There have been many investigations on non-biodegradable materials acting as an antibiotic carrier for local drug delivery systems based on polymethyl methacrylate (PMMA) beads. However, the material is non-degradable and non-porous so that less than 5% of the encapsulated drug is released. In order to obtain better release of the antibiotics, greater porosity of the beads would be required. Adding fillers could increase the bead’s porosity, thus improving the antibiotic release from the beads. The purpose of the study is to optimize release kinetics of gentamicin from handmade beads by adding fillers such as glycine and sodium chloride in different concentrations. Terms of percolation theory will qualitatively be applied in interpreting the final results. Model beads were made by blending the antibiotics (gentamicin) with powdered PMMA, prepared with the inclusion of glycine and different concentration of sodium chloride in 100% monomer. To determine the gentamicin release, beads were placed in phosphate buffered saline (PBS) and aliquots were taken at designated times to measure the gentamicin concentration. Addition of glycine yielded 16 % release of the total amount of gentamicin incorporated in 24 hours. Subsequent addition of sodium chloride resulted in an increased gentamicin release, with little or no difference in gentamicin release once 16 g or more sodium chloride was added (gentamicin release 100% of the amount incorporated). In conclusion, addition of glycine and sodium chloride resulted in an increased release of gentamicin; however, the combination without sodium chloride seemed to have an inhibitory effect on the gentamicin release. Substantial progress in treating these diseases has been made by the invention of antibiotic-loaded polymethylmethacrylate (PMMA) beads providing the carrier for local delivery of antibiotics in the treatment of infection in orthopaedic practice, such as chronic osteomyelitisStudies have been conducted to identify any substances that can increase the porosities of the antibiotic beads but have not yielded optimal results. Therefore, further investigation is needed and sodium chloride, which has a larger particle size than glycine particle size, was chosen as filler and its effect evaluated in creating porosity.4. Due to the controlled pharmacokinetics, the antibiotic is released from bone cement beads by way of diffusion, which is dependent on the material properties of the beads.Antibiotic release from bone cement is a complex process and important variables include: type of antibiotic, type of bone cement and the mixing conditions4. Initially however, antibiotics adhering to the surface of the PMMA beads will dissolve rapidly to create a so-called burst-release, which is followed by prolonged release of antibiotics from the matrix by diffusion. Clearly, prolonged release depends on the porosity of bone cement4. The effects of porosity and pore size distribution on water permeability and drug release are included in so-called percolation models6.Release of antibiotics is also strongly influenced by the mixing technique. This study used antibiotic powder and soluble fillers. First, PMMA powder is mixed together, monomer methacrylate is then added, allowed to moisturise and again blended using spatula for two minutes to obtain a free flowing soft paste. This leaves intact as many large crystals as possible to create more porous mixture to increase antibiotic elution rate. The PMMA matrix is structured to provide optimum interaction between the carrier matrix and the antibiotic6.Application of the principles of percolation is necessary to design bead systems with an optimal antibiotic release. In essence a “percolating cluster” can be described as a function of relative volume ratios of one or more components in a matrix and when their concentration exceeds a certain threshold, a system with interconnecting pores is obtained yielding optimal antibiotic release8. Gentamicin is an aminoglycoside and in bead shape constitutes an effective drug delivery system for local antibiotic therapy in bone and soft-tissue infections, and gentamicin concentrations at the site of the infection exceed the minimum inhibitory concentration (MIC) for the infecting organisms. Moreover, MIC values for gentamicin are far lower as resistant bacteria have a MIC level of 4 μg/mL10. The purpose of the study is to optimize the release kinetics of gentamicin from handmade beads by adding glycine and sodium chloride. Results will be qualitatively interpreted according to the terms of percolation theory.In order to enhance the porosity of PMMA bone cement, various fillers, such as dextran, glycine, sodium chloride or a second antibiotic have been added5. Percolation models have been used to characterize the behaviour of porous matrices. The application of the principles of percolation theory is useful to design controlled release porous matrices. “Percolating cluster” can be described as a function of relative volume ratios of one or both components, built by the component particle that are in contact with each other. When the concentration occurs in an infinite ideal system it is known as “percolation threshold” and represents a critical value in each system and can be determined as a critical porosity, therefore, the release kinetics is expected at this point12.Some research has been performed to study the effect of pore size, porosities and pore size distribution on water uptake and drug