the dynamic range to be 2.4 to 6 orders of magnitude. However, when the beads are distributed on a more realistic functionalized area (bottom rows), the results are dramatically different.For all three cases, we find for the idealized case (top rows) that et al. [Sϕmax and that ϕstat ≫ ϕmax. Hence, the analysis predicts that statistical sampling fluctuations prevent the assessment of the true average value of ϕ from a single sensor measurement. The assumption of iid magnetic beads may be an oversimplification, but the above overall conclusion that poor statistical sampling prevents general assessment of ϕ from the measurement on a single sensor chip remains valid even for a smaller functionalized area as the signal fluctuations are dominated from contributions near the sensor edges. Due to the large value of ϕstat, a realistic increase of the number of sensors will therefore not significantly improve the situation. A lowering of the relative importance of the statistical fluctuation could be achieved by increasing the sensor area and by limiting magnetic beads to be bound either inside or outside the sensor area.For Graham et al. , we findet al. [Sϕmax indicates that the signal is weakly dominated by contributions from magnetic beads outside the sensor area. Inserting ϕstat in For Martins et al. , a monolet al. [Sϕmax. Due to the large aspect ratio of the sensor and the small magnetic bead size, our calculations predict that the statistical sampling fluctuations are very small (ϕstat ≪ ϕmax) and hence that the sensor signal, assuming negligible noise, has a dynamic range of up to five orders of magnitude. Experimentally, the authors find quite small standard deviations in their dose-response curve, which covers 2.2 orders of magnitude in the signal. According to the considerations above, statistical sampling fluctuations are negligible on this scale, so signal variations are likely due to sensor noise and/or variations in experimental conditions between experiments.For Gaster et al. , the sigWe have described the statistics of the magnetoresistive sensor signal as function of the functionalized area for beads magnetized by a homogeneous in-plane magnetic field. We have presented a general theoretical framework to (1) estimate the relative importance of sample-to-sample fluctuations of the sensor signal from a finite number of beads, and (2) assess the ability to determine a reliable value of the bead coverage from a single sensor measurement.We have exemplified and discussed the signal from of a square sensor with a square functionalized area centered on the sensor area. We have studied the effect of the size of the functionalized area for a fixed size of magnetic beads. The signal level grows with the size of the functionalized area until it covers the sensor area, after which the signal decreases. The statistical sampling fluctuations of the signal increase with the size of the functionalized area  and remain constant when the functionalized area extends beyond the sensor area. We also investigated the effect of the size of the magnetic bead for two sizes of the functionalized area. The statistical sampling fluctuations are found to be unimportant when the magnetic beads are limited to the sensor area, and the bead size can be chosen to maximize the dynamic range of the sensor signal or to obtain maximum sensitivity to the binding of a single binding event. When the functionalized area is allowed to extend beyond the sensor area such that beads contribute both positively and negatively to the sensor signal, the picture changes dramatically and statistical sampling fluctuations are likely to play a key role for the sensor signal fluctuations and the resulting obtainable dynamic range. Further, we have discussed limitations imposed by the simplifying assumptions and indicated how the effect of, e.g., the magnetostatic field of the sensor can be considered for a more detailed analysis of specific sensor designs.et al. [et al. [et al. [Finally, we have used the theoretical framework to analyze three cases from the literature. For Graham et al. , we foun [et al. , we foun [et al. , we founThe results demonstrate how the presented theory can be used to take all relevant sensor parameters into account—including the sensor noise, the statistical sample-to-sample fluctuations and the discrete nature of the magnetic beads—to assess the applicability of a sensor design and operation. We note that the presented general theoretical framework can be modified to deal with other experimental geometries and field excitation schemes, for example, where magnetic beads are magnetized using the field from the sensor bias current  or on-chFuture work involves using the framework to optimize operating conditions (magnetic bead size and magnetic field) to maximize performance of existing designs and to compare to more elaborate experimental studies.S1 Tablew and a total length ℓw. R denotes the radius of the magnetic beads used in the studies. z0 = h + R and Dimensions of the GMR sensor and magnetic beads used in the indicated literature case studies. The sensors have a width (PDF)Click here for additional data file.


Culex sp. and Psorophora ferox) collected at Brejeira farm, Canaan dos Carajas, Para state in northern Brazil. Eight of the viruses were distantly related to Piura virus, hereafter designated as Brejeira virus; the other 4 were similar to Wallerfield virus.The complete genome was determined for 12 viruses isolated from 8 different pools of mosquitoes ( During the past decade, a growing number of novel insect-specific viruses have been detected in naturally infected mosquitoes . These iNegevirus. Negeviruses were isolated from mosquitoes and phlebotomine sand flies collected in North and South America, Africa, and Asia , State of Pará, Northern Brazil, and represent low-passage viruses in C6/36 cells. Viruses were provided by the Department of Arbovirology and Hemorrhagic Fevers, Pan American Health Organization, World Health Organization Collaborating Center for Arbovirus Research.Mosquito pools were separated by species (1–30 mosquitoes), kept in 2-mL Eppendorf tubes containing D-PBS and one 3-mm metal bead (Tungsten Carbide Beads), and macerated using the TissueLyser II (Qiagen) at 25 Hz for 1 min. Mosquito macerates were centrifuged at 3,000 rpm, and the supernatant was used for inoculation in the C6/36 cells, in an attempt to isolate the virus. The supernatant of infected C6/36 cells was harvested after 75% to 90% cytopathic effect production and used for RNA extraction.RNA was extracted using the QIAamp Viral RNA minikit. Full-length genomes were obtained using the Ion Torrent PGM (Life Technologies), as previously described . The seqAll genomes were assembled and compared to viral sequences in the NCBI database using BLASTx, and 12 Negevirus-like viruses were identified. Maximum pairwise nucleotide identities between four Wallerfield viruses (this study), showed 97.4% to 97.2% on the nucleotide level with a Wallerfield virus (NC023440) isolated in Trinidad and Tobago, suggesting the detection of four isolates of one novel strain from Brazil.The Brejeira virus (a new virus from the Amazon region) was mostly related to Piura virus (JQ675607), with maximum pairwise nucleotide identities between 68.0% and 67.2% on the nucleotide level, suggesting the detection of eight isolates of one novel Negevirus-like species identified in Brazil.Similar to other Negevirus-like sequences, four domains were predicted for the first open reading frame (ORF1), a methyltransferase domain, an rRNA methyltransferase domain, a helicase domain, and an RdRp domain. No conserved domains were identified for ORF2 and ORF3.Culex, Aedes, and Anopheles, as well as sand flies of the genus Lutzomyia)  .In this scenario, it is important to assess how these viruses interact with the environment and contribute or discourage the transmission of other arboviruses of interest to human and/or animal health.KM350504 through KM350507  and KM350508 through KM350515 (eight different Brejeira viruses).The complete sequences have been deposited in GenBank under the accession numbers


