In this letter, we treat a rod-shaped virus as a free homogenous nanorod and identify its confined acoustic vibration modes that can cause strong resonant microwave absorption through electric dipolar excitation with a core-shell charge distribution. They are found to be the n = 4N-2 modes of the longitudinal modes of the nanorods, where N is an integer starting from 1 and n is the mode order quantum number. This study was confirmed by measuring the microwave absorption spectra of white spot syndrome virus (WSSV), which is a rod-shaped virus. This is also the first study to identify the “dipolar-like” mode in a rod-shaped nano-object. Our study is not only an important step to achieve rapid and sensitive detection of rod-shaped viruses based on their microwave spectroscopic features and a non-contact method to measure the Young’s modulus of rod-shaped viruses, but also is critical to formulate an efficient epidemic prevention strategy to deactivate viruses with the structure-resonant microwaves. This resonant microwave absorption process is through the dipolar coupling of electromagnetic waves with the specific “dipolar” vibration modes of the same frequency in the spherical viruses, assisted with the inherent core-shell charge separation of the nano-sphere-like virions4. Since it is a resonant conversion of a photon into a phonon of the same energy, this energy transfer is highly efficient and thus provides a new strategy for virus epidemic prevention in open public. However not all viruses are nano-sphere-like, it is thus highly desirable to explore the “dipolar-like” resonant energy transfer effect in other viruses. Besides the spherical shapes, the rod-like shape is the other common shape for viruses. Up to now, there is no study to identify the “dipolar-like” mode in a rod-shaped nano-object. In this letter, by treating a rod-shaped virus as a free homogenous nanorod, we experimentally identify the confined acoustic vibration modes that can cause strong resonant microwave absorption. They are found to be the longitudinal modes of the nanorods with a mode order quantum number n equivalent to 4N-2, where N is an integer starting from 1. This theoretical analysis was confirmed by measuring the microwave absorption spectra of the white spot syndrome virus (WSSV), which is a rod-shaped virus. Our study is not only an important step to achieve rapid and sensitive detection of rod-shaped viruses and provides a non-contact method to measure their elastic properties, but also is critical to formulate an epidemic prevention strategy to deactivate rod-shaped viruses with efficient structure-resonant microwaves.By treating icosahedral viruses as nano-spheres, a recent study has shown highly efficient structure-resonant energy transfer from microwave to the confined acoustic vibration modes of spherical viruses5 proposed the theory of confined vibration modes in spherical particles derived from a stress-free boundary condition. The confined acoustic modes of spherical nano-particles were not observed until 19866. In 1992, E. Duval7 proposed the selection rules for far-infrared and Raman transitions of a solid sphere. According to selection rules, only dipolar modes, which are the spheroidal modes with l = 1 where l is the angular momentum quantum number, can be excited by far-infrared due to the electric-dipolar transition. However, there was no experimental observation of dipolar modes until two recent reports8 on the terahertz (THz) absorption in TiO2 nanopowders and CdSe/CdTe core-shell type-II quantum dots. The type-II quantum dot study proved the existence of the resonance dipolar interaction between THz photons and the spheroidal dipolar phonons in nano-spheres. The same study also indicated the critical role of the specific core-shell spatial charge separation for the excitation of the resonance dipolar interaction. Taking advantage of the core-shell-like charge separation structure in a virus, this finding was then applied to spherical viruses by treating one icosahedral virus in serum as a single free homogeneous nano-sphere. T.-M. Liu et al. observed the resonance microwave absorption in spherical viruses through confined acoustic vibrations4. The measured microwave absorption peak frequencies agreed well with those of the dipolar modes with the quantum numbers l = 1, n = 0 & 1, and were size-dependent of the studied viruses. This observation of resonance microwave absorption of viruses, combined with the matured microwave technology, could provide a promising physical mechanism for future development of rapid non-contact identification, quantification, manipulation, and selective destruction of viruses without the need of affinity biosensors. Recently this structure-resonant energy transfer effect from microwaves to viruses, based on the dipolar mode transition, was shown to be efficient enough so that airborne virus was inactivated with reasonable microwave power density safe for the open public1. This effect was demonstrated by measuring the residual viral infectivity of influenza A virus after illuminating microwaves with different frequencies and powers. Nevertheless, so far there is no experimental report related to dipolar transition through infrared/THz wave/microwave excitation of the confined acoustic phonon modes of nanorod/nano-cylinder or the rod-shaped viruses, and there is no study reported on the dipolar–like modes of rods or cylinders. It is thus highly desirable to identify the “dipolar-like” confined acoustic phonon modes in rod-shaped viruses not just for fundamental physics studies, for the non-contact assessment of the virus Young’s modulus, but also for its potential applications on virus deactivation.In 1882, H. Lamb9 researched the vibrations of solid cylinders, and in 1886 C. Chree10 studied infinitely long circular rods with traction-free circumferential boundaries. In 1964, G. W. McMahon11 published his experimental results for natural frequencies of solid cylinders with free boundaries. In 1972, J. R. Hutchinson12 studied the axisymmetric free vibrations of finite length rods using a series solution to the three-dimensional theory of elasticity and further13 included nonaxisymmetric vibrations of a solid rod in 1980. Based on previous researches, recently H. R. Hamidzadeh and R. N. Jazar14 categorized the confined vibrational modes of a cylinder as follows: 1) breathing mode, where any point on the cross section of the cylinder vibrates harmonically in its radial direction; 2) torsional mode, associated with transverse displacement of the circumference of the cylinder; 3) axial/longitudinal/extensional mode, associated with axial vibration; 4) bending and axial shear/transverse mode, where the cross section of the cylinder remains the same; 5) lobar mode, where both radial and transverse displacements vary with sin(nθ)14. These modes have been found to be the dominant vibration modes of a cylinder or a rod-like object. The analysis of the motion for any point in a cylinder can be approximated by summing the effects of the dominant modes.The vibrations of a cylinder have been studied with a long history. In 1876, L. Pochhammern equivalent to 4N-2 could be corresponding to the dipolar-like mode in a nano-rod for the microwave excitation under a core-shell charge separation structure, where N is an integer starting from 1. Figure In this study, we apply the above-mentioned free boundary condition approximation to rod-like viruses in serum. After examining all different modes, we found that  of the cylinder is much smaller than that of length (L), and the boundary conditions are free. Based on the equation of motion of a three-dimensional elastic body15u in the 3 cylindrical coordinates r, θ, z with the form To explore the electric dipolar transition with confined acoustic vibrations in a rod-like object, here we assume that the nanorod (or cylinder) is isotropic, solid and elastic. The radius  of baculovirus genomes19. To predict the microwave absorption peak frequency with the electric dipolar excitation through the confined “dipolar-like” modes, one needs the effective longitudinal sound velocity VL of WSSV. Recently based on Brillouin scattering measurement, the effective VL of the wet Satellite Tobacco Mosaic Virus (STMV) was determined to be 1920 m/s22. With more genomes packed inside the capsid, the effective VL of the EnteroVirus 71 (EV71) was determined to be 2450 m/s3 by using microwave resonant absorption spectroscopy. Based on Eq. . WSSV is a widespread disastrous viral pathogen of cultured shrimps and causes high economic losses. WSSV also attacks crabs, crayfish, and many other crustaceansd on Eq. , the con3. For spherical viruses, dipolar transition with resonant microwave absorption is through the excitation of l = 1 dipolar modes with n = 0 and 1 with a peak frequency ratio of 1:2, very different from the 1:3:5:7 ratio of the rod-like viruses.The microwave absorption spectroscopy of the first set of WSSV virion sample was performed by adopting the same microwave absorption measurement system as described in ref. Q of the resonance modes. In a previous microwave absorption study on Perina nuda viruses (PnV)4, it was found that the hydration levels on the capsid surface of viruses can affect the bandwidth of microwave resonant absorption induced by the confined acoustic vibrations. This was evidenced by a pH dependent study, where the enhancement of the surface hydrophilicity resulted in a rise of Q. It is thus possible that this wide bandwidth is related to the hydration levels on the capsid surface of viruses. Another factor which can affect the bandwidth will be the purity of the samples, which would result in inhomogeneous broadening of the resonance linewidth. It was known that host-factor (vitellogenin and hemocyanin) contaminations were frequently observed on WSSV virions16. Hemocyanin is a host factor present in shrimp hemolymph which involved in host immune response against pathogen. To avoid possible host factor contamination to affect the measured bandwidth, we revised our purification method and prepared the second set of WSSV virion samples. To provide a better spectral resolution and to investigate the possible bending mode issue, to be discussed later, we thus employed a vector network analyzer with a microwave measurement frequency range only up to 20 GHz but with a 0.1 GHz frequency resolution. The microwave absorption spectroscopy of the second set of WSSV virion samples was performed in a biosafety cabinet while the sample temperature was kept at 25 °C, by using a microfluidic channel adhered on a coplanar waveguide (CPW) circuit and a portable vector network analyzer (Anritsu MS2028C) with a bandwidth up to 20 GHz, as described in Methods and as shown in Fig. The measurement on microwave absorption spectra indicated a wide bandwidth and low quality factor 3 20, the effective Young’s modulus of WSSV can be estimated to be ~5.4 GPa. To check on the shape effect, we have developed a capsule model and to compare the effect of the capsule shape (versus a cylinder) to the measured longitudinal mode frequency. As shown in Fig. 23, while packing the high-density DNA/RNA cores was known to result in shell stiffening23. The densely packed viral cores were hard to be approached by using the AFM nanoindentation method. Our measured relatively high elastic constant could thus be the result of the densely packed viral DNA with a size of 300 kbp inside the nucleocapsid. Please notice that our reported value is the effective value considering the whole virus structure, not just the soft capsid part. This value is almost on the order of but lower than the previously measured Young’s modulus of individual Type 1 collagen fibrils24, which was found to be in the range from 5 GPa to 11.5 GPa.Figure One might wonder the possible interaction of bending mode vibration with microwaves. Bending mode of a cylinder can also be called transverse mode or flexural mode. The bending mode is associated with no cross-sectional distortion, while bending in the axial direction occurs. The odd mode-number bending modes move the center of the rod toward x (or y) while move both ends toward −x (or −y), following the coordinate system shown in Fig. In summary, cores of most viruses have inherent negative charges due to the phosphate groups in genomes. In contrast, the amino acids of viral capsids or envelopes have complex charge distributions on the surface. Such a core-shell charge separation structure paves the road for the dipolar coupling between microwaves and confined acoustic vibrations. The microwave resonant absorption processes would thus occur in viruses when the confined acoustic vibration moves the charges and changes their dipole moments. By treating icosahedral viruses as nano-spheres, a recent study has shown highly efficient structure-resonant energy transfer from microwave to the confined acoustic vibrations in spherical viruses, resulting in efficient deactivation of flu virus with low microwave power safe for the open republic. Except for spheres, rods and filaments are among the other shapes most commonly