releaseSoluble fillers, like glycine, sodium chloride and Kollidon can be used to increase porosities. The soluble filler is a part of percolation systems; therefore, it is able to change the condition from non-percolating to percolating during the solution. After dissolving the filler will cause the antibiotic release, assuming the soluble filler is located around the antibiotic particle see  and C. GThe particle size distributions of gentamicin sulphate, PMMA powder Simplex-P bone cement, crystalline glycineand sodium chloride were measured by laser diffraction spectrometry  using a 200 mm lens. The powders were dispersed at a pressure of 5,00 bar with a Sympatec Rodos dry disperser.The mixing was performed as follows: 8 g PMMA powder  was first mixed with 0.2 g of gentamicin sulphate powder , 0.6 g crystalline glycine  and different amounts  of sodium chloride  in a ceramic bowl for two minutes. Once the antibiotic powder and soluble fillers were fully blended, the monomer (methacrylate/MMA) was poured over the powder, allowed to become soaked and again blended according to the manufacturer instructions using a spatula for two minutes to yield a free flowing paste. Subsequently, beads with an approximate diameter of 12 mm were hand-rolled, for which 30 min were available until complete polymerization.0C. At designated time intervals , 0.5 µL aliquots of the gentamicin-PBS solution were taken and their gentamicin concentrations were measured using an o-phtaldialdehyde reagent, made and stored for 24 hours in a dark environment. The gentamicin sample, ophtaldialdehyde and 2-mercapto ethanol were mixed in equal proportions and stored for 30 minutes at room temperature. The o-phtaldialdehyde reacted with the gentamicin and a chromophoric product was obtained. The absorbance was measured at 332 nm13 using a Spectronic 20 Genesys spectrophotometer. . The percentages gentamicin released out of the total amount incorporated were calculated for all cements used.The beads were placed in 10 ml of PBS and incubated at 37in vitro study was measured. As can be seen, the 50% distribution of the gentamicin particles is 18.1 µm and PMMA powder is 15.5 µm  around the beads. Local antibiotic concentration remained therapeutic for 14 days but became sub-therapeutic at the next measured time point of 28 days. The PMMA beads are left in place for 3 to 4 weeks and then surgically removed10 with consideration for patient safety reasons15.Local antibiotic delivery systems have improved the management of complex wounds in musculoskeletal surgery. When a thorough debridement is augmented with high sustained local antibiotic concentration, the progression of bone and soft tissue contamination to become a regional infection may be prevented. Antibiotic-PMMA beads are often used to sterilize and temporarily maintain dead space following debridement surgery.Beads were loaded with the combination of gentamicin and glycine, thus without sodium chloride, showed no enhancement of the gentamicin release at all. In this experiment, addition of sodium chloride was influencing the gentamicin release greatly. For the combination of gentamicin-glycine-sodium chloride in different concentration , gentamicin release could be seen up to 336 h. Remarkably, increasingly higher doses of sodium chloride seemed to have gradually decreased the gentamicin release after peak level at 168 h. It seemed that in this dose, sodium chloride also had an inhibitory effect on the gentamicin release. Sodium chloride has been used to enhance the porosity of acrylic bone cement, and this resulted in an increased released rate of antibiotics in elution studies. The glycine data from this study and the sodium chloride data supported the novel concept of using an inert, inexpensive, soluble filler to increase the elution of antibiotic from PMMA while using a smaller total antibiotic load. By using large amounts of filler such as glycine or sodium chloride, the porosity of handmade beads could be increased to the point that gentamicin would elute in a concentration closer to those seen from commercially prepared beads, and also excessive sodium choride would not cause negative effect in-vivo. Initial rapid release of gentamicin by glycine without added sodium chloride was thousand times lower than gentamicin release with the lowest dosage of sodium chloride. This substance was safe and well tolerated. Moreover, higher dose of antibiotics mixed with monomer did not show good results because the beads would be bulky and would not release the antibiotics.A combination of glycine and sodium chloride will cause the total available gentamicin to be eluted sooner, shortening the duration of activity. Gentamicin is the most common additive because it has, amongst other features, a good spectrum of concentration-dependent bactericidal activity, thermal stability and high water solubility. Interestingly, when used in combination with gentamicin with the new substance, a synergistic effect appeared.The effect of adding sodium chloride on the material properties of PMMA has not been studied. A 0.6 g dosage would be expected to compromise the strength and endurance properties of the PMMA.We conclude that it is possible to optimize the gentamicin release kinetics from handmade PMMA beads by adding glycine and sodium chloride. The gentamicin release is somewhat comparable with the commercial beads, which can be obtained with the addition of glycine and a high dosage of sodium chloride.