Gen. Virol.2012, 93, 2076–2097.Turnell, A.S.; Grand, R.J. DNA viruses and the cellular DNA-damage response. 4.Oncogene2005, 24, 5986–5995.Marriott, S.J.; Semmes, O.J. Impact of HTLV-I tax on cell cycle progression and the cellular DNA damage repair response. 5.ACS Chem. Biol.2006, 1, 217–226.Smith, J.A.; Daniel, R. Following the path of the virus: The exploitation of host DNA repair mechanisms by retroviruses. 6.J. Gen. Virol.2014, 95, 991–1004.Higgs, M.R.; Chouteau, P.; Lerat, H. 'Liver let die': Oxidative DNA damage and hepatotropic viruses. 9.Expert Rev. Mol. Med.2006, 8, 1–11.Sinclair, A.; Yarranton, S.; Schelcher, C. DNA-damage response pathways triggered by viral replication. 12.Int. J. Cancer2006, 118, 3030–3044.Parkin, D.M. The global health burden of infection-associated cancers in the year 2002. 33.Virology2007, 368, 219–226.Davy, C.; Doorbar, J. G2/m cell cycle arrest in the life cycle of viruses. 119.Cancer Res.2002, 62, 7075–7082.Duensing, S.; Munger, K. The human papillomavirus type 16 e6 and e7 oncoproteins independently induce numerical and structural chromosome instability. 135.J. Virol.2010, 84, 8007–8020.Boichuk, S.; Hu, L.; Hein, J.; Gjoerup, O.V. Multiple DNA damage signaling and repair pathways deregulated by simian virus 40 large T antigen. 138.et al. Spectrum of epstein-barr virus-related diseases: A pictorial review. Jpn. J. Radiol.2009, 27, 4–19.Maeda, E.; Akahane, M.; Kiryu, S.; Kato, N.; Yoshikawa, T.; Hayashi, N.; Aoki, S.; Minami, M.; Uozaki, H.; Fukayama, M.; 163.Science1999, 284, 641–644.Ballestas, M.E.; Chatis, P.A.; Kaye, K.M. Efficient persistence of extrachromosomal kshv DNA mediated by latency-associated nuclear antigen. 207.Hepatology1989, 9, 322–327.Miller, R.H.; Kaneko, S.; Chung, C.T.; Girones, R.; Purcell, R.H. Compact organization of the hepatitis b virus genome. 208.Nature1980, 286, 535–538.Edman, J.C.; Gray, P.; Valenzuela, P.; Rall, L.B.; Rutter, W.J. Integration of hepatitis b virus sequences and their expression in a human hepatoma cell. 209.Pathol. Biol.2010, 58, 267–272.Wei, Y.; Neuveut, C.; Tiollais, P.; Buendia, M.A. Molecular biology of the hepatitis B virus and role of the x gene. 214.Mutat. Res.2004, 554, 305–318.Mathonnet, G.; Lachance, S.; Alaoui-Jamali, M.; Drobetsky, E.A. Expression of hepatitis b virus x oncoprotein inhibits transcription-coupled nucleotide excision repair in human cells. 215.Cell Cycle2011, 10, 3554–3565.Knoll, S.; Furst, K.; Thomas, S.; Villanueva Baselga, S.; Stoll, A.; Schaefer, S.; Putzer, B.M. Dissection of cell context-dependent interactions between hbx and p53 family members in regulation of apoptosis: A role for hbv-induced hcc. 216.BMC Infect. Dis.2005, 5, e95.Bolukbas, C.; Bolukbas, F.F.; Horoz, M.; Aslan, M.; Celik, H.; Erel, O. Increased oxidative stress associated with the severity of the liver disease in various forms of hepatitis b virus infection. 219.Cancer Sci.2006, 97, 683–688.Wang, H.C.; Huang, W.; Lai, M.D.; Su, I.J. Hepatitis b virus pre-s mutants, endoplasmic reticulum stress and hepatocarcinogenesis. 220.Hepatogastroenterology2008, 55, 2028–2032.Gwak, G.Y.; Lee, D.H.; Moon, T.G.; Choi, M.S.; Lee, J.H.; Koh, K.C.; Paik, S.W.; Park, C.K.; Joh, J.W.; Yoo, B.C. The correlation of hepatitis b virus pre-s mutation with cellular oxidative DNA damage in hepatocellular carcinoma. 223.Virology2012, 426, 73–82.Hodgson, A.J.; Hyser, J.M.; Keasler, V.V.; Cang, Y.; Slagle, B.L. Hepatitis b virus regulatory hbx protein binding to ddb1 is required but is not sufficient for maximal hbv replication. 224.Int. J. Biochem. Cell Biol.2011, 43, 1664–1667.Iovine, B.; Iannella, M.L.; Bevilacqua, M.A. Damage-specific DNA binding protein 1 (ddb1): A protein with a wide range of functions. 237.et al. Genome-wide survey of recurrent hbv integration in hepatocellular carcinoma. Nat. Genet.2012, 44, 765–769.Sung, W.K.; Zheng, H.; Li, S.; Chen, R.; Liu, X.; Li, Y.; Lee, N.P.; Lee, W.H.; Ariyaratne, P.N.; Tennakoon, C.; 238.Lancet Infect. Dis.2005, 5, 558–567.Shepard, C.W.; Finelli, L.; Alter, M.J. Global epidemiology of hepatitis c virus infection. 239.J. Hepatol.1992, 15, 382–386.Zignego, A.L.; Macchia, D.; Monti, M.; Thiers, V.; Mazzetti, M.; Foschi, M.; Maggi, E.; Romagnani, S.; Gentilini, P.; Brechot, C. Infection of peripheral mononuclear blood cells by hepatitis c virus. 240.J. Virol.2002, 76, 5974–5984.Egger, D.; Wolk, B.; Gosert, R.; Bianchi, L.; Blum, H.E.; Moradpour, D.; Bienz, K. Expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex. 241.J. Virol.2003, 77, 5487–5492.Gosert, R.; Egger, D.; Lohmann, V.; Bartenschlager, R.; Blum, H.E.; Bienz, K.; Moradpour, D. Identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons. 257.J. Virol.2013, 87, 5523–5539.Maqbool, M.A.; Imache, M.R.; Higgs, M.R.; Carmouse, S.; Pawlotsky, J.M.; Lerat, H. Regulation of hepatitis c virus replication by nuclear translocation of nonstructural 5a protein and transcriptional activation of host genes. We have noted a number of errors in the references of this manuscript. Please find below the list of corrected references:


Preterm birth is a major risk factor for morbidity and mortality among infants worldwide, and imposes considerable burden on health, education and social services, as well as on families and caregivers. Morbidity and mortality resulting from preterm birth is highest among early  and moderate (28–32 weeks) preterm infants, relative to late preterm infants (33–36 weeks). However, substantial societal burden is associated with late prematurity due to the larger number of late preterm infants relative to early and moderate preterm infants.The aim in this study was to characterize the burden of premature birth in Canada for early, moderate, and late premature infants, including resource utilization, direct medical costs, parental out-of-pocket costs, education costs, and mortality, using a validated and published decision model from the UK, and adapting it to a Canadian setting based on analysis of administrative, population-based data from Québec.Two-year survival was estimated at 56.0% for early preterm infants, 92.8% for moderate preterm infants, and 98.4% for late preterm infants. Per infant resource utilization consistently decreased with age. For moderately preterm infants, hospital days ranged from 1.6 at age two to 0.09 at age ten. Cost per infant over the first ten years of life was estimated to be $67,467 for early preterm infants, $52,796 for moderate preterm infants, and $10,010 for late preterm infants. Based on population sizes this corresponds to total national costs of $123.3 million for early preterm infants, $255.6 million for moderate preterm infants, $208.2 million for late preterm infants, and $587.1 million for all infants.Premature birth results in significant infant morbidity, mortality, healthcare utilization and costs in Canada. A comprehensive decision-model based on analysis of a Canadian population-based administrative data source suggested that the greatest national-level burden is associated with moderate preterm infants due to both a large cost per infant and population size while the highest individual-level burden is in early preterm infants and the largest total population size is in late preterm infants. Although the highest medical costs are incurred during the neonatal period, greater resource utilization and costs extend into childhood. Preterm birth, defined as birth before the completion of 37 weeks gestation, is a majMorbidity and mortality resulting from preterm birth is highest among early  and moderate  preterm infants. The morThe primary objective of this study was to characterize the burden of prematurity in Canada over the first ten years of life—as characterized by healthcare resource utilization, direct medical costs, indirect costs associated with lost productivity, and mortality—to describe trends in utilization patterns from infancy and into childhood, and across gestational-age categories. These costs are characterized both as cost per individual preterm infant, and scaled to the Canadian population level by extrapolating individual costs to the number of preterm infants born each year in Canada, and the corresponding gestational age distribution.Data from longitudinal, administrative population-based databases from Québec, Canada were used to meet this objective. The methodology presents a Canadian adaptation of a previously developed burden of illness model from the United Kingdom (UK) estimating the long-term costs of preterm birth throughout childhood in England and Wales, based on® Excel. Data from the RAMQ was stored in a SQL database (Microsoft® SQL version 10.50.1600.1), and analysis was conducted using R 2.13.1.Resource utilization parameters were populated using Régie de l'assurance maladie du Québec (RAMQ) physician billing data from Québec, Canada linked to MED-ÉCHO hospital discharge abstract databases. A retrospective population-based design was used to establish and follow a birth cohort of all premature infants born during 1996–1997 until age ten. The RAMQ insures all provincial health plan registrants in Québec  for necessary medical and hospital services and their databases include: 1) claims[Epidemiological and resource utilization parameters were stratified by gestational age category, and overall results are a weighted average of gestational age-specific results and cost parameters, based on the relative distributions of early, moderate, and late preterm infants. Following live birth, infants who did not die in the delivery room went on to either admission to a neonatal care facility or discharge directly home. Following hospital discharge, a single model state was used to describe time until age two to account for increased medical costs incurred during early childhood. Following age two, costs were accrued annually until age ten. Level of disability was incorporated in a sensitivity analysis to characterize costs associated with special education requirements. Disabilities included motor function , vision and hearing impairment, and cognitive abilities, consistent with the definitions used within the VICSG cohort. ChildreThe probability of live discharge from the neonatal intensive care unit, by gestational age, were taken from a study by the Canadian NICU Network during 1996–1997. AdditioCosts were discounted at 5% annually.Several additional costs associated with preterm birth were considered in exploratory sensitivity analysis. The primary analysis did not incorporate these elements because empirical data were not available to calculate the relevant parameters, which were instead estimated based on assumption and