found in viruses. In this work, we investigated the dipolar-like confine acoustic modes in rod-like viruses with a core-shell charge separation. As a result, we found that the selection rule of the confined acoustic-mode for microwave resonant absorption varies with the shape of viruses. By using 300 nm long baculoviruses as our test target, we proved our hypothesis that longitudinal modes with a mode order quantum number n = 4N-2 can induce resonant microwave absorption. This is not only the first study to reveal the “dipolar-like” confined acoustic modes in a nano-cylinder, but also provide a non-affinity way to detect and distinguish a rod-like virus from a spherical virus. This microwave resonant absorption spectroscopy could serve as a convenient way to measure the elastic constant of a virus. Our finding will also help to formulate a strategy for non-contact low-power virus deactivation.et al.16, we infected healthy Procambarus clarkii crayfish using inoculated hemolymph prepared from WSSV-infected Penaeus monodon. After one week, the intact virions in the crayfish hemolymph were purified as described previously19, by using sucrose gradient ultracentrifugation . The final pellet of the intact WSSV virions was dissolved in Tris buffer . Characterized by real-time PCR, the number density of the virus was 5.5 × 1011 genome copies/ml and 1.7 × 109 genome copies/ml for the first and the second set of virion samples. From the TEM image  were injected with diluted (1:500 in PBS) virus stock, and 5~9 days later, all tissues except for the organs in the cephalothorax were cut into small pieces and homogenized in TESP buffer . Instead of ultracentrifugation, the virus was first centrifuged for 5 min at 3500 × g at 4 °C, the supernatant was collected and further centrifuged at 30,000 × g for 30 min at 4 °C. The supernatant and the pink upper layer of the pellet were gently removed, and the remaining gray/white pellet was then resuspended with TM buffer . The purity of the WSSV virion samples was checked using SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) and Western blotting, and the integrity of the virions was confirmed by negative staining TEM. By using this purification method, host factors (e.g. hemocyanin) contaminations were significantly reduced in the second set of samples.For the first set of samples, host factors (vitellogenin and hemocyanin) contaminations were observed on the SDS-PAGE gel11 and the transmission S21 parameter of the CPW circuit, the microwave attenuation spectra can be evaluated as described previously in ref. The microwave absorption spectroscopy of the second set of WSSV virion sample was measured by using a microfluidic channel adhered on a coplanar waveguide (CPW) circuit and a portable vector network analyzer (Anritsu MS2028C) with a bandwidth up to 20 GHz. This CPW circuit substrate used the proprietary woven glass reinforced ceramics  as the dielectric material with copper layers on two sides. The substrate and metal layers are around 1.5 mm and 35 μm in thickness. The dielectric constant of the dielectric material is 3.33–3.43 and the dissipation (loss) factor of that is below 0.0035 at 10 GHz. A mechanical engraving approach was used to define two slots on one side of the substrate. The width of slot and signal lines were 250 and 800 μm, respectively. A simple microfluidic channel was then adhered on top of the CPW circuit Fig. . For mic


Mucosal HIV-1 transmission predominantly results in a single transmitted/founder (T/F) virus establishing infection in the new host despite the generally high genetic diversity of the transmitter virus population. To what extent HIV-1 transmission is a stochastic process or driven by selective forces that allow T/F viruses best to overcome bottlenecks in transmission has not been conclusively resolved. Building on prior investigations that suggest HIV-1 envelope (Env) features to contribute in the selection process during transmission, we compared phenotypic virus characteristics of nine HIV-1 subtype B transmission pairs, six men who have sex with men and three male-to-female transmission pairs.All recipients were identified early in acute infection and harbored based on extensive sequencing analysis a single T/F virus allowing a controlled analysis of virus properties in matched transmission pairs. Recipient and transmitter viruses from the closest time point to transmission showed no signs of selection for specific Env modifications such as variable loop length and glycosylation. Recipient viruses were resistant to circulating plasma antibodies of the transmitter and also showed no altered sensitivity to a large panel of entry inhibitors and neutralizing antibodies. The recipient virus did not consistently differ from the transmitter virus in terms of entry kinetics, cell–cell transmission and replicative capacity in primary cells. Our paired analysis revealed a higher sensitivity of several recipient virus isolates to interferon-α (IFNα) which suggests that resistance to IFNα cannot be a general driving force in T/F establishment.With the exception of increased IFNα sensitivity, none of the phenotypic virus properties we investigated clearly distinguished T/F viruses from their matched transmitter viruses supporting the notion that at least in subtype B infection HIV-1 transmission is to a considerable extent stochastic.The online version of this article (doi:10.1186/s12977-016-0299-0) contains supplementary material, which is available to authorized users. The identification of host and viral determinants that govern human immunodeficiency virus type 1 (HIV-1) transmission and infection is essential for the development of a targeted prevention approach. The observation that virus populations in acute infection are very homogeneous  contrast+ T-cells but not monocyte-derived macrophages (MDMs) 69], PGT1+ T cells with stimulated, CD8-depleted PBMCs. The TCID50 on PBMCs and the co-receptor usage on MT-2 cells of the obtained virus isolates were determined as described previously [Autologous virus was isolated by co-culturing patient CD4eviously .env (nucleotides 5978–9171 based on HXB2) was PCR amplified with Platinum Taq High Fidelity Polymerase (Life Technologies) according to the manufacturer’s instructions. Primers for amplification were 5′allspl (5′-AAGAAGCGGAGACAGCGACGAAGA-3′) and MascN. The PCR cycle conditions were: 94 °C for 2 min followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 68 °C for 3.5 min, followed by a final extension at 68 °C for 10 min. PCR products were gel purified and cloned into the pcDNA3.1/V5-His-TOPO expression vector (Life Technologies). Transformation into E. coli XL10 Gold (Agilent) was done according to the manufacturer’s instructions. Control of plasmid insert orientation via env PCR, plasmid preparation, Env-pseudovirus generation in small scale for functionality test and subsequent functionality screen on TZM-bl cells were performed as described [env plasmid at a ratio of 3:1 using polyethylenimine (PEI) as described [For the generation of Env-pseudoviruses, viral RNA from plasma or virus isolates was isolated using the Rneasy kit (Qiagen) and reverse transcribed into cDNA using Superscript III (Life Technologies) and primer MascN. Subsequently, full-length escribed . Furtherescribed ) and theescribed . 48 h afEnv plasmids were sequenced with the Illumina MiSeq v2 50 cycles kit. For preprocessing and de novo assembly, CLC Genomics Server version 6.5 trim and mapping algorithms were used to trim 23 nucleotides from the 5′ and 3′ ends, followed by end quality trimming to obtain reads with an average Phred score >30 and free of ambiguous nucleotides. Contigs were generated using the de novo assembly algorithm with automatic bubble and word size parameters specified. HIV-1 contigs were then positively selected by aligning them to the HXB2 reference.de novo assembly—using the methods mentioned above; and (2) sample specific consensus sequences—called from CLC Genomic Workbench Extract Consensus Sequence algorithm for BWA mem alignments of sample specific reads (plasma or primary virus isolate) to a Zurich HIV-1 subtype B reference, with “Threshold = 100” and “Post-remove action = Split into separate sequences” specified. Haplotypes were generated for gp120 using QuasiRecomb [Patient plasma or primary virus isolates were sequenced with the Illumina MiSeq v2 300, 500 or 600 cycles kit. Reads were pre-processed using prinseq-lite.pl . PrinseqsiRecomb  with the5 stimulated CD8-depleted PBMCs in 100 μl culture medium per well. 50 μl culture supernatants were harvested and 50 μl fresh medium was added back on days 4, 5, 6, 7, 8, 10, 12 and 14 post infection (p.i.). At day 14 p.i. culture supernatants were assayed for p24 antigen by using an in-house p24 antigen enzyme-linked immunosorbent assay (ELISA) as described previously [50 was determined. Retrospectively, p24 antigen was measured by ELISA of the dilution containing the same virus inoculum (125 TCID50 per well as determined with day 14 p.i. supernatants of the same experiment) from all other harvesting days. Because the virus inoculum was not washed out after infection, the residual input p24 concentration was measured and subtracted from all test results.To assess the in vitro replicative capacity on PBMCs, virus isolates were serially diluted in cell culture medium and 100 μl virus dilution was added in quadruplicates per dilution on 96-well plates containing 2 × 10eviously , 87 and 4 MDMs per well were infected in triplicates with virus isolates at an MOI of 0.05, spinoculated onto MDMs  and unbound virus was washed away. On day 1, 7 and 14 p.i. 50 μl culture supernatant was assayed for p24 antigen by ELISA.To assess the in vitro replicative capacity on MDMs, 8 days after monocyte isolation 96-well plates containing 7 × 105 stimulated CD8-depleted PBMCs in 100 μl of cell culture medium were pre-treated with 1000 U/ml of IFNα-2a  for 4 h at 37 °C. Virus isolates were then added at a TCID50 of 80 per well (as determined on the same pool of stimulated CD8-depleted PBMCs) in 100 μl cell culture medium to 6 wells of IFNα-2a treated and 6 wells of untreated PBMCs. Virus inoculum was washed out on day 1 p.i. Culture supernatants were harvested, assayed for p24 antigen by ELISA and culture wells fed with new medium on days 1, 3, 5, 7, 9, 11 and 13 p.i. In addition, on every day until day 8 (after that no new infection of PBMCs occurs) new IFNα-2a was added onto the culture wells. In this manner a final assay concentration of 1000 U/ml was maintained in the culture wells throughout the experiment as IFNα is known to have a short half-life.To assess the in vitro replicative capacity in the presence of IFNα, 96-well plates containing 2 × 102 − p241) * (T2 − T1)/ln(p242/p241). The sum of AUCs from successive time points then gave the total AUC.As a measure of replicative capacity, we determined the AUC of the p24 production in ng/ml with following formula: AUC =  were derived by fitting pooled data from 2 independent experiments to the log inhibitor vs. response variable slope fit in GraphPad Prism. The MPN was defined as the average percent neutralization reached at the highest plasma concentration of 1:40. If 50 % inhibition or neutralization was not reached at the highest or lowest inhibitor or plasma concentration, a greater-than or less-than value was recorded and used for statistical tests.Pseudoviruses carrying the murine leukemia virus and the HIV-1 SF162 Envs were used as negative and positive controls for all plasma samples, respectively. The inhibitor concentration or reciprocal plasma dilution causing 50 % reduction in viral infectivity (ICEntry kinetics was probed as previously described . Briefly5 cells per well) and transfected with the env plasmids and the NLinGluc vector [Cell–cell transmission and free virus infection was tested as described , 53. 293c vector , 89 for 3 transfected cells were seeded to 1.5 × 104 A3.01-CCR5 cells per well (in triplicates per env plasmid). After 65 h incubation at 37 °C gaussia luciferase production was quantified .To assess cell–cell transmission, 6 h after transfection the supernatant was removed and the 293-T cells were resuspended in 2 ml culture medium (without DEAE-dextran). 5 × 104 A3.01-CCR5 cells per well in 96-well plates (in triplicates per env plasmid) in the presence of 10 μg/ml DEAE-dextran. After 65 h incubation at 37 °C firefly luciferase production was quantified.To assess free virus transmission, 6 h after transfection the supernatant was replaced with 1 ml fresh culture medium. 48 h later, the virus-containing supernatant was harvested, cleared by centrifugation and stored at −80 °C. Subsequently, 50 μl virus was added onto 5 × 10All statistical analyses were performed in GraphPad Prism 5 (GraphPad Software). Transmitter and recipient viruses were compared by the Wilcoxon matched-pairs signed rank test. When more than one Env-pseudovirus was measured per patient, the median value was taken for statistical tests. Correlation was performed by linear regression analysis. p value ≤0.05 was considered statistically significant.