Pseudomonas aeruginosa, while TplEi neutralizes the toxic effect of TplE to protect bacteria autointoxication. Blocking the interaction of TplE-TplEi could unleash the toxin, causing bacterial cell death. In this study, we applied a crystallographic approach to design a structural-based antimicrobial peptides targeting the interaction of TplE and TplEi. We found that a peptide (designed as “L” peptide based on its shape) derived from TplE can form a crystal complex with TplEi after subtilisin treatment and the crystal structure was solved at 2.2Å. The “L” peptide displays strong binding affinity to TplEi in vitro and can release the TplE toxin to induce bacteria death in vivo. Our findings suggest that as a toxin activator, the “L” peptide could be a possible drug lead for treating P. aeruginosa infection. Our findings provide an example that the T6SS effector and immunity protein could be a potential drug target against bacteria infection.The type VI secretion system (T6SS) secretes numerous toxins for bacteria-bacteria competition. TplE is a newly identified trans-kingdom toxin secreted by the T6SS in  Pseudomonas aeruginosa is one of the most common nosocomial infectious bacteria that causes significant morbidity and mortality in immunocompromised patients or intensive care unit (ICU) patients  or even extensively drug-resistant (XDR) phenotypes, which are resistant to most of the currently used antibiotics. Thus, it is an urgent need to develop novel effective antibacterial agents  has recently garnered more attention than ever because of its widespread occurrence and significance in the ecosystem function and human health  secretion islands (named H1-to H3-T6SS) that act against both prokaryotic and eukaryotic cells , cell membrane-targeting enzymes (Type VI lipase effectors name Tle), nucleases, and NAD(P) glycohydrolase effectors. These effectors participate in inter- and intra-species competition, and they are produced concomitantly with specific immunity proteins that neutralize cognate toxins to prevent autointoxication  generated by treatment with subtilisin. The TplE peptide (denoted the “L” peptide) has a high binding affinity for TplEi E. coli strain Rosetta™ (DE3) competent cells (Novagen) for expression. The bacterial cultures were grown in LB medium at 37°C. The induction was initiated by the addition of IPTG  when the culture reached OD600 = 1.2. The bacterial culture was incubated with shaking at 25°C overnight after the induction. The bacterial cells were then harvested by centrifugation  and re-suspended in lysis buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM imidazole and 4 mM β-mercaptoethanol and disrupted by ultrasonication on ice. The cell debris was removed by centrifugation at 13,000 rpm for 60 min. The clarified supernatant was loaded to Ni-NTA resin (Qiagen). The TplEi and/or TplEi-TplE complex was next loaded on a HiTrap Q HP column  and eluted with the linear gradient of 75–1,000 mM NaCl. Finally, the eluted proteins were concentrated and applied to a Superdex 200 HR 10/30 column  equilibrated with 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 2 mM DTT. The selenomethionine-substituted TplEi-TplE complex was produced by expression in the E. coli methionine auxotrophic strain B834 (DE3) in LeMASTER medium containing L-selenomethionine. The purification procedure for the SeMet derivative was the same as that of the native protein.Plasmid-encoding TplEi and/or TplEi-TplE complex (pETDuet-1 vector with ORF1 encoding TplEi and ORF2 coding TplE) was transformed into in situ limited proteolysis by incubating trace amounts of trypsin and subtilisin from the Proti-ACE Kit (Hampton Research). The SeMet-labeled TplEi were grown in 0.52 M lithium sulfate, 13% PEG8000 after 48 h of incubation. The TplEi-TplE complex crystal appeared 1 month later at 22°C. The optimized condition for the TplEi-TplE complex was achieved by mixing 1 μl of buffer containing 0.1 M sodium HEPES, 20% (w/v) PEG 10,000 and 1 μl of protein solution with trace amounts of subtilisin. Crystals were soaked in reservoir solution supplemented with 10% ethylene glycol for 30–60 s before flash freezing in liquid nitrogen. X-ray diffraction experiments were conducted at beam line BL17U in Shanghai Synchrotron Radiation Facility (SSRF). All diffraction data were processed with the XDS package .The crystallization experiments were performed using the hanging drop vapor diffusion setup at 22°C by mixing 1 μl of buffer and 1 μl of protein solution equilibrated over a 0.3 ml reservoir solution. Crystallization trials of TplEi alone and TplEi-TplE complex were subjected to  Kabsch, . The sof Kabsch, . The ato Kabsch,  and was  Kabsch, . The finThe “L” peptide (TplE residues 82-108) and its mutants Table  derived Isothermal titration calorimetry (ITC) assay was performed with a MicroCalTM iTC200 calorimeter  at 25°C; both the protein and peptides were dissolved with the same buffer . The concentration of TplEi was between 0.03 and 0.04 mM. The concentrations of wild type peptides and mutants were between 1 and 2 mM. Titration was scheduled with 18 consecutive injections of 2 μl of peptide with a 120 s interval between injections, using a stirring rate of 600 rpm. Data acquisition and analysis were performed using MicroCal Origin software (version 7.0).Superdex 200 10/300GL column  was equilibrated with the buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl and calibrated using molecular weight standards, γ-globulin (158 kDa), ovalbumin (45 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa). The purified TplEi and/or TplEi-TplE complex (~1 mg/ml) was loaded onto the column running at a flowrate of 0.15 ml/min.E. coli BL21 (DE3) strains harboring plasmids expressing periplasmic-targeted TplE, TplE-TplEi and the “L” peptide were grown overnight and serially diluted in LB medium at 10-fold. A 2 μl bacterial dilution was spotted onto LB agar plate containing 0.045 mM IPTG. Images were taken after 24 h growth.Cell-toxicity assay was carried as previously described with minor modification  was recently solved , we calculated the buried area by “L” peptide (1173.6Å2), accounting for 46.4% of the total surface area of the peptide, and the ΔG value (the solvation free energy gain upon formation of the interface) was −19.6 kcal/mol. By comparison, the surface area buried by the intact TplE in the TplE-TplEi complex is 2,735.5 Å2, which is more than double that of the “L” peptide. However, the ΔG of the TplE-TplEi complex is −21.4 kcal/mol, which is not significantly different from that of the TplEi-“L” peptide complex. These results suggest that the region corresponding to the “L” peptide is crucial for the TplE-TplEi interaction, which is consistent with our proteolysis and crystallographic results.As shown in Figures The structure of TplEi alone is very similar to that observed in the peptide-bound form Figure , with onKd) between A/B peptides and TplEi and evaluated their binding affinity in vitro. Intriguingly, while the dissociation constant (Kd) is ~125 nM between the A peptides and TplEi the binding affinity of the shorter B peptides decreased with a Kd = 478 nM  of the “L” peptide could abolish the binding with TplEi, regardless that a large area of the peptide is involved in the interaction with the immunity protein. To further elucidate the mechanism underlying the “L” peptide binding, we mutated several key residues of TplEi interacting with the “L” peptide, and measured the binding affinity of the “L” peptide to these TplEi mutants using ITC. The side chain of K100 of the “L” peptide forms four hydrogen bonds with TplEi, among which only one involves side chain interaction with S215 of TplEi, whereas the other three involve backbone atoms. Therefore, we introduced S215A mutation to TplEi, and found that the “L” peptide bound the S215A mutant with the Kd = 7.8 μM, indicating nearly 62-folds reduced binding affinity comparing to the affinity to the wild type TplEi  pairs is analogous to toxin-antitoxin (TA) systems, the exploitation of which has received considerable attention as a strategy for developing antibacterial drugs  attached to the TplE “L” peptide could potentially overcome this drawback to provide a novel therapeutic agent (Fosgerau and Hoffmann, P. aeruginosa and other bacteria. The TplE “L” peptide represents a novel drug candidate for a T6SS-based therapeutic strategy against P. aeruginosa.The TplE “L” peptide is an example of a toxin-mimicking peptide that binds to TplEi and disrupts the TplE-TplEi interaction, releasing the active TplE toxin. However, further work is needed to assess the potential of the “L” peptide for treating The atomic coordinates and structure factors have been deposited in the Protein Data Bank with the accession codes 5H7Z for TplE and 5H7Y for TplE peptide-TplEi.SC, YS, and XG designed the study. SC, YS, and XG wrote the paper. XG and ZM purified and crystallized protein and determined structure. XG and ZM performed and analyzed ITC assay, Cell-toxicity assay. BQ designed construct for expression of protein. All authors reviewed the results and approved the final version of the manuscript.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.


Aedes spp.). This arbovirus was initially isolated from a rhesus monkey in the Zika forest in Uganda in 1947. ZIKV infection is commonly associated with unclear symptoms. However, in about 20% of cases, it can cause self-limited symptoms such as fever, joint pain, rash, and conjunctivitis for several days (Zkia virus (ZIKV) is a flavivirus from the flaviviridae family, transmitted to humans predominantly by infected mosquitoes  probability of the sexual transmission of a male patient was infected in southeastern Senegal in 2008, to his wife . 3) On FThe isolation of the infectious virus from semen and saliva , 8, and ZIKV can be transmitted from infected men to their sex partners through vaginal, anal, and oral sex; however, sexual transmission from women to others has not been stated . Altoget


We report 26 complete genomes of Zika virus (ZIKV) isolated after passaging the Zika virus strain FLR in mosquito (C6/36) and mammalian (Vero) cell lines. The consensus ZIKV genomes we recovered show greater than 99% nucleotide identify with each other and with the FLR strain used as input. Flavivirus genus within the Flaviviridae family and has a single-stranded positive-sense RNA genome that is approximately 10.8 kb in length. Like many flaviviruses, ZIKV is an arbovirus, cycling between mosquito and mammalian hosts (Zika virus (ZIKV) belongs to the The ZIKV FLR strain was originally isolated from the blood of a human in Barranquilla, Colombia, in December 2015 and was passaged twice on C6/36 cells prior to BEI deposit and a third time at BEI Resources (NR-50183) , as described previously , 4. Nextde novo assembled. BLASTN searches of these contigs were performed against the GenBank nonredundant nucleotide database . The mean nucleotide identity across strains was calculated to be 99.999% (range 99.995% to 100%).Sequencing reads were demultiplexed by barcode, trimmed, and then database . FollowiIn summary, we identified very few differences between the various ZIKV FLR strains at the consensus level, despite the different plaque phenotypes that were observed. These sequence data can increase our understanding of variations that may contribute to host specificity.MF574552 through MF574577 under BioProject identification number PRJNA314889.The consensus sequences for these viruses were validated manually and annotated with VIGOR  prior to