expert opinion.Excess prenatal costs were included in addition to costs associated with the infant following birth. These costs were defined as those associated with additional resource utilization incurred by women identified as high-risk for preterm labor and were based on published sources and expert opinion  was undertaken to assess the impact of uncertainty in model input parameters on potential variability of overall total cost results. The epidemiological parameters that were assumed to be consistent with those reported previously were assumed to follow the described distributions. For de The distributions of survival and disability at ages two and ten, respectively, stratified by gestational age at birth, are shown in Figure Figure The total economic burden of prematurity by category of expenditure  is reported in Table At the individual level, for all gestational age categories, the largest contributor to total costs was the cost associated with the neonatal intensive care unit stay, followed by medical costs incurred between discharge and age two. Across categories of expenditure, individual-level costs tended to be highest for early preterm infants prior to age two, followed by moderate preterm infants and late preterm infants. Prior to age two, costs were similar between moderate and preterm infants, and substantially lower for late preterm infants. The most substantial cost differences were in neonatal hospitalization which ranged from $3,768 in late preterm infants to $53,308 in early preterm infants. After age two, costs were comparable across all age categories, although this implies that costs incurred by surviving children were highest for early and moderate preterms as the denominator was all live births, and there was notably higher mortality following live births for the earlier gestational age categories.In this study, a decision model was used to capture trends in survival, resource utilization, and indirect costs over the first ten years of life for preterm infants in Canada. A rigorous and comprehensive decision model, originally developed for the UK, was adapted to the Canadian setting by updating unit costs to Canadian values, and quantifying resource utilization by age and gestational age at birth category using a population-based real-world administrative data source. The results of this study allow for potential interventions to delay or prevent preterm birth, or to prevent morbidity in preterm infants to be contextualized with respect to the overall burden.A recent study published by Landry et al. reported resource utilization for infants born in Québec from 1983–1992, although this was restricted to infants with respiratory complications, and utilization and costs were not further stratified by gestational age at birth. The stuConsistent with the medical literature, a dramatic improvement in survival in moderate and late preterm infants relative to early preterm infants was observed. Not surprisingly, neonatal intensive care costs were the largest contributor to overall medical costs amongst all preterm infants. Neonatal costs associated with moderate preterm infants were found to be similar to early preterm infants. When considered in exploratory sensitivity analysis, education costs were an important cost driver, and were highest in late preterm infants, due to the larger number of survivors and the larger proportion attending a mainstream primary school, relative to earlier preterm categories with higher prevalence of severe disability, associated with not attending a mainstream school (Additional fileThe general trend in overall costs was that early and moderate preterm infants tended to incur similar costs, with much lower costs observed in late preterm infants. Based on model structure, there are two main determinants of costs incurred: the proportion surviving and the level of resource utilization incurred by survivors. Amongst surviving infants, early preterm infants tended to have the greatest medical resource utilization amongst all categories of utilization and at all ages Figure . HoweverA key strength of this study is the high quality and comprehensive nature of the data used to populate model parameters. The RAMQ data describe population-based resource utilization for all preterm infants born in the province of Québec during 1996 and 1997 over their first ten years of life. These resource utilization data were combined with a published model describing the epidemiology, survival, and disability trajectories of preterm infants, and unit costs for health resources were updated to 2012 values using inflation factors. Thus, the Canadian adaptation of the model provides an up to date and comprehensive estimate of the overall economic burden of prematurity in Canada. In addition, these results are potentially generalizable beyond Canada to countries with similar trends in pediatric treatment patterns and relative costs of health resources.Limitations to the approach include the fact that prescription medication costs were excluded from the analysis as the RAMQ data only include prescription records for a subset of individuals with medication coverage. The assumption was made that for preterm infants during childhood, the costs associated with medications would be substantially less important than those associated with hospitalizations and outpatient visits. In addition, in order to maximize length of available follow-up, the analysis was based on a cohort of infants born during 1996–1997, and, as such, their patterns of care may not reflect current treatment practices. Results were scaled to the population of Canada based on the assumption that clinical outcomes and resource utilization in Québec, and unit costs for medical resources from Ontario would be generalizable to the rest of Canada. While health care in Canada is delivered at the provincial level, it was assumed that individual provinces would not vary substantially with respect to pediatric clinical outcomes, resource utilization, and unit costs, such that the observed values for Québec and Ontario could serve as a suitable approximation for other provinces. Ontario, the largest Canadian province was selected as the most applicable province for selecting unit costs. While model parameters associated with survival and long-term disability trajectory were taken from UK and Australian sources where not available for Canada, where values were available for multiple sources (e.g. probabilities of live discharge from hospital for gestational age 23–35 weeks were available for both Canada and the The strength of an economic model is dictated by the strength of evidence used as model inputs. The primary, core analysis was based on inputs for which empirical data were available, including the incidence of premature birth and gestational age distribution in Canada, and survival and resource utilization and costs associated with preterm infants in Québec. While the highest quality evidence available was used in the primary analysis and sensitivity analyses, where empirical evidence was lacking, expert opinion evidence was used as sensitivity analysis, for prenatal resource utilization, special education associated with disability, and construction of neonatal facilities. For the inclusion of excess healthcare utilization for prenatal care, it was assumed that 50% of preterm births would have been associated with such care, due to a paucity of published estimates. In adapting the model to a Canadian setting, it was assumed that the values assumed within the UK model describing distribution of disability, and requirements for special needs education would be relevant for Canada.Future extensions of this work include the assessment of temporal trends in care to project expected updates to utilization estimates, and to compare the costs of preterm infants to those incurred by full-term infants in order to estimate an incremental cost of prematurity in addition to the absolute costs presented here. In addition, it would be of interest to expand the burden of illness model to compare differences in economic burden with respect to specific medical conditions relative to prematurity and pediatric populations, such as respiratory morbidity. Finally, the incorporation of quality-of-life estimation and empirical estimation of out-of-pocket expenses and lost productivity costs in Canadian families in addition to survival, resource utilization, and economic outcomes could provide a more inclusive view of the burden of prematurity throughout childhood, and would allow for a more comprehensive comparison of the overall burden experienced during childhood by preterm infants born at varying gestational ages. The model described here allows for numerous “what if” scenarios to be considered in future consideration of additional research questions.Premature birth results in significant infant morbidity, mortality, healthcare utilization and costs in Canada. The results of this study allow for potential interventions to delay or prevent preterm birth, or to prevent morbidity in preterm infants to be contextualized with respect to the overall burden. A comprehensive decision-model based on analysis of a Canadian population-based Canadian administrative data source suggested that substantial costs per infant are observed in early and moderate preterm infants, but when scaled to the national level, late preterm infants contribute a substantial burden due to the relatively larger population size. Although the highest medical costs are incurred during the neonatal period, higher resource utilization and costs extend into childhood.P. Vo is an AbbVie Inc employee and may hold stock or options in AbbVie Inc.K. Gooch is an AbbVie Inc employee and may hold stock or options in AbbVie Inc.This study was funded by AbbVie, North Chicago, IL.KG developed study objectives and reviewed and provided significant feedback on the manuscript. KMJ designed and built the model, and wrote the first draft of the manuscript. EK performed statistical analysis of RAMQ data. EO contributed to literature review, and manuscript writing. AL and PV provided significant feedback on the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/14/93/prepubSupplementary data input tables.Click here for file