Mycobacterium tuberculosis (M. tuberculosis), contains an abundant cell wall glycolipid and a crucial virulence factor, trehalose-6,6’-dimycolate (TDM). TDM causes delay of phagosome maturation and thus promotes survival of mycobacteria inside host macrophages by a not fully understood mechanism. TDM signals through the Monocyte-INducible C-type LEctin (Mincle), a recently identified pattern recognition receptor. Here we show that recruitment of Mincle by TDM coupled to immunoglobulin (Ig)G-opsonised beads during Fcγ receptor (FcγR)-mediated phagocytosis interferes with phagosome maturation. In addition, modulation of phagosome maturation by TDM requires SH2-domain-containing inositol polyphosphate 5’ phosphatase (SHP-1) and the FcγRIIB, which strongly suggests inhibitory downstream signalling of Mincle during phagosome formation. Overall, our study reveals important mechanisms contributing to the virulence of TDM.The causative agent of tuberculosis,  Mycobacterium species [Mycobacterium tuberculosis (M. tuberculosis), the main causative agent of tuberculosis (TB) in humans, TDM is a potent virulence factor involved in establishing the intracellular niche crucial for mycobacterial survival and growth in macrophages [M. tuberculosis with purified TDM is sufficient to temporarily restore ability to delay phagosome acidification and fusion with lysosomes, thereby promoting bacterial survival [M. tuberculosis [M. tuberculosis as mice deficient in activating FcγRs were more susceptible to M. tuberculosis infection than WT ones. In contrast, mice lacking the inhibitory FcγRIIB were more resistant to experimental TB [The mycobacterial glycolipid trehalose-6,6’- dimycolate (TDM) is an abundant component of the cell wall of all  species . In Mycorophages . Reconstsurvival . Simplifsurvival –4. The gsurvival ,6. Minclsurvival ,8. Upon survival –12. Howesurvival . Both Misurvival . Mycobacrculosis . Phagocyental TB .By employing IgG-opsonised beads as model to study FcγR-mediated phagosome biogenesis, we show herein that co-engaging Mincle and FcγR by its ligands, TDM and IgG, respectively, trigger an inhibitory cross talk, which decelerates maturation of phagosomes containing IgG-opsonised beads. TDM also requires both, SH2-domain-containing protein tyrosine phosphatase (SHP-1) and the inhibitory FcγRIIB to delay phagosome maturation. Thus, our study reveals that TDM uses an innate immune receptor along with inhibitory signalling to down-regulate phagosome biogenesis.Simplified bead models to study phagosome biogenesis revealed the ability of TDM from both, mycobacterial and closely related nocardial species, to decelerate bead phagosome maturation –4. We thTogether, these results show that TDM is able to delay maturation of phagosomes containing IgG-opsonised beads, which mediate phagocytosis through FcγR. These results extend the previously observed ability of TDM to interfere with phagosome maturation in macrophages to FcγR-mediated phagosome biogenesis ,3.-/- BMDM whose lysosomes had been labelled with dexTR were incubated with IgG and IgG TDM beads. TDM-mediated inhibition of IgG bead-phagosome maturation was completely abolished in the absence of Mincle 30 min following internalisation  under the license V312-72241.123–3 (24-2/07) or 102-8/09.BMDMs were differentiated from bone marrow cells of WT, Mincle or FcγRIIB KO C57BL/6 mice and the Raw 264.7 macrophage cell line was maintained as previously described .M. tuberculosis  as previously described [7 glycolipid-coated magnetic beads were incubated with 100 μg/ml mouse monoclonal anti-BSA antibody  for 1 h at RT or overnight at 4°C. Presence of TDM was confirmed by TLC as previously described .Magnetic Tosyl-activated beads  covalently coated with sterile endotoxin-free bovine serum albumin  were coated with TDM from escribed . 5×107 g7 cells/T75 flasks. Macrophages were incubated in D10 with opsonised beads at a multiplicity of 5 beads/cell. for 30 min at 37°C. Bead-phagosomes were isolated using magnetic separation as previously described [For β-Galactosidase assay, Raw 264.7 macrophages were seeded at 10escribed . Levels escribed .5 cells per well in triplicates on microscope chamber slides  in D10 supplemented with 100 μg/ml Dextran Texas Red (dexTR)  for 3 hours at 37°C and BMDMs were chased with fresh media, overnight at 37°C. Where indicated, macrophages were also incubated with 200 nM Syk inhibitor  or 11 μM SHP-1 inhibitor  for 30 min at 37°C in D10 prior to bead addition. Macrophages were incubated in D10 in technical triplicates with opsonised beads at a multiplicity of 5 beads/cell. Phagocytosis was allowed to proceed for the indicated time in D10 at 37°C. Cells were fixed with 2% PFA for 15 min at RT. Chamber slides were mounted with an aqueous-based mounting medium  for CLSM analysis. Fluorescence images were obtained with a Zeiss LSM 510 confocal microscope using the 543nm laser line with a 63X 1.3 NA objective set at pinhole diameter equivalent to 1–1.5 Airey units. Images were analysed using the Zeiss LSM software. Images were taken at 20x magnification. Following removal of the fluorescent background, percentages of beads co-localizing with lysosomes were determined by manually counting beads positive for dexTR staining in at least 50 macrophages per replicate.For CLSM, BMDMs were seeded at 107 BMDMs were transfected with 20 μg/ml SHP-1 siRNA  or control scramble siRNA  following manufacturer's instructions. The assessment of SHP-1 expression knock-down was performed by Western blot using specific SHP-1 antibody .SHP-1 expression was knocked-down with specific siRNA using the Amaxa Mouse Macrophage Nucleofactor Kit (Lonza Group). 10Data are presented as means ± interquartile ranges and are combined from 2–6 independent experiments done in triplicate. Kruskal-Wallis test followed by Dunns post-test was used for statistical analysis when multiple groups were analyzed. Mann-Whitney test were used for statistical analysis when two groups were analyzed.S1 FigRaw 264.7 macrophages were treated with or without either anti-FcγR antibody (2.4G2) or control isotype for 10 min followed by the addition of IgG beads (ratio 3:1) for 5 min. Non-internalised beads were differentiated from internalised beads using an anti-mouse Cy3 secondary antibody under non-permeabilising conditions. The graph shows quantifications by CLSM of the percentage of internal beads. Scale bar: 10 μm. Data are expressed as medians % of internalised beads and combined from two independent experiments ± interquartile ranges.(TIF)Click here for additional data file.S2 FigDexTR-labelled BMDMs (A) or Raw 264.7 (B) macrophages were incubated with IgG or IgG TDM beads (ratio 1:5) and phagocytosis was allowed to proceed for 10, 30 or 180 min. The graph (A) indicates the quantification by CLSM of the percentage of bead-phagosomes colocalising with dexTR. Beads were assessed in BMDMs for fusion with dexTR-labelled lysosomes by CLSM. The graph (B) indicates the relative β-galactosidase activity from purified bead-phagosomes. Data are expressed as medians % of dexTR-positive bead-phagosomes (A) or relative β-galactosidase activity (B) from a single experiment each done in triplicates ± interquartile ranges.(TIF)Click here for additional data file.S3 FigCoverslips were either coated with TDM  or left untreated before BMDM were seeded and labelled with DexTR. Macrophages were incubated with control BSA beads (ratio 1:5) for 30 min. The graph shows quantification by CLSM of the percentage of bead-phagosomes colocalising with dexTR. Data are expressed as medians % of dexTR-positive bead-phagosomes done in triplicates ± interquartile ranges.(TIFF)Click here for additional data file.S4 FigDexTR-labelled BMDMs macrophages were incubated with control BSA or TDM BSA beads (ratio 1:5) and phagocytosis was allowed to proceed for 30 min. The BSA used for coating beads in all experiments is endotoxin free. The graph indicates the quantification by CLSM of the percentage of bead-phagosomes colocalising with dexTR. Beads were assessed in BMDMs for fusion with dexTR-labelled lysosomes by CLSM. Data are expressed as medians % of dexTR-positive bead-phagosomes done in triplicates ± interquartile ranges.(TIF)Click here for additional data file.S1 FileExperimental procedures: 1. Assessment of bead phagocytosis; 2. Isolation of bead-phagosomes; 3. SHP-1 expression analysis by Western blot(DOC)Click here for additional data file.