Actinidia chinensis in Auckland, New Zealand. The complete genome of ASbLV is 8,192 nucleotides long, excluding the 3ʹ poly(A) tail, contains four open reading frames, and is most closely related to Caucasus prunus virus (56% nucleotide sequence identity), a member of the genus Prunevirus. Based on the demarcation criteria of the family Betaflexiviridae, ASbLV is a new member of the genus Prunevirus.We report the complete genome sequence of a novel virus, tentatively named “actinidia seed-borne latent virus” (ASbLV), isolated from The online version of this article (10.1007/s00705-017-3701-x) contains supplementary material, which is available to authorized users. Actinidia (commonly known as kiwifruit), whose members occur naturally in China, is composed of over 50 species. Of these, A. deliciosa and A. chinensis are the most widely cultivated for fruit production. Kiwifruit was first introduced into New Zealand from China in the early 1900, and New Zealand is now one of the biggest kiwifruit producers in the world.The genus A. chinensis vines grown from seeds in a home garden in Auckland, New Zealand, showed virus-like symptoms of vein chlorosis and mottling. Symptomatic leaf samples from five vines were collected, and total RNA was extracted from three bulked leaves per vine, using an RNeasy Plant Mini Kit  according to the manufacturer’s instructions. The RNA from each vine was tested by reverse transcription polymerase chain reaction (RT-PCR) for a range of viruses, including those previously reported to infect kiwifruit. RT-PCR was performed with various generic and specific primers, using the Invitrogen SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase . Four of the five vine samples were positive for actinidia virus A using species-specific primers [Citrivirus) but it was distinct from those of all trichoviruses and vitiviruses. A primer pair that amplifies a 425-bp region of the CLBV coat protein gene [In 2013, A. chinensis leaf tissue was homogenised in RLT buffer (QIAGEN) modified according to MacKenzie et al. [free Kit (Life Technologies), followed by host ribosomal RNA (rRNA) removal using a Ribo-Zero rRNA Removal Kit for plant leaf  according to the manufacturer’s instructions. The rRNA-depleted RNA was fragmented and used to synthesise double-stranded cDNA as described in the cDNA Rapid Library Preparation Method Manual, GS Junior Titanium Series, May 2010 . The sample was sequenced on a 454 GS Junior System (Roche 454 Life Sciences) following the manufacturer’s protocols. A total of 189,817 reads averaging 433 bp in length were obtained from the GS Junior sequencing run.To determine the complete genome sequence of the novel virus, symptomatic e et al. . Total nThe GS Junior reads were mapped to reference sequences of actinidia virus B, apricot vein clearing associated virus, Caucasus prunus virus, cherry rusty mottle associated virus, CLBV and prunus virus T. The best reference mapping was to Caucasus prunus virus (CPrV) where the GS Junior reads mapped to the CPrV genome at positions approximately 21 to 1,685 bp and 3,598 to 7,870 bp. Repeated rounds of mapping the GS Junior reads to the consensus sequences extended the contig ends until the complete genome of the novel virus was obtained in a single contig, apart from a small region at the 5ʹ end. Homopolymer regions were resolved by Sanger sequencing of amplicons generated using specific primer sets designed from the contig sequence. Overall, 5,272 GS Junior and Sanger reads were assembled with a mean coverage level of 278 . The remaining 5ʹ end of the genome was determined using a GeneRacer Kit (Life Technologies) as per the manufacturer’s instructions. All sequence analysis was performed using Geneious 10.0.6 .Actinidia virus consists of 8,192 nucleotides (nt) (GenBank accession number MF440375), excluding the 3ʹ poly(A) tail .The complete genome sequence of the novel Prunevirus, subfamily Trivirinae includes two species, Apricot vein clearing associated virus and Caucasus prunus virus. Members of both species have been found to infect trees of the genus Prunus. The genomes of these viruses contain four ORFs. ORF1 encodes a large polypeptide that functions as a replicase, ORF2 encodes the 30K-type MP, ORF3 encodes the CP, and ORF4 encodes the NB. Members of this genus show affinities with members of the genus Citrivirus in the replicase and MP genes. However, the 3ʹ portion of their genome differs from that of citriviruses by possessing an NB ORF and showing different phylogenetic affinities in the CP gene; citriviruses are closer to the triple gene block group, whereas prunevirus are closer to the 30K-type group [The genus pe group .Actinidia virus were compared separately with sequences of other viruses belonging to the family Betaflexiviridae. Phylogenetic analysis of aa sequences was performed using Geneious 10.0.6. The neighbour-joining (NJ) method