O. aries-tc/ZAF/11/OBP-115) isolated from experimentally vaccinated cattle. Full-genome sequencing and phylogenetic analyses show that the bluetongue virus 9 strain 9/B. taurus-tc/ZAF/15/Onderstepoort_B02b is a reassortant virus containing segments from both BTV-9 and BTV-8.This is a report of the complete genome sequences of plaque-selected isolates of each of the five virus strains included in a South African commercial trivalent bluetongue virus (BTV) attenuated live virus vaccine, a BTV-4 field strain isolated from Rustenburg, South Africa, in 2011, and a bluetongue reassortant (bluetongue virus 4 strain 4/ Culicoides biting midges (Diptera: Ceratopogonidae) . The genome of BTV is composed of 10 segments of double-stranded RNA (dsRNA) that collectively encode seven structural proteins and five nonstructural proteins . Sequencing templates were prepared using full-length amplification of cDNAs  is a noncontagious disease of ruminants that is transmitted by gonidae)   are available on GenBank as accession numbers KT317665 to KT317674. The full-genome sequences of the bluetongue virus 9 strain 9/B. taurus-tc/ZAF/14/Onderstepoort_B02b that is a reassortant between BTV-9 and BTV-8 has been deposited in GenBank as accession numbers KT885075 to KT885084.The full-genome sequences of bluetongue virus 3 strain 3/Labstr/ZAF/14/OBP-115, bluetongue virus 8 strain 8/Labstr/ZAF/14/OBP-115, bluetongue virus 9 strain9/Labstr/ZAF/14/OBP-115, bluetongue virus 10 strain10/Labstr/ZAF/14/OBP-115, and bluetongue virus 11 strain11/Labstr/ZAF/14/OBP-115 have been deposited in GenBank under the accession numbers


Staphylococcus aureus. Hybrid coatings containing silver, copper and zinc cations were prepared through radical polymerization via sol-gel method and applied on glass slides or into the wells of polymethylmethacrylate plates. A 10 μl droplet of several viruses such as human immunodeficiency virus type 1 (HIV-1), influenza, dengue virus, herpes simplex virus, and coxsackievirus was added to coated and uncoated slides or plates, incubated usually from 5 to 240 min and followed by titer determination of recovered virus.Healthcare-acquired infections by pathogenic microorganisms including viruses represent significant health concern worldwide. Next to direct transmission from person-to-person also indirect transmission from contaminated surfaces is well documented and important route of infections. Here, we tested antiviral properties of hybrid coating containing silver, copper and zinc cations that was previously shown to be effective against pathogenic bacteria including methicillin-resistant 10TCID50, n = 3) already after 20 min of exposure to coatings, than on coated polymethylmethacrylate plates with 75–100 %  and 98–100 %  HIV-1 titer reduction after 20 and 120 min of exposure, respectively. Slower virucidal kinetics was observed with other enveloped viruses, where 240 min exposure to coated slides lead to 97 % (dengue), 100 % (herpes simplex) and 77 % (influenza) reduction in virus titers. Interestingly, only marginal reduction in viral titer after 240 min of exposure was noticed for non-enveloped coxsackie B3 virus.Scanning electron microscopy analysis showed better adhesion of coatings on glass surfaces, which resulted in 99.5–100 % HIV-1 titer reduction (3.1 ± 0.8 logOur hybrid coatings showed virucidal activity against HIV and other enveloped viruses thus providing further findings towards development of broad-spectrum antimicrobial coating suitable for surfaces in healthcare settings. Healthcare–associated infections (HAIs) by pathogenic bacteria, viruses and other microorganisms constitute significant cause of morbidity and mortality worldwide. Although exact statistical data are lacking, 1.7 million HAIs were estimated to occur in 2002 in U.S. hospitals alone –7, microHybrid materials are very conveniently prepared by sol-gel method, which can be briefly described as a special process of glass and ceramics manufacture at room or slightly elevated temperature. This technique became an independent and a very comprehensive discipline, documented by numerous books –15 and rFrom metal nanomaterials with biocidal properties, silver and copper received the most attention. Next to well documented bactericidal activities both silver and copper showed potent virucidal properties . SeveralVirucidal activity of copper in the form of copper oxide was evaluated in variety of materials such as fibers, latex, filter matrices and other polymeric materials , 34. Cop2 and has been shown to effectively inactivate influenza A virus  versus time.Coated or uncoated (in control experiment) 18 × 18 mm cover glasses were placed into 6-well plate and 10 μl droplet of 1.66 × 105 TCID50/ml of HIV-1 was placed in the center of wells (coated with photopolymerization and uncoated) in 96-well plate and covered with lid. At 2, 5, 10, 20, 30, 60, and 120 min 90 μl of 1X PBS was added to the well, mixed three times by pipetting up and down and removed from the well. All experiments in 96-well plates were performed in triplicate. Recovered virus was immediately titrated according the protocols mentioned below and virus titer reduction was expressed in percentage.Virus droplet (10 μl) of 1.66 × 10The determination of all virus titers were performed in triplicate from twofold serial virus dilutions and calculated according the method by Reed and Muench . Methods2, the supernatant was removed, cells washed with 1X PBS, fixed with 1 % glutaraldehyde and incubated with X-galactosidase staining solution. After 2 h of incubation infected wells that developed blue loci were counted as positive. Alternatively, after 48 h of incubation the firefly luciferase luminescent assay was performed and luminescence was measured in Victor X3 plate reader . Wells with relative light units above background (mean of luminescence of negative wells plus two standard deviations) were considered positive.TZM-bl indicator cell line was used to quantitate HIV titers . Twofold2, cells were fixed with 4 % paraformaldehyde and permeabilized with 0.2 % Triton X-100. Then, cells were washed with 1X PBS and incubated overnight at 4 °C with dengue virus type 2 serotype-specific mouse monoclonal antibody, which was harvested from HB-46 cells. Wells were washed three times with 1X PBS, incubated 90 min with Cy3-labeled donkey anti mouse IgG (Jackson Immunoresearch Europe) and documented using fluorescence microscope Olympus IX-81 with camera . ImageJ software (NIH) was used for image analysis and evaluation of positive and negative wells.Immunofluorescence staining was used to visualize infected cells . Briefly2, the avicel solution was removed from the wells, the cells were washed with 1X PBS, fixed with 4 % paraformaldehyde, permeabilized with 20 mM glycine/0.5 % Triton X-100 for 20 min and incubated for 1 h with 1:2500 dilution of monoclonal influenza A antibody against nucleocapsid  in 10 % normal horse serum and 0.05 % Tween 80 (both Sigma-Aldrich). Mouse secondary antibody (1:5000 dilution) in 10 % normal horse serum and 0.05 % Tween 80 was applied for 1 h and followed by 30 min of incubation in TrueBlue  until blue plaques developed in infected wells.Immuno-stained plaque assay using low-viscosity overlay medium developed by Matrosovich et al.  was adap2 the same procedure was followed as with influenza titer determination with following changes in the virus specific antibody dilutions. HSV-1 infected cells were incubated with 1:800 dilution of anti-HSV type I and II monoclonal antibody and coxsackievirus-infected cells were incubated with 1:1000 dilution of anti-coxsackievirus B3 antibody .Twofold serial dilutions of herpes simplex and coxsackie B3 virus were added to 20,000 Vero cells and 30,000 HeLa cells, respectively. Both cell lines were plated day before in DMEM with 10 % FBS and following day the medium was replaced with serum-reduced DMEM with 2 % FBS. After 1 h of incubation, the cells and virus mixture was overlaid with 1:1 mixture of 2.4 % Avicel RC/CL and DMEM with 2 % FBS. After three days of incubation in 37 °C in 5 % COSix uncoated and twelve coated glass covers  were incubated with 500 μl of 1X PBS for 15 sec and for 4 h. After incubation, 50 μl of liquid were transferred in triplicate on HeLa and Vero cells, incubated for 72 h and cytotoxic effect was analyzed by XTT colorimetric assay. Briefly, 50 μl of 50:1 mixture of XTT labeling reagent (1 mg/ml) and PMS electron-coupling reagent (0.383 mg/ml) was added to the wells and incubated for 4 h in 37 °C in 5 % CO2. Formation of orange formazan dye was measured in Victor X3 plate reader and wells with absorbance lower than cutoff that was calculated as mean of absorbance of wells without virus plus two standard deviations were considered infected.p-value less than 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism v.6.05 .Data were analyzed using a one-way analysis of variance test and followed by the Bonferroni’s multiple comparison test. All differences with a -1 for type 1, and 1655 – 1750 cm-1 for type 2. The infrared absorption of bimodal frequency distribution occurred due to the conjugation effect between -C = O and nearby -C = C- and hydrogen bonds. The characteristic stretching vibration peak of -C = C- occurred at 1633 cm−1 and the characteristic stretching vibration peak of BPO initiator occured at 1787 - 1767 cm-1. In Fig. 2 in the process of TEOS and TMSPM co-hydrolysis.Figure The surfaces of hybrid coatings were analyzed using SEM on two types of substrates; PMMA plates Fig.  and glasWe observed a number of clearly visible pores in size of few nm on the coated PMMA sample Fig. , in contThe cytotoxicity of washings from hybrid coatings with silver, copper and zinc prepared by heat and photopolymerization were evaluated in Vero and HeLa cell lines at two time points Fig.  