Coronaviridae-wide conserved domains, suggesting that the GDS04 strain belongs to the same species as HKU2.We identified from suckling piglets with diarrhea in China a new bat-HKU2–like porcine coronavirus . The GDS04 strain of this coronavirus shares high aa identities (>90%) with the reported bat-HKU2 strains in  Several pathogens are thought to be responsible for porcine diarrhea, including porcine epidemic diarrhea virus (PEDV) . We inoculated each animal with 5 mL of excreta through the oral route. After 2 days, all inoculated animals exhibited similar clinical symptoms, including diarrhea and dehydration. We randomly selected 2 inoculated pigs in each group and performed necropsies on days 3 and 5 postinoculation, respectively. We filtrated homogenate of small intestine and intestinal contents, using the resultant supernatant for RNA extraction as described previously . The full length of the PEAV genome is 27,171 nt (excluding the poly-A tail); this size is similar to that of bat-like HKU2 strains of coronavirus (http://www.ncbi.nlm.nih.gov/nuccore/NC_028824.1). Both viruses are bat-associated viruses. However, the 2 strains are distributed in relatively different branches (O-ribose methyltransferase). These nonstructural proteins are Coronaviridae-wide conserved domains in replicase polyprotein pp1ab. A threshold of >90% in aa sequence identity suggests that 2 viruses are of the same species; our findings suggest that the GDS04 strain belongs to the same species as HKU2.Our phylogenetic analysis based on the whole genome of GDS04 and representatives of 4 established coronavirus genera, including the human and bat-like coronaviruses, demonstrated that GDS04 clusters with bat-like coronaviruses . Accordibranches . Based oFurthermore, we designed specific primers of the n gene for the detection of the GDS04 strain. By using reverse-transcription PCR with these primers, we found that 97 out of 308 clinical intestinal or fecal samples were positive for GDS04. We collected all clinical samples from 25 farms in Guangdong Province during February–April 2017 and used 20 samples collected from healthy vaccinated piglets as negative controls.In summary, we report preliminary data on our detection of a new coronavirus-like virus, PEAV. PEAV is thought to be responsible for the most recent diarrhea endemic in pig herds in southern China. Virus isolation and serologic testing are underway. The outbreak of the newly discovered virus arose among swine with severe diarrhea in swine breeding farms in southern China, suggesting the regional outbreaks of diarrhea could contribute to the emergence of new pandemic viruses. Extensive surveillance for GDS04 PEAV is required to define its epidemiology and evolution.


To the Editor: Previous studies have reported avian influenza virus (AIV)–positive serum samples obtained from Adélie (Pygoscelis adeliae), chinstrap (Pygoscelis antarcticus), and gentoo (Pygoscelis papua) penguins , Arctowski Base , and Bernardo O’Higgins Base  and identified 5 serum samples positive for influenza. We also collected 513 cloacal swabs from Adélie, chinstrap . This virus was subtyped as an H5N5 and named A/chinstrap_penguin/Antarctica/B04/2015 (H5N5). Analysis of its cleavage site confirmed this was a typical low pathogenicity AIV (LPAIV) containing cleavage motif PQRETRGLF  in Chile (Stercorarius maccormicki), might represent natural reservoirs that play a role in the introduction and maintenance of AIVs into Antarctica.The timing of arrival of migratory birds that breed in Antarctica  overlaps with that of the penguins as they return to colonies for breeding and nesting during the summer in the Southern Hemisphere. These birds share a habitat, enabling close contact  and an unidentified shore bird at Delaware Bay . This finding suggests a plausible entryway of this gene into Antarctica from South America through the Atlantic or Pacific-American flyway, which are common routes used by shore birds, such as the ruddy turnstone, white-rumped sandpiper , and red knot  (The chinstrap penguin neuraminidase segment clustered within a Eurasian N5 clade that includes sequences from 2001–2010 Figure 3.As previously suggested for H11N2 viruses from Antarctica, our data supports the idea that these AIVs are evolutionarily distinct from other AIVs (The positive animals we identified originated from a single location on the Antarctic Peninsula, which suggests recent introduction of this AIV H5N5 in the colonies sampled. Antarctica is refuge for most penguin colonies, including the near-threatened emperor penguins. Previous reports suggested that AIV could have caused Adélie penguin chick death (These data provide novel insights on the ecology of AIV in Antarctica. Our findings also highlight the need for increased surveillance to understand virus diversity on this continent and its potential contribution to the genetic constellation of AIV in the Americas.Phylogenetic analyses of hemagglutinin and neuraminidase gene segments of avian influenza virus H5N5 obtained from chinstrap penguin in Antarctica in 2015, showing North American and Eurasian origins.


This paper presents a deterministic bead-in-droplet ejection (BIDE) technique that regulates the precise distribution of microbeads in an ejected droplet. The deterministic BIDE was realized through the effective integration of a microfluidic single-particle handling technique with a liquid dispensing system. The integrated bead dispenser facilitates the transfer of the desired number of beads into a dispensing volume and the on-demand ejection of bead-encapsulated droplets. Single bead–encapsulated droplets were ejected every 3 s without any failure. Multiple-bead dispensing with deterministic control of the number of beads was demonstrated to emphasize the originality and quality of the proposed dispensing technique. The dispenser was mounted using a plug-socket type connection, and the dispensing process was completely automated using a programmed sequence without any microscopic observation. To demonstrate a potential application of the technique, bead-based streptavidin–biotin binding assay in an evaporating droplet was conducted using ultralow numbers of beads. The results evidenced the number of beads in the droplet crucially influences the reliability of the assay. Therefore, the proposed deterministic bead-in-droplet technology can be utilized to deliver desired beads onto a reaction site, particularly to reliably and efficiently enrich and detect target biomolecules. Over the recent decades, bead-based assay methodologies have had a noticeable impact on the detection of biological and chemical analytes134Inkjet-based particle dispensing has been widely used as it enables on-demand and noncontact delivery to various substrates and containers without the risk of cross-contamination. In this technique, the printing suspension is prepared through dilution to control the number of particles in a dispensed droplet10111314151618In this paper, we propose a plug-in bead dispenser that uses a deterministic bead-in-droplet ejection (BIDE) mechanism through which a bead is actively transferred into the dispensing liquid volume via a microfluidic pneumatic valve20The plug-in bead-dispensing module is composed of a liquid cartridge and a plug-in bead dispenser. The cartridge comprises female sockets, liquid reservoirs, detachable caps, and solenoid valves , and theThe deterministic BIDE mechanism  consistsThe plug-in bead-dispensing module comprises a liquid cartridge and a plug-in dispenser. The acrylic cartridge is machined via computer numerical control (CNC) milling. The four female sockets at the front of the cartridge are 5.0 mm deep, with outer and inner diameters of 7.0 and 3.0 mm, respectively. A through-hole path (diameter 1 mm) was fabricated at the center of each socket to form the liquid channel connecting the liquid reservoirs and the dispenser. To prevent the sedimentation of the introduced beads because of gravity, the female socket connected to the inlet reservoir has a through-hole path that is smaller (diameter 0.5 mm) than that of the other sockets. Two three-way air solenoid valves  were mounted on the left and right sides of the liquid cartridge. One of the valves applies a pressure pulse to actuate the microfluidic pneumatic valve in the dispenser, and the other applies a pressure pulse to dispense the liquid.A replica molding techniqueA polystyrene bead suspension  was diluted with a phosphate-buffered saline solution  containing 0.5% (v/v) Tween 20 (Sigma-Aldrich) surfactant to reduce bead aggregation and nonspecific binding between the PDMS surface and polystyrene beads. For the dispensing test, suspensions of 50,000, 200,000, and 600,000 beads/mL were prepared. To effectively visualize the dispensed beads in the ejected droplet, a suspension of fluorescent beads  in deionized (DI) water with 1% (v/v) Tween 20 was used. For the streptavidin–biotin binding test, streptavidin-coated polystyrene beads  were mixed with the DI water, and suspensions of 100,000 and 600,000 beads/mL were used, and a 100-μM Cy3-labelled biotin solution (Bioneer) was diluted with DI water to prepare 4, 8, 16, 32, 64, and 128 nM biotin solutions.in-situ monitoring system, which includes a high-speed camera  with a X20 objective lens  and a 150 W halogen light source , and an t source . A pneumBefore all experiments, liquid reservoirs of the dispensing module and microfluidic liquid channels were filled with the intact PBS solution through micropumping and without any bubble trapping that could cause malfunctioningTo investigate the capturing performance of biotin molecules as a function of the number of beads in a given reaction volume, we first dispensed droplets encapsulating different numbers of streptavidin beads on the cover glass via the proposed bead dispensing system to dispense single beads and the manual pipetting to dispense multiple beads, respectively. After evaporation of the droplet, the streptavidin beads remained; then, the Cy3-labelled biotin solution was manually pipetted onto the streptavidin beads. The streptavidin bead surface was enriched with biotin molecules through internal flow and diffusion during evaporation. Finally, the buffer droplet was put on the reacted bead to wash excessive residue not bound to the streptavidin bead, following which the emitted fluorescence signal was measured. This procedure was repeated with 4, 8, 16, 32, 64, and 128 nM biotin solutions and with different numbers of streptavidin beads. Two hundred nanoliters of streptavidin bead suspension with different concentrations of 100,000 and 600,000 beads/mL was manually pipetted to load different numbers of beads on each cover glass; on average, 12.4  and 79.4  beads were loaded at these two concentrations, respectively.in-situ monitoring using a high-speed camera . We first defined the required times for each operation mode. Images from the high-speed camera shown in Trapping time is defined as the time until the trapping of a following bead after the release of a trapped bead. Therefore, trapping time depends on the flux of the influent beads and is thus a function of the flow rate and bead concentration. We measured the trapping time at various bead concentrations at the optimized operation condition . To reduce