was used to construct the phylogenetic trees with the Jukes-Cantor model, and the statistical significance of the branches was evaluated with 1000 bootstrap replications. Phylogenetic analysis of the deduced aa sequences of the different genes showed that the novel Actinidia virus clusters with the members of the genus Prunevirus . Comparison of the novel Actinidia virus Pol, MP and CP aa sequences with those of the other members of the family Betaflexiviridae showed sequence identities ranging from 20 to 47%, 8 to 52% and 6 to 64%, respectively; seven viruses with the highest identity are shown in Table Actinidia virus showed the closest similarity to that of CPrV, with 56% nt sequence identity. This is well below the threshold value (less than 80% identity at the aa level in the CP or replicase genes) used as the molecular criterion for species demarcation, and above the value (less than 45% identity at the nt level) used for genus demarcation throughout the family Betaflexiviridae.The complete genome sequence and the deduced aa sequences encoded by ORF1, ORF2, ORF3 and ORF4 of the novel Betaflexiviridae [Based on the Pol, MP and CP genes, the new virus is most closely related to, but distinct from, CPrV. The genetic difference in the NB gene suggests a possible recombination event. Genome recombination has been frequently identified in members of the family iviridae , 7.Prunevirus, the newly discovered virus should be considered a member of the genus Prunevirus, which we tentatively name “actinidia seed-borne latent virus” (ASbLV). The original vine and three more vines (tested later) from the same property were co-infected with actinidia virus A (genus Vitivirus) and/or actinidia virus 1 (family Closteroviridae). Although the plants in which the virus was detected were symptomatic, ASbLV was also detected in asymptomatic vines as a single infection. Assessment of the biological properties of this virus is challenging due to the difficulty in transmitting the virus to herbaceous indicators. The impact of ASbLV is currently being investigated by Plant and Food Research (P&F) scientists. Other viruses belonging to the family Betaflexiviridae reported in Actinidia sp. include actinidia citrivirus, actinidia virus A, actinidia virus B and apple stem grooving virus (ASGV). All of these viruses except ASGV have been reported only in kiwifruit. To date, no efficient vectors have been reported for these viruses, which are thought to be transmitted only by grafting [Based on the genome organisation and the degree of sequence similarity to known members of the genus Supplementary material 1 (PDF 29 kb)Supplementary material 2 (FASTA 8 kb)Below is the link to the electronic supplementary material.


Cryptococcus neoformans and C. gattii, have been commonly designated as either an opportunistic pathogen for the first species or as a primary pathogen for the second species. Such a distinction has been based on epidemiological findings that the majority of patients presenting meningoencephalitis caused by C. neoformans are immunocompromised while C. gattii infection has been reported more often in immunocompetent patients. A recent report, however, showed that GM-CSF (granulocyte-macrophage colony-stimulating factor) neutralizing antibodies were prevalent in the plasma of “apparently immunocompetent” C. gattii patients with meningoencephalitis. Because GM-CSF is essential for differentiation of monocytes to macrophages and modulating the immune response, it is not surprising that the lack of GM-CSF function predisposes otherwise healthy individuals to infection via inhalation of environmental pathogens such as C. gattii. Since the test for anti-GM-CSF autoantibodies is not included in routine immunological profiling at most hospitals, healthy patients with GM-CSF neutralizing antibodies are usually categorized as immunocompetent. It is likely that a comprehensive immunological evaluation of patients with C. gattii meningoencephalitis, who had been diagnosed as immunocompetent, would reveal a majority of them had hidden immune dysfunction. This paper reviews the relationship between GM-CSF neutralizing antibodies and the risk for C. gattii infection with CNS involvement.The two etiologic agents of cryptococcal meningoencephalitis,  Cryptococcus neoformans and C. gattii are sibling species (85%–90% genomic identity) [C. neoformans is worldwide in distribution and though it can cause disease in otherwise healthy individuals, a majority of the reported cases have been from patients whose immune system have been compromised by various causes such as HIV infection, corticosteroid treatment and other immunosuppressive underlying conditions [C. neoformans has been widely known as an opportunistic pathogen. Epidemiological deviancy in C. neoformans infection, however, has been reported among patients in the Far East countries: China, Korea and Japan. In these countries, most of the cryptococcosis patients were caused by C. neoformans and were HIV sero-negative with or without any apparent predisposing underlying condition [C. gattii in HIV+ patients have been reported mostly in tropical and subtropical regions of the world such as Africa [The etiologic agents of cryptococcosis, dentity)  that caudentity) ,3 Epidemnditions . This isondition ,7. Crypts Africa .C. gattii infects apparently healthy hosts with no known predisposing risk factor more frequently than immunocompromised individuals regardless of geographic regions and is therefore