Immediat50/ml and 2987 ± 1721 TCID50/ml, respectively. Moreover, recovered virus titer approximately matched initial input of HIV-1 (3320 TCID50/ml) after similar dilution in 0.49 ml of PBS without exposure to the uncoated glass coverslip. Decrease in HIV-1 titer was already noticeable after 5 min exposure to hybrid coatings. The decrease was smaller in the case of glass coverslips with hybrid coatings prepared by heat polymerization with 79 % HIV-1 titer reduction on average, than in the case of glass coverslips with hybrid coatings prepared by photopolymerization where the reduction was 98 %. Similarly, 20 min exposure of HIV-1 on glass coverslips with hybrid coatings prepared by photopolymerization exhibited better virucidal activity that resulted in 100 % inactivation of HIV-1 on two out of three coverslips than coverslips coated by heat polymerization where 10 % of residual HIV-1 activity remained.As a first step in evaluating the virucidal activity of hybrid coatings we coated six glass coverslips by heat polymerization, six by photopolymerization and six were left uncoated. We applied 10 μl of HIV-1 on the coverslip and incubated three coverslips for 5 min and three coverslips for 20 min. The Table Next we examined the anti-HIV-1 activity of hybrid coating when applied on poly(methyl methacrylate) surface. Figure 10TCID50 reduction after 30 min exposure, but followed with only additional 1.1 log10TCID50 decline in 4 h. Different course of exposure experiment to hybrid coatings was observed in the case of herpes simplex virus. Similarly 1.0 log10TCID50 decrease of HSV-1 titer was achieved in first 30 min, but complete inactivation was accomplished after 4 h. Only small decrease (0.7 log10TCID50) of influenza virus titer when compared with untreated control was achieved after 4 h of exposure to hybrid coating. Even smaller virucidal effect of hybrid coatings than against influenza virus was observed against coxsackie B3 virus, where titers of recovered virus from coated coverslips were on average only 0.2 log10TCID50 lower than titers of recovered virus from uncoated coverslips.To assess the virucidal potential of hybrid coatings against other enveloped, nonenveloped, RNA and DNA viruses we selected dengue virus, coxsackievirus, influenza virus and herpes simplex virus. Droplet of 10 μl of each virus was applied on glass coverslips, incubated up to 4 h, titer of recovered virus was determined as specified in Methods section depending on the virus, and plotted in the same graph as titer of recovered virus from untreated glass coverslips Fig. . Very quStaphylococcus aureus and its methicillin-resistant variant [Development of broad-spectrum antimicrobial surface coating would represent significant help in the battle against hospital-acquired infections. We recently reported new type of hybrid coating containing silver and copper with strong antibacterial effect against variety of commonly occurring bacteria in hospital including  variant . To findOur general observation is that the properties of prepared hybrid coatings depend more on the sol synthesis and the subsequent curing method and less on the types of substrate. This fact was further confirmed by the examination of chemical and mechanical resistance on different types of substrate , 49. TheI+, CuII+ and ZnII+ ions are more toxic than their corresponding nanoparticles Ag, CuO, ZnO [I+ and CuII+ amount occurred only after first washing cycle and after fifty washing cycles there still remained 40 % of AgI+ and 20 % of CuII+ ions in the coating [Thorough toxicological analysis of potential adverse effects is essential for successful implementation of hybrid coating in health care settings. In general, AgCuO, ZnO . We obse coating . CertainI+, CuII+ and ZnII+ ions supports hypothesis that these ions have direct inhibition effect on HIV-1 virions. It is generally accepted that silver and copper ions can exert their biocidal power by directly lysing membranes or by binding to thiol groups of proteins [II+ ion can inactivate 3 thiol groups [The antiviral activities of hybrid coating were initially tested against HIV-1 on glass coverslips prepared by the same sol-gel method, which differed only in polymerization process that was either heat- or photoinitiated. Coatings prepared by either curing method showed strong anti-HIV-1 activity after 5 min and further intensified after 20 min. The bigger decrease in residual HIV-1 activity after 20 min than after 5 min of exposure of HIV-1 to hybrid coating containing Agproteins , 51, 52.proteins , 54 and proteins –58 effecproteins –61, respproteins , vacciniproteins , HSV [64proteins  but exacproteins  showed tproteins  and inhiproteins . Strong proteins . They shl groups .Next we coated PMMA 96-well plates and confirmed quick and increasing anti-HIV-1 effect during prolonged incubation, albeit we achieved on average only 99 % reduction in HIV-1 titer after 2 h of exposure to hybrid coatings. Better results with glass coverslip experiments can be explained by better adhesion of coatings to glass surfaces as documented by SEM analysis and primarily by the fact that HIV-1 droplet on glass coverslip was covered with another coverslip and thus was more tightly distributed on larger coated surface.10TCID50 [Finally, to determine the hybrid coating ability to inactivate other viruses, we tested four additional viruses representing double-stranded DNA virus, negative and positive sense single-stranded RNA virus and non-enveloped virus. Interestingly, we observed different time course of virus titer reduction for these viruses. Positive single-stranded RNA enveloped dengue virus type 2 had initially quick titer reduction in first 30 min, but it was not followed by complete inactivation even after 4 h of exposure. There are limited data in the literature about anti-dengue activity of coatings with or without metal ions and nanoparticles. Dengue virus type 2 infection was not suppressed by nanoscale silica platelets hybridized with silver nanoparticles but introduction of anionic sodium dodecyl sulfate efficiently reduced titer almost about three log10TCID50 . Recentl10TCID50 . Binding10TCID50 , but the10TCID50 . Quick d10TCID50 reduction in titer in the first half hour was followed by complete inactivation after 4 h. HSV entry is complex event involving five glycoproteins and three alternative cellular receptors with heparan sulfate being the most important but not essential [50 = 25 μg/mL [In case of double-stranded DNA HSV-1 we have seen different course of virus titer reduction than with dengue virus type 2. Initial one logssential . Baram-Pssential . Moreove25 μg/mL . Based o25 μg/mL , 34, 73.10TCID50 reduction after 4 h of exposure. Inhibition of influenza A was previously reported with silver nanoparticles, but the strongest inhibitory effect was achieved at rather high concentration of 50 μg/ml of silver nanoparticles [10TCID50 during 40 min filtration [The effect of hybrid coating against influenza A virus lead to only 0.7 logarticles . Copper ltration . Mechaniltration .Picornaviridae family as coxsackie viruses, exhibited only nonsignificant decrease after passing through copper oxide-based filter [Hybrid coating exerted only negligible effect on non-enveloped positive single-stranded RNA coxsackie B3 virus. Similarly, previously reported rhinovirus type 2, member of the same d filter . These rIn summary, we have characterized virucidal effect of hybrid coatings containing silver, copper and zinc cations against different viruses representing double-stranded DNA virus, negative and positive sense single-stranded RNA virus and non-enveloped virus. Our experiments showed good virucidal effect against enveloped viruses especially HIV and together with previously reported excellent antibacterial effect this hybrid coating has potential to provide antimicrobial protection on surfaces and materials in healthcare settings.The data sets supporting the results of this article are included within the article.Not applicable.Not applicable.