operating time, we followed “trap–release–dispense–load” mode sequence rather than the “trap–release–load–dispense” sequence . This chTo the best of our knowledge, this is the first report of a deterministic bead-in-droplet technology to accurately control the encapsulated bead number at low bead numbers  and within a fixed droplet volume. By repeatedly switching the pneumatic valve, the desired number of beads was loaded sequentially and ejected onto a cover glass. Beads in a dispensed droplet were aggregated during its evaporation, and the phenomenon facilitated the detection and counting of dispensed beads . For effAs a practical application of our bead dispensing system, affinity-based capturing and enrichment of biotin molecules was demonstrated using streptavidin-coated beads in an evaporating droplet. During droplet evaporation, collision and binding between biotin molecules and streptavidin beads occur effectively through diffusion and internal flowA deterministic bead-in-droplet dispensing system with a plug-in microfluidic bead dispenser was developed through the effective integration of on-demand droplet dispensing and microfluidic particle trap-and-release technologies. For easy and reversible integration of the dispensing module, the plug-and-socket connecting method was used. Furthermore, the operating conditions  of the system were optimized, and minimal consumption of a 30 μL bead suspension was realized to facilitate economical operation. The entire process of the deterministic BIDE mechanism was automated using a programmed sequence, without any visual observation, for increasing the practicability of the proposed technique. Using a 600,000 beads/mL bead suspension, bead-encapsulated droplets were continuously ejected every 3 s for an optimized interval  without any failure to dispense single beads. In addition, the encapsulated number of beads in a dispensing droplet was deterministically regulated by appropriate switching of the microfluidic valve. Finally, an ultralow, bead-based streptavidin–biotin binding test via droplet evaporation demonstrated the novel, sensitive, and reliable detection of target molecules by using the proposed technique. With advances in synthesis and functionalities of beads, the proposed bead dispensing system can be applied to improve various parameters  of bead-based applications.How to cite this article: Kim, H. et al. Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead–based applications. Sci. Rep.7, 46260; doi: 10.1038/srep46260 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We find that the stack order of the bilayer heterostructure plays a key role in the interfacial coupling strength, and the coupling at the LSMO(top)/LNO(bottom) interface is much stronger than that at the LNO(top)/LSMO(bottom). Moreover, a strong spin glass state has been observed at the LSMO/LNO interface, which is further confirmed by two facts: first, that the dependence of the irreversible temperature on the cooling magnetic field follows the Almeida-Thouless line and, second, that the relaxation of the thermal remnant magnetization can be fitted by a stretched exponential function. Interestingly, we also find an exchange bias effect at the LSMO/LNO bilayer below the spin glass freezing temperature, indicating that the exchange bias is strongly correlated with the spin glass state at its interface.We study the magnetic properties of an epitaxial growth bilayer composed of ferromagnetic La The dl LaNiO3 , 15. Fortransfer . Peng ettransfer , 17. Whetransfer . Therefo0.7Sr0.3MnO3 and paramagnetic metal LaNiO3 bilayer with a width of dozens of nanometers. As reported in previous works, LNO is the only member of the perovskite nickelates family lacking any magnetic order in its bulk form [In this paper, we report the experimental results of the relatively thick ferromagnetic half-metal Laulk form , 20. Thr2-terminated SrTiO3 (100) substrate which was set-etched with buffered HF acid. The samples were deposited by pulsed laser deposition (PLD) that could be monitored in situ, assisted with reflection high-energy electron diffraction (RHEED). The deposition was done using a 248-nm KrF excimer laser at a temperature of 725 °C, and oxygen pressure of 100 mTorr. After the growth, the samples were in situ annealing for 1 h in an oxygen pressure 300 Torr. In this work, we prepared four different types of samples using 25-nm LSMO and 35-nm LNO single layer films, a LNO (35 nm)/LSMO (25 nm) bilayer where LNO is the top layer and a LSMO (25 nm)/LNO  bilayer reversing the deposited sequence. The structure quality and orientation of the samples were analyzed by X-ray diffraction (XRD) using Cu Kα radiation. The surface morphology of the substrate was measured by atomic force microscopy (AFM). The magnetic properties of the samples were measured using vibrating sample magnetism (PPMS-VSM) and an applied magnetic field parallel to the sample plane.To obtain high quality epitaxial films, all the samples were grown on an atomically flat TiOc-axis orientation properties. In the LNO/LSMO and LSMO/LNO bilayers, the diffraction peaks of the LSMO layer are inconspicuous and may be overlapped with those of the substrates and the LNO layers. The perpendicular c-axis lattice parameters of the LNO and LSMO single layers are calculated as 3.841 and 3.865 Å respectively, which is smaller than their bulk values. Thus, both the LNO and LSMO layers deposited on STO substrates sustain an in-plane tensile strain. It is obvious that the peaks of LNO in bilayers have a slight shift to the right compared with those in the corresponding single layer, which is caused by the additional tensile strain by the LSMO layer.All the samples exhibit epitaxial growth, for instance, the RHEED patterns of the STO substrate and the LSMO/LNO bilayer at the end of growth are shown in Fig. MS) of 360 emu/cm3. The coercivity of the LSMO single layer is 115 Oe. The coercivity slightly increases to 160 Oe for the LNO/LSMO bilayer with LNO on the top, and increases dramatically to 401 Oe for the LSMO/LNO bilayer with LNO at the bottom. Moreover, the saturation magnetization of the LNO/LSMO bilayer is almost same as the LSMO single layer and clearly decreases for the LSMO/LNO bilayer. This indicates that the stack order of bilayer heterostructure plays a significant role in the interfacial coupling strength. However, Peng et al. report that the LNO/LSMO bilayer with LNO on the top exhibits strong interfacial coupling, which is not obvious in our experiment [HC increases and the MS decreases as the LNO layer thickness increases, as shown in Fig. The hysteresis loops of the four samples measured at 5 K are shown in Fig. periment , which oHC and decrease of MS, we measure the temperature dependent magnetization (M–T) curves from 0.05 to 1 kOe for the bilayer of LSMO (25 nm)/LNO (35 nm) with an in-plane magnetic field, shown in Fig. TP) before reducing monotonically. The negative magnetization in the ZFC curves measured in low applied fields may originate from an intrinsic effect of uncompensated spins [Tirr) also appears to be a bifurcation between the ZFC and FC curves. Based on these phenomena, we analyze the result usually observed for the several commonly known magnetic systems, such as spin glass [TP and Tirr are very close to each other for all applied magnetic fields, and TP shifts to low temperatures quickly as the measurement field increases. This is characteristic of the spin glass state and indicates that the spin glass is suppressed by a strong magnetic field [Tirr on field cooling should follow the Almeida-Thouless (AT) line [In order to clarify the origination of the strong coupling resulting from the increase of  nm)/LNO 5 nm withed spins . The irrin glass , 23, spiin glass , 25, andin glass . Here, Tic field . AccordiAT) line , 28:Fig.J is the width of the distribution of the exchange interaction and TF is the zero field spin glass freezing temperature. The linear fit to the experimental data is shown in the inset of Fig. TF as 203 K. As Ding et al. reported, the spin glass state in the (FM) LSMO and (G-AFM) SrMnO3 bilayer exhibits relaxation of the thermal remnant magnetization (RTRM) below the spin glass freezing temperature [TF, and apply the stretched exponential function to fit the decay curves at different temperatures:where the parameter Δperature . Accordiω is the relaxation frequency, 8.5 × 10−5 s−1, and C is the exponential factor, 0.34 [n is determined to be 0.826 at 180 K and 0.656 at 150 K, which is similar to the values of LSMO/SMO [TF, the relaxation characterization is not observed. From these results, we suggest that the magnetic relaxation and glassy behavior are most prominent near the freezing temperature of TF in the LSMO/LNO bilayer.where the parameter or, 0.34 . In Fig.LSMO/SMO . As the eg orbits of the Mn and Ni ions should occupy the x2–y2 in LSMO/LNO and LNO/LSMO bilayers [z2–r2 orbits [The spin glass state in the LSMO/LNO bilayer can be linked to the competition at the interfacial magnetic moment. Because the tensile stress can be existent in both LNO and LSMO layers when they are grown on the STO substrate, the bilayers . Neverth2 orbits . This va2 orbits . OtherwiHEB and HC dependent on different cooling fields. It can be seen that the HEB increases rapidly to 35 Oe as the cooling field increases to 1 kOe, and then decreases monotonically to 5 Oe as the cooling field reaches to 70 kOe. The HC has a similar trend. It should be noted that there is competition between the spin glass order and the Zeeman coupling, and, in fact, a strong enough magnetic field can destroy the spin glass state entirely [HEB and HC for the LSMO/LNO bilayer are shown in Fig. HEB decreases rapidly with increasing temperature, finally vanishes at the blocking temperature (TB) of 120 K. As TB gets smaller than the freezing temperature TF of the spin glass, we suggest that the exchange bias effect in the LSMO/LNO interface is supported by the emergence of the spin glass state. As previously reported, in a range of diverse materials such as LSMO/SMO, the existence of the spin glass state is known to lead an exponential temperature dependent decay of HEB and HC [HEB and HC by the phenomenological formula:The magnetic hysteresis loops of the LSMO/LNO bilayer measured at 5 K after ±5 kOe field cooling from room temperature are shown in Fig. entirely . The tem and HC , 33–35. T1 and T2 are constant. The perfect fitting results give further support that exchange bias in the LSMO/LNO bilayer is controlled by the spin glass state.where In summary, the interfacial coupling strength can be influenced by the deposition sequence of the bilayer. The coercivity of the bilayer with LNO at the bottom is much higher than that with LNO on the top, indicating that there is a stronger coupling between the interfacial LSMO/LNO bilayer. This strong coupling is due to the presence of a spin glass state in LSMO/LNO bilayer, which is supported by the field dependence of the irreversibility and the magnetic relaxation. Moreover, the temperature and cool field dependence of the exchange bias is attributed to the competition between the spin glass state, thermal disorder, and Zeeman coupling. The interface engineering of the PM/FM heterostructure is promising for inducing many novel physical phenomena.