widely regarded as a primary pathogen [C. neoformans [C. gattii infrequently infects such patients compared to C. neoformans even in the C. gattii endemic regions of the world [C. neoformans includes strains of serotype A, D and AD which are generally unable to assimilate D-proline as a nitrogen source and glycine as a carbon source. C. gattii on the other hand can utilize D-proline as a nitrogen source and glycine as a source for both carbon and nitrogen [Several studies have confirmed that pathogen ,14,15,16oformans ,18, C. ghe world ,13,16. Dnitrogen ,20. Thesnitrogen , which initrogen  and the nitrogen .C. gattii infection in apparently healthy individuals is the result of increased environmental exposure to the fungus. Such speculation has been based on the over-representation of C. gattii infection among the Australian Aboriginal people living in rural areas [C. gattii such as Eucalyptus and other trees, organic debris and soil is likely higher than the population living in urban areas. Analysis of the risk factors for C. gattii infection among patients in British Columbia, Canada showed that, in addition to environmental exposure, certain underlying medical conditions to be of significant risk [C. gattii infection include preexisting oral steroid use, emphysema, chronic bronchitis, chronic obstructive pulmonary disease, sarcoidosis and pneumonia [It has been speculated that al areas ,23 whereant risk . These uneumonia . Environneumonia .C. gattii infection in individuals with these risk factors have not been evaluated. Countless numbers of tourists and inhabitants are exposed to the environment of C. gattii endemic regions such as Vancouver, Canada, Southeast Asia and South America each year but the number of patients afflicted by C. gattii has only been a fraction of the exposed populations: less than one case per million per year in Australia [C. gattii disease may be triggered by exposure, inhaled C. gattii cells in healthy individuals are readily eliminated by the host defense systems unless certain predisposing risk factors are present in these patients. Such endogenous risk factors may also contribute to increased persistence of dormant C. gattii or be required to trigger reactivation of dormant C. gattii to cause infection. GM-CSF neutralizing antibody appears to be one such host-dependent risk factor. Defining C. gattii as a primary pathogen may become obsolete as more subtle immune dysfunctions are recognized in otherwise healthy patients with C. gattii meningoencephalitis.The host specific mechanisms explaining the increased incidence of ustralia  and Coloustralia  to 5.8 custralia . This suet al., detected anti-GM-CSF autoantibodies which inhibited GM-CSF signaling in the plasma of four previously healthy HIV-negative patients suffering from cryptococcal meningoencephalitis [n = 64), diseased controls  or cryptococcosis patients with recognized immunodeficiency (n = 36). These results suggested that GM-CSF neutralizing antibodies could be a previously hidden risk factor for cryptococcal meningoencephalitis in HIV negative and otherwise healthy individuals. Of the seven cryptococcal strains (four from current and three from the archived cases) isolated from GM-CSF autoantibody positive patients, three had been reported as C. neoformans, one as C. gattii and three as Cryptococcus without identification at species level. The three Cryptococcus unspeciated strains had been isolated from the immunocompetent patients whose plasma samples had been archived and the strains had been maintained as lyophilized cultures.In 2013, Rosen phalitis . This fiC. gattii and one healthy volunteer, but none of the 20 patients infected by C. neoformans . The reason for higher association of GM-CSF autoantibodies with C. gattii infection than with C. neoformans infection is not clear. However, the higher prevalence of C. gattii than C. neoformans infection among patients with anti-GM-CSF autoantibodies could offer some clues to address the major differences in host’s immune responses to these two etiologic agents of cryptococcosis.Does this imply that GM-CSF neutralizing antibodies are a risk factor exclusively for infection due to C. neoformnans has been recognized since the early 1990s when GM-CSF neutralizing antibody increased mortality of C. neoformans infected mice with rapid progression of meningoencephalitis [−/− mice) failed to clear the fungus, which led to higher cryptococcal burdens in pulmonary cryptococcosis because GM-CSF was required for early influx of macrophages, CD4 and CD8 cells into the lung [Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of the family of glycoprotein cytokines that mediates the survival, proliferation, differentiation and function of hematopoietic cells ,32,33. Iphalitis . Corrobothe lung . Additiothe lung ,43.et al., as “idiopathic PAP” [Furthermore, cryptococcosis is reported more frequently than other fungal disease in patients with acquired pulmonary alveolar proteinosis (PAP) . When auhic PAP” . This dihic PAP” , surfacthic PAP” . While ihic PAP” ,52,53, thic PAP” ,55. To ahic PAP” ,57.Cryptococcus. The etiologic agent of cryptococcosis in this patient could have been C. gattii rather than C. neoformans. The patient was working in a lumberyard in Massachusetts when he was diagnosed with cryptococcosis [C. gattii but the lumberyard could have been handling trees imported from regions where C. gattii is endemic. This case is reminiscent of the first autochthonous case of