Apis mellifera L.) and varroa mite (Varroa destructor) samples from Europe and the USA. The virus has a naturally poly-adenylated RNA genome of about 6500 nucleotides with a genome organization and sequence similar to the Tymoviridae , a predominantly plant-infecting virus family. Literature and laboratory analyses indicated that the virus had not previously been described. The virus is very common in French apiaries, mirroring the results from an extensive Belgian survey, but could not be detected in equally-extensive Swedish and Norwegian bee disease surveys. The virus appears to be closely linked to varroa, with the highest prevalence found in varroa samples and a clear seasonal distribution peaking in autumn, coinciding with the natural varroa population development. Sub-genomic RNA analyses show that bees are definite hosts, while varroa is a possible host and likely vector. The tentative name of Bee Macula-like virus (BeeMLV) is therefore proposed. A second, distantly related Tymoviridae-like virus was also discovered in varroa transcriptomes, tentatively named Varroa Tymo-like virus (VTLV).Around 14 distinct virus species-complexes have been detected in honeybees, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee ( Apis mellifera L.) are hosts to a large number of viruses  for a 10-fold dilution series of an external standard of known concentration. The regression slopes were also used to determine the reaction efficiencies of the different assays  in 20 μL reaction volumes containing 10 μL of a 1/10 dilution of random-primed cDNA , 0.4 μM EU) was assembled from Illumina sequence data derived from RNA of 100 varroa mites from 10 colonies of apiary #878 [www.ebi.ac.uk). The 823 BeeMLV contigs (median length of N50 = 520 bp) were scaffolded using the SeqMan program  and finally edited manually. The assembled Illumina sequence was confirmed by chain-termination sequencing [16) for cDNA synthesis, followed by RT-PCR with a virus-specific forward primer. The sequences of the USA variant of bee Macula-like virus (BeeMLVUSA) and of varroa Tymo-like virus (VTLV) were assembled from 454 and Illumina sequence data available from Trinity, a composite varroa transcriptome database (Genbank accession pending) of varroa samples with a variety of biological and geographic origins. The BeeMLV 5′ terminus was approximated by the consensus of the EU and USA assemblies.The sequence of the European variant of bee Macula-like virus (BeeMLVary #878  and OASEary #878  made avaquencing  of clonee” and “f”) to those of other Macula-, Marafi- and Tymoviruses (g”) as described above, except that the evolutionary distances were computed using the Maximum Composite Likelihood method [p-values calculated by MEGA6 from CLUSTAL-Omega alignments of the eight BeeMLV and two VTLV assemblies. All ambiguous positions were removed for each sequence pair.The phylogenetic position of BeeMLV and VTLV within the Tymoviridae was determined by aligning the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP) amino acid sequences  in Pennsylvania in 2001 . The sequblished . Subsequublished . Finallydatasets . Since Vemolymph , and hasBombyx mori Macula-like latent virus (BmMLV) and Culex Tymo-like virus (CuTLV), and has three overlapping open reading frames (ORFs). The first ORF encodes a large polyprotein containing several non-structural proteins, such as a methyl transferase (MTR), a proline-rich region (PRR), believed to act as a hinge between the MTR and P-Pro domains [Tymoviridae, cleaving the large ORF1 polyprotein into an N-terminal protein containing the MTR, PRR and P-Pro functions and a C-terminal protein containing the replicative proteins [The first task was to assemble the complete genome sequence, which was achieved using a combination of chain termination  and Next domains , a papai domains ; an NTPaproteins ,30. The proteins ,27.Many of the plant Tymoviridae also have an additional, large ORF in the 5′ end of the genome coding for a movement protein  wEU), KT162925 (BeeMLVUSA) and KT162926 (VTLV).A number of varroa and honeybee transcriptome sequence libraries from the USA were screened for BeeMLV sequences. The virus was not found in honeybee transcriptomes from an extensive survey of colonies affected, or not, by Colony Collapse Disorder , and onTymoviridae, but outside the Tymovirus, Marifivirus and Maculavirus genera , bee viruses X and Y , Arkansas bee virus (ABV) and Berkeley bee picorna-like virus (BBPV). However, much is known about the physical, chemical, and biological properties of these viruses (2(3) = 26.94; P < 0.005), i.e., colonies that were positive in spring, or became positive during summer, were more likely than not also positive in autumn. Among the autumn samples there was also positive association between the prevalence in adults and pupae (χ2(1) = 20.24; P < 0.005) but not between either adults or pupae and mites. Both the seasonal and adult-pupae associations are less clear for the pooled apiary samples, although this may simply be a statistical consequence of having only 1/10th the amount of data to analyze.A screen of cDNA samples from the 2002 French honeybee virus survey of 360 colonies in 36 apiaries throughout France ,8 showedAn independent extensive virus survey found that BeeMLV was also common and abundant in Belgian honeybee colonies . HoweverThe next question to address was whether bees and mites are true infection hosts of the virus, or merely carriers of passively acquired virus particles. The presence of a sub-genomic RNA, for expression of the second ORF, makes it possible to differentiate active and passive states, since only actively replicating virus produces sub-genomic RNA. We designed two separate qPCR assays in the RdRp region, 5′ of the putative start of the sub-genomic RNA  and two Tymoviridae [Tymoviridae, with its genus status to be resolved when similar insect-specific Tymo-like viruses are reported. The phylogenetic relationships between the three genera are in any case rather indistinct, with a few other assigned and unassigned species floating in between the groups, attesting to a considerable degree of homoplasy in the data , and perhaps also to a degree of flexibility in genome organization, replication and translation strategies , but none of these are by themselves conclusive. Establishing host status is easier for bees than for mites, as illustrated by the sub-genomic analyses. Varroa mites are intimately connected with honeybees, feeding almost continually on the bee haemolymph; in many ways little more than a small bladder of honeybee blood, with all the honeybee viruses that would contain. Nucleic acids extracted from varroa contain significant levels of honeybee-derived nucleic acids [The other main point of discussion is the origin and primary host of the virus, which ideally should be reflected in its name. There are a number of indirect indicators that this virus may be more closely linked to ic acids . The preic acids  and the ic acids , Scandinic acids , all extic acids , and defic acids , tend toic acids ,41 but cic acids ,42,43 suic acids ,45, stabFor these reasons we propose the generic name of Bee macula-like virus (BeeMLV), rather than a host-specific name, reflecting its replication in honeybees and close association with varroa mites, its uncertain origins, and its taxonomic affiliation.


Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.Members of the family  Ribosome profiling is emerging as a powerful technique to monitor translation in living cells at sub-codon resolution. It has particular applicability to virology, with the capacity to identify viral mRNAs that are being translated during infection and to provide new insights into virus gene expression, regulation and host-virus interactions. In this work, we carried out the first ribosome profiling analysis of an RNA virus, using as a model system the murine coronavirus strain MHV-A59, a betacoronavirus in the same genus as the medically important SARS-CoV and MERS-CoV. Parallel ribosome profiling and RNA sequencing of infected-cell time points was performed during the course of MHV replication in mouse tissue culture cells and used to determine virus gene expression kinetics and the relative translational efficiencies of virus and host mRNAs. The sensitivity and precision of the approach permitted us to uncover several unanticipated features of coronavirus translation, giving insights into ribosomal frameshifting, ribosome pausing, and the utilisation of short, potentially regulatory, upstream open reading frames. We also identified some challenges associated with the technique that are of general relevance to the ribosome profiling technique and developed bioinformatic strategies to address these. Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases (kb). Several coronaviruses, including SARS-CoV and MERS-CoV, are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Coronavirus infections are frequent in bats and other mammals  pulse labelling in the presence of actinomycin D provided a similar but slightly different order (mRNA7 > mRNA6 > mRNA5 > mRNA1/mRNA3/mRNA4 > mRNA2)  dripped onto the cells. The cells were scraped extensively to ensure lysis, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4°C, the supernatants recovered and stored in liquid nitrogen.At the appropriate time point, cells were treated with CHX , or HAR  then CHX . Cells were rinsed with 5 ml of ice-cold PBS, the dishes were submerged in a reservoir of liquid nitrogen for 10 s and then transferred to dry ice and 400 μl of lysis buffer  were set up as recommended by the manufacturer and added dropwise to the tissue culture cell growth medium. Cells were harvested 24 h post transfection (h.p.t.) and reporter gene expression was determined using a dual-luciferase assay system kit (Promega). Frameshifting efficiencies were calculated by dividing the Fluc/Rluc ratios of the test samples by the Fluc/Rluc ratio of the in-frame controls.2HPO4, 1.5 mM KH2PO4 (pH 6.7), and 0.1% Tween 20] and probed with mouse monoclonal antibodies raised against nsp9 , N , S (1:500), GAPDH  or a polyclonal rabbit anti-I . Membranes were incubated in the dark with an IRDye-conjugated secondary antibody in PBST . Blots were scanned and bands quantified using an Odyssey Infrared Imaging System (Licor).Proteins were separated by 10%, 12% or 15% SDS-PAGE depending on the molecular weight of the protein of interest and transferred to nitrocellulose membranes. These were blocked for 30–60 min with 5% powdered milk (Marvel) in PBST -methionine. Reaction mixtures were incubated for 30 min at 26°C and stopped by the addition of an equal volume of 10 mM EDTA, 100 μg/ml RNase A followed by incubation at room temperature for 15 min. In ribosomal pausing assays, conditions were the same except that the reaction mixture had a volume of 40 μl and the translational inhibitor edeine was added 3 min after the start of the reaction in order to obtain synchronous initiation . Aliquots of 1.5 μl were withdrawn from the translation reaction mixture at specified intervals and mixed with an equal volume of EDTA/RNase A mixture, as above. In immunoprecipitations, 10 μl of RRL was mixed with either mouse anti-N, anti-S or rabbit anti-I for 30 min at 4°C prior to binding to protein A-Sepharose CL-4B  and subsequent washing. Samples were prepared for SDS-PAGE by addition of 10 volumes of 2X Laemmli’s sample buffer and boiling for 4 min. Proteins were resolved on 10% or 15% SDS-PAGE gels. 14C-labelled molecular weight standards (MW) were from Amersham International (United Kingdom). Dried gels were exposed to a Carestream Kodak Biomax MR film (Sigma-Aldrich) and scanned.pcDNA.3 and pPS0 plasmids were linearized with eviously . RNAs weS1 TableTable of host and virus read counts for the different samples.(DOCX)Click here for additional data file.S2 TableTRSs (UCUAAAC or similar) are indicated in bold. Nucleotides consistent with tandem copies of the pentanucleotide UCUAA are indicated in red  and blue . Note also the high similarity between the sequences at the leader (mRNA1) and mRNA7 junction sites: when polymerase jumping for mRNA7 occurs 5 nt upstream of the canonical site, 17 nt of 3′ sequence are required to distinguish gRNA reads from mRNA7 reads.(DOCX)Click here for additional data file.S3 Tableand ten or more occurrences in repeat 2 are shown. Data are shown for the 5 h p.i. RNASeq libraries.Chimeric reads utilizing the leader TRS were identified by searching for all reads containing the sequence UUUAAAUCUAA (AY700211.1 nt 55 to 65), and classified according to the identity of the following nucleotides at positions +3 to +17. These 15 nucleotides are listed in column 4. The genomic coordinate of the first nucleotide of the 15 is given in column 5. Nucleotides at positions +1 to +2 in the RNASeq read are listed in column 3. The corresponding two nucleotides from the genome are listed in column 2. Also, the 5 nucleotides preceding these in the genome are listed in column 1. The numbers of junction/body chimeric reads containing each sequence are given in column 6 (repeat 1) and column 7 (repeat 2). Only sequences with three or more occurrences in repeat 1 (DOCX)Click here for additional data file.S4 Tablefwd indicates forward primer, and rev indicates reverse primer.Note (DOCX)Click here for additional data file.S1 FigReads were mapped to virus RNA, and host rRNA, mRNA, ncRNA and gDNA databases. Reads mapping to gDNA are expected to derive from unannotated transcripts not present in the mRNA or ncRNA databases, but, since the direction of transcription is not annotated in the gDNA database, such reads constitute a mixture of forward and reverse-sense matches. Reverse-sense rRNA matches in the RNASeq samples are expected to derive from the RiboZero kit which contains complementary sequences to rRNA.(TIF)Click here for additional data file.S2 FigPhasing of 5′ ends of reads that map to host mRNA coding regions as a function of read length. Reads whose 5′ ends map to the first, second or third positions of codons are indicated in purple, blue or orange, respectively.(TIF)Click here for additional data file.S3 FigSee (TIF)Click here for additional data file.S4 FigHistograms of RPF 5′ end positions relative to annotated initiation and termination codons summed over all host RefSeq mRNAs for the RiboSeq libraries. To account for different library sizes, histograms are normalized by the sum of total virus RNA (positive and negative-sense) plus total host mRNA for the library.(TIF)Click here for additional data file.S5 FigHistograms of RPF 5′ end positions relative to annotated initiation and termination codons summed over all host RefSeq mRNAs for the RiboSeq CHX 5 h p.i. time point (repeat 1) as a function of RPF length. RPFs of ribosomes paused during initiation with the initiation codon  in the P-site have 5′ ends that normally map to position −12 (12 nt upstream), or, particularly for longer RPFs (e.g. 30–32 nt), position −13. The smaller peak at 0 is likely an artifact of ligation bias —all RPFs mapping to this position begin with 5′-AUG, whereas RPFs that map to other positions have differing 5′ end nucleotides so that any 5′-end-dependent biases are averaged out when summing over many mRNAs. Termination occurs with the stop codon  in the A-site so that the 5′ ends of RPFs paused during termination normally map to position −17 (15 nt upstream).(TIF)Click here for additional data file.S6 FigHistograms of read 5′ end positions relative to annotated initiation and termination codons summed over all host RefSeq mRNAs for the RNASeq libraries. To account for different library sizes, histograms are normalized by the sum of total virus RNA (positive and negative-sense) plus total host mRNA for the library.(TIF)Click here for additional data file.S7 FigLength distributions for reads mapping to host mRNAs (green), positive-sense virus RNA (orange) and negative-sense virus RNA  for repeat 1. The left panel in each pair shows the absolute read counts. The right panel in each pair shows the distributions normalized to have equal total sums to facilitate comparison of distribution shapes. Differences between host and virus distributions are indicative of contamination. Negative-sense virus read length distributions are only shown at 5 h p.i. and 8 h p.i. as the counts at earlier time points are often too low to assess distribution shape.(TIF)Click here for additional data file.S8 FigSee (TIF)Click here for additional data file.S9 FigReads were counted in windows from 10 to 100 codons upstream  or downstream  of annotated termination codons, and summed over all host mRNAs. The left panel in each pair shows the absolute read counts, allowing comparison of the CDS and 3′ UTR read densities; the density ratio (3′ UTR / CDS) is indicated in purple in each panel. For all RiboSeq samples, 3′ UTR occupancy is very low compared to CDS occupancy, whereas, for RNASeq, 3′ UTR occupancy is typically around 80% of CDS occupancy . The right panel in each pair shows the distributions normalized to have equal total sums so that the shapes of the CDS and 3′ UTR distributions can be compared. For RNASeq, the two distributions have essentially identical shapes. For RiboSeq, differences in the two distributions provide an indicator of the level of non-RPF contamination present in the sample.(TIF)Click here for additional data file.S10 FigSee (TIF)Click here for additional data file.S11 FigUnfractionated RiboSeq RNA prepared for the 5 h p.i. time point of repeat 2 was re-run on a 15% denaturing acrylamide-urea gel and a larger gel slice taken to sample RPFs within the range 28 to ~80 nt. Histograms show the positions of the 5′ ends of reads with a +12 nt offset to map the approximate P-site. RPF distributions were smoothed with a 15-nt running-mean filter. Note the widely varying vertical axis scales—the vast majority of RPFs fall in the size range 25–35 nt. Blue triangles indicate the previously analysed sites of RPF accumulation see .(TIF)Click here for additional data file.S12 Fig(A) Composition of the high MOI libraries (see (B) Comparison of read length distributions for virus and host mRNA (see (C) Phasing of reads mapping to host mRNAs (see (TIF)Click here for additional data file.