The ideal balance between viral oncolysis and immune stimulation is unknown yet very consequential. Cytolysis from virus replication exposes previously unexposed cancer antigens to immune effector cells which are attracted to the area by the virus in the context of a danger response and can attack the tumor; however the immune cells can also remove the virus, and thereby limit viral oncolysis.Oncolytic herpes simplex virus (oHSV) therapy offers great promise at targeting difficult-to-treat solid tumors as evidenced by the first FDA approval of an oncolytic virus, modified HSV-1 talimogene laherparepvec  to treat advanced melanoma [Researchers have explored combination approaches with oHSVs including various chemotherapeutics and low-dose radiation to maximize both viral oncolysis and the effect of the added therapy. In most combination studies with chemotherapy drugs, apoptosis was increased but viral replication was not enhanced -4. Interin vivo thereby prolonging oncolysis possibly by preventing the influx of virus-mediated NK cells that may limit virus replication. 3) Alisertib prevented the increase of monocyte myeloid derived suppressor cells (MDSCs) induced by HSV1716. MDSCs have been shown to be pro-tumorigenic. Taken together, these exciting results demonstrate how a combination strategy may be used to not only improve the effectiveness of oncolytic virotherapy but also increase the efficacy of the combined drug. Further studies are needed to determine if there is one principal mechanism resulting in the increased antitumor effect or if each of these findings (and perhaps others not yet described) combined are responsible for the outcomes. Additionally, the generalizability of these findings to other models will be interesting to investigate.In a recent issue of Oncotarget, Currier et al. describe the synergistic effects of combining Aurora A Kinase inhibitor alisertib (MLB8237) with HSV1716, a first-generation oHSV currently in phase I clinical trial in children with non-central nervous system solid tumors (NCT00931931), in neuroblastoma and malignant peripheral nerve sheath cell lines [In addition to maximizing virus replication and the effect of an added agent, a critical challenge remains exploiting the immune system to create a sustained anti-tumor response. The combination of HSV1716 and alisertib removed unfavorable MDSCs which are known to promote pro-tumorigenic M2 macrophages. Additional opportunities exist with adjuvant immunotherapies such as natural killer (NK) cell therapy and checkpoint molecule inhibitors. Recently, researchers demonstrated synergy between bortezomib, a proteasome inhibitor, and oHSV in causing inflammatory necroptotic tumor cell death [


Culicoides-borne orbiviruses, such as bluetongue virus (BTV) and African horse sickness virus (AHSV), are important pathogens that cause animal epidemic diseases leading to significant loss of domestic animals. This study was conducted to identify Culicoides-borne arboviruses and to investigate the associated infections in local livestock in Yunnan, China.Culicoides were collected overnight in Mangshi City using light traps during August 2013. A virus was isolated from the collected Culicoides and grown using baby hamster kidney (BHK-21), Vero, Madin-Darby bovine kidney (MDBK) and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by polyacrylamide gel (PAGE) analysis. A full-length cDNA copy of the genome was amplified and sequenced. Serological investigations were conducted in local cattle, buffalo and goat using plaque-reduction neutralization tests.Tibet orbivirus (TIBOV). Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), sub-core-shell  proteins revealed that DH13C120 clustered with TIBOV, and the amino acid sequences of DH13C120 virus shared more than 98% identity with TIBOV XZ0906. However, outer capsid protein VP2 and outer capsid protein VP5 shared only 43.1 and 79.3% identity, respectively, indicating that the DH13C120 virus belongs to TIBOV, and it may represent different serotypes with XZ0906. A serosurvey revealed the presence of neutralizing antibodies with 90% plaque-reduction neutralization against TIBOV DH13C120 in local cattle (44%), buffalo (20%), and goat (4%). Four-fold or higher levels of TIBOV-2-neutralizing antibody titers were detected between the convalescent and acute phases of infection in local livestock.We isolated a viral strain (DH13C120) that caused cytopathogenic effects in BHK-21, Vero, MDBK and C6/36 cells. Suckling mice inoculated intracerebrally with DH13C120 showed signs of fatal neurovirulence. PAGE analysis indicated a genome consisting of 10 segments of double-stranded RNA that demonstrated a 3–3–3–1 pattern, similar to the migrating bands of Culicoides. This study provides the first evidence of TIBOV infection in livestock in Yunnan, China, and suggests that TIBOV could be a potential pathogen in livestock.A new strain of TIBOV was isolated from  Orbivirus within the family Reoviridae has a genome consisting of 10 segments of double-stranded (ds) RNA. It contains 22 recognized virus species and 10 unclassified viruses [Equine encephalosis virus (EEV), Epizootic hemorrhagic disease virus (EHDV), Palyam virus (PALV) and Peruvian horse sickness virus (PHSV) are important pathogens causing epidemic diseases in animals. In these orbiviruses, although PHSV is mosquito-borne, most of these viruses are carried by Culicoides [Culicoides is considered a potent vector of important animal arbovirus diseases, which cause major economic losses in domestic animals [The viral genus licoides . Therefo animals –6.Anopheles maculatus mosquitoes collected in 2009 from Motuo County, Tibet, China [Culex fatigan mosquitoes and Culicoides specimens collected from Guangdong and Yunnan, respectively [Culicoides. Sequence analysis of its entire genome and evidence of DH13C120 infection in local livestock were obtained from samples collected in the southwest border area of Yunnan Province in 2013.The TIBOV was first isolated from t, China . Subsequectively . Here, wCulicoides, killed by freezing at −20 °C for 20 min and identified by using morphologic characteristics, were pooled into groups of approximately 100 insects and stored in liquid nitrogen until viral isolation.Specimens were collected during August 2013 from the suburb of Manbing Town in Mangshi City, Dehong Prefecture, Yunnan Province. Samples were collected overnight using light traps . Captured Culicoides pools, which included midges that were engorged or not engorged, were removed from liquid nitrogen and immediately homogenized and centrifuged as reported previously [Aedes albopictus (C6/36) cells  were inoculated at 28 °C. The cells were observed daily (days 1–7 post-inoculation) for cytopathic effects (CPEs).eviously . The supAliquots of 30 μL cell culture material  were inoculated intracerebrally into 1-day-old suckling mice, and 30 μL of Dulbecco’s modified Eagle’s medium (DMEM) was similarly applied in the control group, with eight suckling mice in each group. Symptoms (or the time of death) in the virus-treated neonatal mice were recorded each day for 14 days post-inoculation.To reveal the number of dsRNA segment and the genome pattern, viral RNA was extracted using RNAiso Plus  according to the manufacturer’s protocols, and then separated on RNA-PAGE as described previously –11.The virus genome was amplified by full-length amplification of cDNA (FLAC), as described previously , 12, 13.Escherichia coli DHα5 cells (TaKaRa). Single colonies were cultured and sequenced by the Shenzhen Huada Genome Institute, Shanghai, China. We performed 2 sequencings from the same RNA extraction and these sequences (that include the differences) were cover 2.5 times.An anchor primer, PC3-T7 loop , similar to that described by Maan et al. , was lighttp://www.directoryofshareware.com/preview/clustalx/) and MegAlign (DNASTAR Inc.). Phylogenetic and evolutionary analyses were conducted using MEGA 5.1 (http://www.megasoftware.net/) based on the neighbor-joining technique and 1,000 bootstrap replications [Initial sequence assembly and analyses were conducted using the DNAStar software package . Homology and alignment analysis was performed using Clustal X (ver. 2.1)  . The neutralizing antibody titer was identified as the highest serum dilution that reduced the number of virus plaques in the test by ≥ 90%. The samples were considered to be positive when titers were ≥ 20. A ratio of TIBOV PRNT 90 titer between two samples of a same animal ≥ 4 was considered to confirm the presence of a TIBOV infection [Serum samples were tested for neutralizing antibodies against TIBOV isolate DH13C120 by 90% plaque-reduction neutralization tests (PRNT 90) using standard methods. Samples were tested with serial two-fold dilutions from 1:10 to 1:1,280. Diluted samples were mixed with equal volumes of culture medium 1× MEM (pH = 7.4) containing DH13C120 (100 PFU) and incubated at 37 °C in a 5% COnfection .Culicoides were processed to isolate viruses in BHK-21, Vero and C6/36 cells. A CPE was observed with one pool (DH13C120) collected from Dehong. Isolate DH13C120 caused CPEs in BHK-21, Vero, MDBK and C6/36 cells at 48, 48, 144, and 120 h post-infection, respectively. The characteristic CPE in BHK-21, Vero and C6/36 cells infected with DH13C120 was rounding and desquamation, whereas that in MDBK cells showed vacuolization and desquamation.Suckling mice inoculated intracerebrally with DH13C120 showed signs of tremor and stiff necks at 48 h, and died within 72 h.Separation by PAGE revealed that DH13C120 is a 10-segment dsRNA virus, with a segment pattern of 3–3–3–1 , and 4 nt in the terminal sequences were reverse complementary. The 5′ and 3′ UTRs of DH13C120 comprised 3.80% of the total genome; the G + C content of DH13C120 was 42.59%, similar to Culicoides-borne Orbivirus species; and an Arg–Gly–Asp (RGD) motif [A full length cDNA copy of the DH13C120 virus genome was obtained by FLAC and sequencing, with the length of segments 1–10 ranging between 3,740 nucleotides (nt) (Seg-1) to 788 nt (Seg-10) (Table D) motif –17 was oFengkai virus), which was above the threshold of 91% used for species determination by Attoui et al. [The sub-core-shell (T2) (VP3) protein of DH13C120 is encoded by segment 3. Phylogenetic analvysis showed that DH13C120 has a close relationship with TIBOV XZ0906, YN12246  and Fengkai virus. Amino acid (aa) sequence analysis of the T2 protein revealed 96.3–99.9% sequence similarity between DH13C120 and the TIBOV species  revealed that the DH13C120 virus isolated from Culicoides in Yunnan and the Fengkai virus isolated from mosquitoes in Guangdong  had sequence similarities of 96.8%/97.1% nt/aa, whereas XZ0906 isolated from mosquitoes in Tibet displayed similarities of 53.9%/43.1% nt/aa  protein of TIBOV isolated from China  (VP1), T2 (VP3), and T13 (VP7) , and those transmitted by ticks  or mosquitoes . Phylogenetic analysis showed that DH13C120 has a close relationship and similar coding pattern to other Culicoides-borne Orbivirus species. The Pol, T2, and T13 proteins of DH13C120 share 58–71.8%, 57.6–77.8%, and 40.6–68.2% aa sequence identity with the Culicoides-borne Orbivirus species, respectively, and 47%, 38%, and 23.6% aa identity with mosquito-borne YUOV, respectively.To clarify the taxonomic position of DH13C120 with other A total of 135 serum samples were collected from cattle, buffaloes and goats in Mangshi City, Jiangcheng and Shizong County of Yunnan. Neutralizing antibodies against DH13C120 virus were detected by PRNT 90 assays. Most of the positive samples were collected in Jiangcheng County, with the highest seroprevalence (90%) in cows. Additionally, positive sera were collected in Mangshi City (27%) and Shizong County (10%), but positivity rates in both areas were much lower than those in Jiangcheng County  [St Croix River virus). These data suggested that DH13C120 virus is more or most related to viruses vectored by Culicoides spp.Previous studies have shown that the noncoding region (NCR) comprises 5.03–5.695% of ne group . Analysi in EEV) , 22, butCulicoides-borne Orbivirus species, such as BTV, EHDV, and AHSV, a highly conserved RGD motif was present at position 168–170 of VP7 , which could play a role in the attachment of core particles to Culicoides cells [Fengkai virus, reflecting its closer overall similarity to BTV and EHDV, and potentially implicating Culicoides spp. as biological vectors.For es cells –17. An ROrbivirus were first isolated from ticks, birds, rats, and livestock in the Yadong and Cuona areas of Tibet, with typical CPE in BHK-21 cells and lethal illness in suckling mice. Antibodies against the Orbivirus isolates were detected in local residents with a seroprevalence of 26%, suggesting that the new Orbivirus isolates could potentially be a novel animal pathogen in China [Culicoides in Yunnan, Guangdong Province and Tibet [Culicoides in Yunnan, and high levels of neutralization antibodies were detected in cattle serum from three areas in Yunnan Province. Moreover, neutralization antibodies against DH13C120 were detected in target animals at all three monitoring sites, with the highest positivity rate being 92%. In addition, neutralization antibody titers that increased more than four-fold were detected in target cattle, and sheep inoculated with DH13C120 experienced fever (data not shown), suggesting that DH13C120 can infect livestock and is a potential pathogen leading to livestock disease.In 1995, 17 strains of in China , 30. Recnd Tibet . In the Culicoides collected in cattle corrals, and neutralization antibodies were detected in animals from the same areas. Seroconversion in animals occurred from June to September, which matched the seasonal peak of Culicoides numbers. These results suggest that Culicoides is the main vector of this new serotype of TIBOV, and cattle form the natural host reservoir. The cattle in these localities have high economic value, and most cattle are bred together in herds numbering over 100. The density of Culicoides was high in these cattle corrals, facilitating circulation of the new serotype. To understand the impact of this new TIBOV serotype, further studies are required to characterize its dissemination and pathogenicity.In this study, DH13C120 was isolated from Culicoides, and provides the first evidence of TIBOV DH13C120 strain infections in local livestock bred in the southwest border area of Yunnan, China. The results suggest that TIBOV isolated from Culicoides in yunnan, might be a pathogen causing livestock disease.This investigation represents the isolation of TIBOV from


We tested blood samples from domestic and captive farmed animals in Minnesota, USA, to determine exposure to severe fever with thrombocytopenia syndrome virus and Heartland-like virus. We found antibodies against virus nucleoproteins in 10%–18% of samples from cattle, sheep, goats, deer, and elk in 24 Minnesota counties. Phlebovirus (Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease in China, caused by a novel bunyavirus in the genus Many bunyaviruses can infect animals  antibodies cross-react with HLV NP and decided to use SFTSV NP antibody detection kits for detecting antibodies against SFTSV- or HLV-like viruses. A standard ELISA reagent kit, developed by Jiangsu Centers for Disease Control (>10 (Antibodies were detected in serum samples from 64 (15.5%) of 414 cattle, 10 (10.9%) of 92 goats, 6 (12.5%) of 48 sheep, 35 (11.8%) of 296 white-tailed deer, and 7 (18.0%) of 39 elk (17.9%) . Thirty->10 . Specifi>10 .Our data show that both domestic and captive farmed animals in Minnesota were exposed to SFTSV- or HLV-like virus, as evidenced by the presence of antibodies reactive against the NP of SFTSV at a prevalence ranging from 10% to 18%. Because SFTSV and HLV are closely related  (Orthobunyavirus is transmitted by mosquitoes, and rodents and foxes can be infected as amplifying hosts during seasonal outbreaks (Vertebrate animals are amplifying hosts for many arboviruses, which have seasonal epidemics (–SFTS epidemics in China are seasonal and occur from late March through early November. Infection rates among humans and animals rise in early March, peak in August, and decrease after November (Evidence that a novel phlebovirus infects domesticated and captive farmed animals as shown in this study validates the concern that an SFTSV- or HLV-like emerging pathogen could pose a serious public health threat in the United States. Epidemiologic studies with a broader scope need to be conducted to elucidate viral ecology, and effective measures must be adopted to control this virus before it spreads among humans.


The background-subtracting measure k = 6 gave the highest host prediction accuracy  with reasonable computational times. Requiring a maximum dissimilarity score for making predictions (thresholding) and taking the consensus of the 30 most similar hosts further improved accuracy. Using a previous dataset of 820 bacteriophage and 2699 bacterial genomes, Viruses and their host genomes often share similar oligonucleotide frequency (ONF) patterns, which can be used to predict the host of a given virus by finding the host with the greatest ONF similarity. We comprehensively compared 11 ONF metrics using several  It is widely recognized that the ‘uncultured majority’ of bacteria and archaea (prokaryotes) dominate biomass in many ecosystem, control important global biogeochemical cycles, and significantly impact the health of humans, animals, and crops . Much leTwo basic but critical components of understanding the biology and impact of viruses on their hosts are characterizing the extant diversity of viruses and understanding what hosts different viruses infect. For many ecosystems, knowledge of these two components is still quite limited. The long-standing approach for assessing viral diversity has been isolation of new viruses using cultured host strains. The isolation approach while it also directly informs what host the virus can infect is low throughput and requires robust growth of the target host strain, which often is not possible for many bacteria and archaea . Virus iMetagenomic sequencing and assembly of viral contigs, however, provide a powerful, high throughput and culture-independent means to identify new viruses ,10. SeveSeveral computational approaches have been recently developed to discern which host(s) particular viruses likely infect based on sequence analysis. These different methods have recently been reviewed and evaluated in  and fallAbundance profiling and sequence composition methods differ from the aforementioned approach in that they are not dependent on sequence homology. These methods show promise in that they exhibit a strong signal for predictions, but as implemented to date they have yielded lower accuracies compared to homology methods (e.g. 25% at the genus level) . The abuk-mers) with their hosts. Since virus replication is dependent on translational machinery of its host, selection on the virus to adopt the codons used by its hosts is thought to cause amelioration of virus nucleotide composition to that of its host , but found at a much higher accuracy of >80% across the genus, family, and order levels. It is unclear if the large difference in reported accuracies between Edwards et al. and Roux et al. is caused by the different distance measures or virus and host datasets used (see Discussion), and this warrants further investigation.ONF differences between host and viral sequences have been most frequently evaluated as the Euclidean or Manhattan distance between the ve hosts , and recve hosts ,21. Edwa . Thend model –24. Despk-mer lengths for predicting virus–host interactions. To evaluate prediction accuracy, we used a benchmark dataset of 1427 RefSeq virus isolate genomes available at the National Center for Biotechnology Information (NCBI) for which the host on which each was isolated is reported. We then computed ONF distances/dissimilarities of ∼32 000 possible bacterial and archaeal host genomes at NCBI. In addition to making predictions by simply selecting the n hosts with lowest dissimilarity to the query virus, we evaluated a consensus method, imposed a dissimilarity threshold for making predictions and rank-sum test approaches for picking the predicted host taxonomy. Overall the k-mer length. Application of the In this study, we have conducted a comprehensive evaluation of 11 oligonucleotide distance/dissimilarity measures over various Genus species strain_name, Genus sp. strain_name, Genus species serovar serovar_name). This smaller dataset therefore consisted of 352 pairs of viral genomes and the genome of the specific host on which they were isolated. The taxonomy of the hosts on which the 1427 viruses were isolated was collected from NCBI. Host predictions were made using a database of 31 986 complete and draft bacterial and archaeal genomes downloaded from NCBI on 5/5/2015, and their taxonomies were also collected. The accession numbers and taxonomies for viral and host genomes used are provided in the supplemental table, Supplemental_table_virus_and_host_genomes.xlsx. The viral contigs and host genomes and their associated taxonomies used previously in Roux et al.  was used to generate a list of genera that were isolated from marine habitats. This list was used to select members from the 31 986 host genomes that belong to those marine genera. We further removed host genomes for particular species that are primarily found in non-marine habitats such as Pseudomonas aeruginosa and Bacillus subtilis.For some analyses, the viral isolates and host genomes were restricted to those that primarily are found in marine habitats. NCBI genome files frequently do not indicate the source habitat from which viruses or hosts were isolated, so habitat information curated for microbial genomes at Integrated Microbial Genomes  of the measures tested are provided in the Supplementary Material available online at NAR, including supplemental references for distance measures. We briefly describe the different measures here. For measures that do not consider background k-mer frequencies, we used several common methods for computing the distance between two vectors, in this case observed k-mer frequencies of each pair of host and viral sequences: Euclidean distance (Eu), Manhattan distance (Ma), Chebyshev distance (Ch), JS)  . The bacocument} , Hao  have significantly lower dissimilarity scores compared to all other taxa at that level . If the Bonferroni corrected p-value for the taxon tested was sufficiently small at the 5% significance level, this taxon was predicted as a potential host of the query virus.A few methods were implemented to select the predicted host of a query virus based on pairwise distance/dissimilarity measures of the viral sequence to all possible host sequences. The simplest was to select the host with the lowest distance/dissimilarity to the query virus (i.e. greatest similarity). Alternatively a consensus method was taken. The hosts with the www.earth.lsa.umich.edu as stated above. Relevant accession numbers for virus and host genomes are found in the supplemental file, Supplemental_table_virus_and_host_genomes.xlsx.All genome sequences used in our analyses are publically available online via NCBI, iPlant, or Eu, Ma, Ch, JS—and those that take into account background k-mer frequencies of the host and virus—Hao, Teeling, EuF and Willner. We first used a highly specific set of 352 genomes of viruses and the hosts on which they were isolated . To initially survey the discriminatory power and specificity of ONF distance/dissimilarity measures for virus-host prediction, histograms of ONF distance/dissimilarities, over several k-mer lengths, for the known 352 virus-host pairs were compared to 352 randomly selected virus-host pairs to simulate non-interacting pairs. The random simulation is reasonable as most of virus-host pairs should not interact. To model the background of the sequences, we used Bayesian information criterion (BIC) to estimate the MC order for the 352 phage sequences as in  distance measure in predicting virus host taxonomy. Among the dissimilarity measures with background correction, Teeling that can be as high as 0.89 and 0.90 when the k-mer length is 6 and 9, respectively.To better assess the discriminatory power of the 11 dissimilarity measures, receiver operating characteristic (ROC) curves were constructed using each measure for various entclass1pt{minimak-mer lengths 4–9. For simplicity, results for k-mer length of 4, 6 and 9 are reported (Table k-mer length 4 (tetranucleotide frequency), Ma distance yielded the highest accuracy at several taxonomic levels . At the genus level, Hao and Teeling outperformed Ma, and at the domain level Ma was tied with the best accuracy with four other measures—Hao, Teeling and EuF—at 97%.Following this initial performance test, we assessed the ability of the 11 measures to predict the correct host taxonomy for the complete set of RefSeq 1427 virus isolate genomes, for which their host is known, from among 31,986 possible bacterial and archaea genomes downloaded from NCBI. Pairwise ONF distances/dissimilarities were computed between all virus and host genomes, and the host with the lowest distance/dissimilarity score (i.e. highest similarity) to the query virus was selected as the predicted host. Prediction accuracy was scored for each level of taxonomy using k-mer length was increased to 6. Except for Ch and JS, prediction accuracy improved at all taxonomic levels except the domain level where accuracy decreased nominally for some measures. At this k-mer length, the background corrected methods in general performed better than non-corrected methods. For example, genus level accuracies ranged from 24% to 33% for corrected methods versus 14% to 24% for non-corrected methods. Among the background corrected methods, Eu and Ma had only slightly higher accuracies (98% versus 97%) . Because the distribution of taxa among the possible ∼32 000 hosts was very unbalanced, the accuracy for selecting a random host at each taxonomic level was empirically calculated. In general, the host prediction accuracy by choosing a random host increased with increasing taxonomic level (from genus to domain) again due to the unbalanced nature of the virus and host databases. For example at the domain level, the accuracy for picking the correct host is very high (95%) because the databases are dominated by bacteria and bacterial viruses. When comparing ONF prediction results, this means that the fold increase in prediction accuracy with over random selection increased with decreasing taxonomic level (from phylum to genus) .All prediction accuracy results in Table k = 6) from among the ∼32 000 possible host genomes. Prediction accuracy generally increased with contig length  when the sequencing error rate was 0.05. For reference, the error rates of Illumina and 454 platforms are ∼0.001 or 0.01, respectively  and host prediction accuracy was assessed when picking the predicted host with the lowest dissimilarity h Figure . Accurac} Figure . Predictectively , so sequk-mer lengths, host prediction accuracies at most taxonomic levels were often relatively low (often below 80%), so we explored approaches for improving prediction accuracy. Rather than simply selecting the predicted host as the one that had the lowest ONF distance/dissimilarity to the query virus, a maximum consensus method was applied. The predicted host taxon was selected as the most frequent taxon among the n hosts with the lowest distances/dissimilarities to the query virus. Using k-mer length 6, prediction accuracy generally increased at all taxonomic levels using this consensus approach for n < 30 . Having established that the et al. 2015 presented a comprehensive comparison of different methods for host prediction including virus-host abundance covariation, sequence