We developed an active virus filter (AVF) that enables virus enrichment for single virus infection, by using insulator-based dielectrophoresis (iDEP). A 3D-constricted flow channel design enabled the production of an iDEP force in the microfluidic chip. iDEP using a chip with multiple active virus filters (AVFs) was more accurate and faster than using a chip with a single AVF, and improved the efficiency of virus trapping. We utilized maskless photolithography to achieve the precise 3D gray-scale exposure required for fabrication of constricted flow channel. Influenza virus (A PR/8) was enriched by a negative DEP force when sinusoidal wave was applied to the electrodes within an amplitude range of 20 Vp-p and a frequency of 10 MHz. AVF-mediated virus enrichment can be repeated simply by turning the current ON or OFF. Furthermore, the negative AVF can inhibit virus adhesion onto the glass substrate. We then trapped and transported one of the enriched viruses by using optical tweezers. This microfluidic chip facilitated the effective transport of a single virus from AVFs towards the cell-containing chamber without crossing an electrode. We successfully transported the virus to the cell chamber (v = 10 µm/s) and brought it infected with a selected single H292 cell. Conventional analysis of viral functions is performed by using virus-infected cultured cells and this method has been considered as the most precise technique of analysis. This method provides information that is an average result of data generated from all of the cells in the population. However, the physiological state and cell cycle stage of each infected cell is different A dielectrophoretic force is a force that is exerted on a polarizable particle under conditions of a non-uniform electric field 2. The electrode is made of Cr/Au. When the focused laser is irradiated to the electrode, the metal electrode is broke, and the bubble is generated. Therefore, the electrodes must be arranged around working space of enrichment and transport parts. A large gap between electrodes enough to manipulation of the virus without crossing the electrode is possible by using iDEP as shown in In insulator-based dielectrophoresis (iDEP), remote electrodes apply an electric field within a fluidic volume while insulating structures are used to distort the electric field thereby producing spatial non-uniformities In this study, we present a virus infection microfluidic chip in which three-dimensional iDEP was used generate an AVF that could enrich for and allow manipulation of viruses in an aqueous solution. This system facilitated the effective transport of a single virus from the periphery towards the cell-containing chamber without crossing an electrode. The ultimate goal of this research was to carry out on-chip infection of a specific cell with a single virus using optical tweezers.E is the electric field strength, r is the particle radius, pε' and mε' are the respective complex permittivities, pε and mε are respective permittivities, pσ and mσ are the conductivities of the particle and the suspending medium, respectively, and ω is the angular frequency of the voltage. RefCM is the Clausius–Mossotti factor related to the effective polarizability of the particle. When high frequencies are applied and the particles are less polarizable than the suspension medium, the particles exhibit “negative” DEP by moving away from regions of high electric field gradient. iDEP systems use both AC and DC electric fields, therefore, the DEP force exerted on a particle for electric field gradient depends on the dielectric properties of the particle and the suspension medium. The DEP force exerted on a particle also depends on the particle shape in addition to size.DEP is the motion of a particle in the medium in which it is suspended due to the presence of a non-uniform electric field. The dielectrophoretic force acting on a spherical particle can be described by the following equation p = ε3 F/m, pσ = 0.8 mS/m m = ε80 F/m, mσ = 10 mS/m. The value of fCM factor was −0.36. Therefore, the virus received negative DEP force. E is often required for the design of the electrodes in a DEP device. From ω is the angular frequency of the applied electrical field E. The expression for the DEP force as a function of the distance between the two electrodes is defined as 0F is the value of F when the distance between the two electrodes L = 0, and c is a constant depending on the configuration of microchannel and electrodes. The DEP force is commonly dependent on the distance between electrodes. Another simulation was therefore performed to investigate the DEP force with respect to the distance between electrodes using a 3D-constricted channel (1L) or 7 mm (2L) and in which single or multiple AVFs were inserted between electrodes set at a distance of 7 mm. The simulation results show that for a planar electrode, the electric field gradient dramatically decays in a cross-section of the microchannel as the distance between electrodes increases. Therefore, the DEP force is very weak for an AVF that is inserted into separated electrodes. One method to increase the DEP force that acts on the particle is to increase the applied voltage. However, an increase in the voltage can result in an increase in the temperature in the microfluidic channel. Intriguingly, the data in A detailed analysis of the factor  channel . NumericBF is the Brownian force [N], vρ is the virus density [g/cm3], r is the virus radius [m], R is the molar gas constant (8.31447 J K−1 mol−1), T is the temperature [K], η is the viscosity of the solution (1.004×10−6 m2/s), and AN is the Avogadro constant [/mol]. We assumed that the virus density was 1 g/cm3, and time resolution t is 0.33 s. From Eq. 4, BF was 8.3×10−24 N. Gravity force of the virus GF is 5.1×10−18 N. When DEPF is larger than B +FGF, the virus inside the microchannel does not adhere to the glass during enrichment of virus by DEP force.Moreover, DEP force is used to prevent the adhesion of the virus to the glass substrate. In the microchannel, forces working to the virus are gravity force, DEP force and Brownian force. The virus does not adhere to the glass in case the DEP force exceeds the Brownian force and gravity force. The Brownian force is given by:Three-dimensional (3D) patterns whose shape is precisely controlled can be fabricated using gray-scale lithography. Gray-scale lithography utilizes locally modulated exposure doses to develop a 3D structure in a photoresist A photograph in p-p, 10 MHz) voltage was applied to the electrodes by a function generator . We used a confocal laser microscope  equipped with a laser manipulation system  to perform iDEP virus enrichment and single virus infection of a specific cell. The virus was tracked through the process of iDEP, transport and infection by visualization of viral fluorescence using the same microscope. The cells of influenza virus attached and unattached cells were assayed by immunostaining using anti-PB1. At 4 h after virus attachment on the cell, the cells were washed with PBS and fixed with 4% paraformaldehyde solution, treated with 0.5% Triton X-100, blocked with 1% BSA. After blocking, the cells were incubated with anti PB1 antiserum for 1 h, washed with 1% BSA/PBS solution and then incubated with anti-rabbit IgG with Cy3 for 1 h at 37°C The influenza viruses (A PR/8) were co-stained with 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine Perchlorate (DiI)  and SYTO 21 live-cell nucleic acid stain  which stain the virus membrane and virus ribonucleoprotein (RNP), respectively We used inverted microscope  having a laser confocal scanning system  for fluorescence observation and a laser manipulation system  for virus manipulation. The laser is focused by objective lens (Magnification: 100) with high numerical aperture (NA: 1.3). The virus is trapped at the laser focal point by Lorentz force because the size of the virus (100 nm) is smaller than that of laser wavelength. Trapping force becomes bigger according to the increase of laser power. However, laser power is limited within 1 W to avoid photo-bleaching of Syto 21 during manipulation. In fact, even if laser power output 1 W, input to a sample is decreased to about 100 mW since an objective lens is passed. In this paper, all transport speed of the virus is 10 µm/s.DEPF of the four analytic surfaces can be ordered according to the constricted microchannel height as follows: 135 µm <15 µm <45 µm <90 µm. The reason why the gradient generation in each height is considered that by increasing the channel height, the gradient of the electric field should decrease as the electric field streamlines are less disturbed at the trapping area. The highest iDEP power observed for the four analytic surfaces was that of 0.3 µN that was observed at a constricted channel height of 90 µm. In the previous study, we estimated the force for transporting the virus by optical tweezers using Stokes’ law. When the size of influenza viruses is 100 nm, the driving force was calculated to be 9.4 fN 6 pfu/mL) was introduced into the AVF. The introduced viruses were enriched by iDEP  in AVF during 10 sec. AVF-mediated virus enrichment can be repeated simply by turning the current ON or OFF as showing In experiment, virus solution . Moreover, Brownian motion of the virus is fast and the virus moves away from the focus plane easily. Therefore, enrichment of the virus by AVF is quite useful by DEP to find the virus before laser transport phase. Moreover, we transported this virus to the cell chamber where it made attachment with the selected H292 cell for infection . InfectiWe should note that electrode allocation of iDEP contributed to the safe and easy transportation of viruses using optical tweezers. Our previous study indicated that the direct irradiation of laser was caused since the pectinate electrodes and transportation path crossed We developed an active virus filter (AVF) that enabled virus enrichment and distribution for single virus manipulation by using 3D insulator-based dielectrophoresis (iDEP). The design of the 3D constricted flow channel enabled the microfluidic chip to produce an iDEP force. We utilized maskless photolithography to achieve precise 3D gray-scale exposure for construction of the constricted flow channel. The use of the AVF achieved enrichment of the influenza virus via a negative dielectrophoretic force. Negative AVF also functions to inhibit virus adhesion onto the glass substrate due to 3D iDEP effect. In this result, we succeeded in infection of a virus to the selected H292 cell.Figure S1Investigation of the relationship between the DEP force and the distance between electrodes by FEM analysis of electric field distribution. Simulations were carried out in which the distance between electrodes was either 4 mm (1L) or 7 mm (2L). The effect of single or multiple AVFs between electrodes set at a distance of 7 mm was also analyzed. The results of FEM analyses are shown.(TIF)Click here for additional data file.Movie S1Enrichment of influenza virus in AVF. Movie showing the iDEP enrichment of influenza virus around the constricted flow channel. We applied sinusoidal wave to the electrodes within the range of amplitude of 20 Vp-p and frequency of 10 MHz. AVF achieved enrichment of influenza virus by the negative dielectrophoretic force repeatedly.(MP4)Click here for additional data file.Movie S2Observation by which an influenza virus is attached to a specific cell by optical tweezers. Movie showing the attachment of a SYTO 21-labeled influenza virus particle to three H292 cells following manipulation with optical tweezers.(MP4)Click here for additional data file.