homology to host genomes, analysis of CRISPR sequences, and ONF methods. They tested 820 bacterial viruses for which their host was reported and made host predictions using 2,699 possible complete bacterial genomes using Euclidean distance and various k-mer lengths. Use of the Eu at k-mer length 6, improving accuracy by 25–99% across the different taxon levels  distance at k-mer length 4 and selecting the most similar host from among the 14 977 host genomes (excluding the source genome from which the virus was recovered). We did a comparable analysis of prediction accuracy with Ma and k-mer length 6 on this same sequence dataset. et al. dataset over Ma, consistent with results above , the relative order of mean virus-host dissimilarities among the virus groups remained the same (myoviruses > podoviruses > siphoviruses), but the significance of differences in distributions between groups changed somewhat  with myoviruses often having the broadest host ranges and podoviruses and siphoviruses typically having relatively narrow host ranges ,42. It ht Figure . Consistt Figure . SiphoviCandidatus Thioglobus singularis and Candidatus Thioglobus autotrophica EF1)   and the ica EF1) ,46. The Thaumarchaeota (16%), members of family Methylococcales (1%) and contigs of unknown taxonomy (11%). For all 18 viral genomes the most similar contig or consensus of the top five most similar hosts were consistently SUP05 group bacterial contigs, demonstrating the utility of ONF methods for correctly predicting virus-host interactions from metagenomes  host genomes found in the human gut and used in the abundance profiling analysis in Dutilh et al. . Bacteroides is the previously inferred host genus for crAssphage from the original study that assembled and discovered this virus. These three genera belong to the phyla Bacteroidetes, Firmicutes and Fusobacteria, respectively. Similarly, rank-sum analysis at the phylum level yielded only two phyla, Bacteroidetes and Fusobacteria, out of eight possible phyla.crAssphage is a viral genome that was assembled from human fecal microbiome metagenomes, was found to be abundant and ubiquitous in humans, but whose true host is unknown . Using aet al.  found thh et al. . Of thesk-mer length used. A few studies have explored the impact of varying k-mer length for a given measure  that may diminish their effectiveness in making predictions . It is possible that the superiority of k-mer patterns that are specific to the closely related genomes. This hypothesis would require further in depth investigation that is beyond the scope and focus of this work. samples ,26–28, set al. when using Euclidean distance and k-mer length 4 , matching of CRISPR sequences, virus-host co-variation, and Eu-based ONF methods. They found that homology searches of viruses against host genomes yielded the highest accuracies, for example ranging from 21% to 62% at the genus level  in the query virus to host. While both ONF and homology searches are host database dependent, fragmented metagenomic viral or host contigs may lack the specific gene(s) or element(s) that allow homology searches to infer their hosts. Alignment-free ONF methods however do not require a specific gene to make a prediction, and for this reason may have an advantage over homology-based methods. Consistent with this, subsampling host genomes even at 10 kb did not appreciably diminish prediction accuracy Figure . We alsoWhile genus level accuracies of ∼30–40% are rather low for a predictive tool, higher taxonomic levels had higher host prediction accuracies, reaching 60–70% at the class and phylum levels. There are a large number of bacterial and archaeal phyla for which viruses that infect them have yet to be identified . There iet al. (k-mer length 4) curiously reported much higher accuracies than found here (60-90% at the genus to order levels without thresholding). Direct comparison of the measures Ma and et al. dataset consists of sequences they report as viruses that were identified from within host genome sequencing projects using their virus finding software VirSorter . In the case of crAssphage, this rank-sum test approach did predict the same host genus (Bacteroides) phylum (Bacteroidetes) as the previously inferred host phylum by co-variance methods . While prediction accuracy began to drop more markedly for smaller contigs, it may still be feasible to make decent predictions on contigs of 5 kb and above. For reference, the metgenomically assembled crAssphage and SUP05 viral genomes were much greater than 20 kb . In the case of predicting the host of viral contigs from metagenomic host contigs, it may be necessary to further assess how accuracy is impacted by both viral and host contig lengths as well as determine relevant threshold levels for This study provides a comprehensive assessment of various distance/dissimilarity measures for use in host prediction methods and specifically recommends the habitats ,12,15,54


Long non-coding RNAs (lncRNAs) are a class of non-protein-coding RNA molecules, which are involved in various biological processes, including chromatin modification, cell differentiation, pre-mRNA transcription and splicing, protein translation, etc. During the last decade, increasing evidence has suggested the involvement of lncRNAs in both immune and antiviral responses as positive or negative regulators. The immunity-associated lncRNAs modulate diverse and multilayered immune checkpoints, including activation or repression of innate immune signaling components, such as interleukin (IL)-8, IL-10, retinoic acid inducible gene I, toll-like receptors 1, 3, and 8, and interferon (IFN) regulatory factor 7, transcriptional regulation of various IFN-stimulated genes, and initiation of the cell apoptosis pathways. Additionally, some virus-encoded lncRNAs facilitate viral replication through individually or synergistically inhibiting the host antiviral responses or regulating multiple steps of the virus life cycle. Moreover, some viruses are reported to hijack host-encoded lncRNAs to establish persistent infections. Based on these amazing discoveries, lncRNAs are an emerging hotspot in host–virus interactions. In this review, we summarized the current findings of the host- or virus-encoded lncRNAs and the underlying mechanisms, discussed their impacts on immune responses and viral replication, and highlighted their critical roles in host–virus interactions. With the rapid development of DNA sequencing technologies, the whole genomes of several species have been mapped and annotated. The first transcriptome analysis performed a decade ago came to a surprising conclusion that only about 2% of the genomic DNA harbors protein-coding genes . In the In recent years, lncRNAs have been found to be critical regulators in various biological processes such as cell differentiation, chromatin modification, pre-mRNA transcription and splicing, and protein translation and translocation –9. UnderFor decades, studies on virus-related host immune responses have been focused mainly on genes or proteins. However, recent studies have shown that lncRNAs may also participate in these biological processes. This review will focus on the lncRNAs involved in host–virus interactions and underlying regulatory mechanisms.Most of eukaryotic lncRNAs are transcribed by RNA polymerase II, whereas a limited number of lncRNAs are transcribed by cellular RNA polymerase III . After tin cis-regulation  is a well-defined positive regulatory component in interleukin (IL)-8 signaling pathway, which can activate the antiviral response. Influenza virus, human immunodeficiency virus (HIV), and other viral infections induce the expression of NEAT1, leading to the formation of nuclear body paraspeckles , 45. Spl4 (Pam3CSK4)  . The subam3CSK4) . AlthougTumor necrosis factor-alpha (TNF-α) is a significant activator of host immune responses to viral infections –51. ReceISGs are induced through the IFN signaling pathway and critical for antagonizing viral infections . To dateSome viruses can induce the expression of the lncRNA BISPR (BST2 IFN-stimulated positive regulator) through the JAK-STAT pathway, such as influenza virus, vesicular stomatitis virus or hepatitis C virus (HCV) –59. BISPA functional lncRNA, called negative regulator of antiviral response (NRAV), is downregulated dramatically during influenza A virus (IAV) infection . Overexpin vivo . The lncRNA CMPK2 can be upregulated significantly by IFN-α or IFN-γ . KnockdoThe lncRNA#32 is located on human chromosome 7p13 and overlaps the 3′-terminus of the HECT, C3, and WW domain containing E3 ubiquitin protein ligase 1 (HECW1) . SilenciTaken together, current understandings propose the nature and breadth of lncRNAs in the regulation of ISGs, which define the first line of defense against pathogens. While a significant baseline has been made, extensive future studies are required to underpin this important aspect of host-pathogen interactions along with their impacts on virus biology and host responses.+ T cells, the lncRNA screening has recently been conducted in CD8+ T cells  and non-coding transcript in CD4 T cells . A total T cells . At the pathways . LimitedHost lncRNAs have been confirmed as positive or negative antiviral regulators in the immune response; surprisingly, a few of host lncRNAs can be induced and hijacked by certain viruses to establish persistent infections. This is likely due to the mutual adaptability of hosts and viruses for millions of years.H1 CD4+ T cells and is considered to be associated with immune response  by forming a ribonucleoprotein complex with NFAT kinases and expression of this lncRNA is significantly altered following HIV-1 infection , 76–79. lncRNA-ACOD1, located near the ACOD1 protein-coding gene, can be induced by various viruses, including Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus (HSV), and vaccinia virus (VACV) . In addiThe existence of virus-encoded lncRNAs has been identified for years , 82. HowA polyadenylated nuclear RNA (lncRNA PAN) expressed by Kaposi’s sarcoma-associated herpesvirus is localized within the cell nucleus and accumulated largely during lytic infection. Several studies demonstrate that PAN represses host gene transcription through a variety of mechanisms. Interferon regulatory factor 4 (IRF4) is a transcription factor that can bind to and transactivate the IL-4 promoter along with PU.1 . HoweverAnother lncRNA Beta2.7, transcribed from the human cytomegalovirus genome, exists at the early stages of viral infection , 89. Betvia the generation of the lncRNA sfRNAs . However, based on the current findings, we believe that lncRNA databases for broader species will facilitate the study on natures and dynamics of lncRNAs-mediated antiviral responses and regulation of the virus life cycle.In conclusion, growing evidence suggests that additional hosts- or viral-origin lncRNAs remain undiscovered, and systematic and novel probing approaches are required to characterize functional lncRNAs and identify clinically relevant lncRNAs with broader antiviral characteristics.X-YM is the major contributor of the review. YL, MNA, YS, YG, HZ and MM participate in the modification of the article. H-JQ conceived and revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.