C. gattii infection in Germany in an apparently immunocompetent healthy male who had been working in sawmills and woodworking factories and exposed to high levels of wood dust, including that of imported tropical trees [Eucalyptus trees as the environmental source of C. gattii in Australia [Since autoantibodies to GM-CSF cause defects in chemotaxis, adhesion, phagocytosis, microbicidal activity and phagolysosome fusion of alveolar macrophages , patientcoccosis . The patal trees . This caustralia .Aspergillus fumigatus [Blastomyces dermatitidis case [Coccidioides case [Histoplasma capsulatum [H. capsulatum [H. capsulatum. The mode of anti-Histoplasma action enhanced by GM-CSF in mice was reported as the sequestration of labile Zn in infected macrophages by inducing metallothioneins in a STAT3 and STAT5 dependent manner [Histoplasma fails to replicate within the macrophages leading to fungal clearance [Aspergillus conidia by maintaining proinflammatory response by alveolar macrophages [Although less frequently reported than cryptococcosis ,63,64,65umigatus ,66,67,68dis case , one Cocdes case , one Mucdes case  and fourpsulatum ,71. GM-Cpsulatum . Neutralt manner ,74. Sinclearance ,74. GM-Crophages .C. gattii than C. neoformans infection? Before attempting to address this question, it is necessary to investigate whether anti-GM-CSF autoantibodies are also prevalent in pulmonary C. gattii infection without CNS involvement since evaluation of GM-CSF autoantibodies and cryptococcosis thus far has only focused on patients with CNS infection. Pulmonary infection without CNS involvement is more prevalent with C. gattii than with C. neoformans infection [C. gattii patients with CNS involvement, it suggests that GM-CSF may play a more important role in dissemination of C. gattii than C. neoformans to the brain from the lung and/or brain immunity against C.gattii than C. neoformans. Little is known about the role of GM-CSF in dissemination of Cryptococci to the brain or brain immunity against the fungus. Our study with GM-CSF−/− mice suggested that a lack of the cytokine function enhances dissemination of both the C. neoformans strain H99 and the C. gattii VG1 strain isolated from the GM-CSF antibody positive Chinese patient [vs. the VGI strain is currently underway in our laboratory and should shed more light as to the role of GM-CSF in dissemination and brain immunity against C. gattii. Although GM-CSF does not appear to influence brain microglial killing of C. neoformans serotype A strains [C. gattii by immune cells in the brain.Why do GM-CSF autoantibodies pose a higher risk for nfection ,16,76. I patient  from the strains , there hPneumocyctis carinii [P. jirovecii) or C. neoformans [et al. [C. gattii infection. There is a possibility that pulmonary infection with C. gattii stimulates GM-CSF production more than C. neoformans and the high cytokine production by C. gattii for a long period of time can cause induction of functional antibodies against the cytokine. Could C. gattii also exacerbate the production of GM-CSF autoantibodies more than C. neoformans in the patients who already are antibody positive? This question cannot be answered since the anti-GM-CSF antibody titers in these patients prior to C. gattii infection are unknown and there are no longitudinal studies on the GM-CSF autoantibody titers during the infection in such patients. Monitoring the health status and the GM-CSF autoantibody titers over time in the healthy Chinese volunteer positive for the cytokine antibodies [Before GM-CSF autoantibody was known as the cause of PAP, a hypothesis that PAP is an abnormal pulmonary response to an unusual infectious agent such as  carinii ,78,79 (noformans  gained soformans . Howeveroformans . Among toformans . In the oformans  as well  [et al. , none of [et al. . This sutibodies  could ofCryptococcus in otherwise healthy individuals. For some unknown reason, the neutralizing antibodies against GM-CSF appear to be a greater risk for cryptococcosis due to C. gattii than C. neoformans. This etiologic agent’s difference in the prevalence of GM-CSF neutralizing antibodies among otherwise healthy cryptococcosis patients may offer an important clue in order to address the differences in the host’s immunological response to the two pathogens. It is important to also include the GM-CSF autoantibody test during immunological profiling of cryptococcosis patients who appear to be otherwise healthy. This is not only for the assessment of subtle underlying immune disorders but also for better clinical management for those positive for GM-CSF antibodies since they may eventually develop PAP after meningoencephalitis.Anti-GM-CSF autoantibodies are apparently an important underlying risk factor for meningoencephalitis caused by


Epidemiologic and phylogenetic analyses show repeated outbreaks derived fromvaccine viruses. African horse sickness (AHS) is a hemorrhagic viral fever of horses. It is theonly equine disease for which the World Organization for Animal Health hasintroduced specific guidelines for member countries seeking official recognitionof disease-free status. Since 1997, South Africa has maintained an AHScontrolled area; however, sporadic outbreaks of AHS have occurred in this area.We compared the whole genome sequences of 39 AHS viruses (AHSVs) from field AHScases to determine the source of 3 such outbreaks. Our analysis confirmed thatindividual outbreaks were caused by virulent revertants of AHSV type 1 live,attenuated vaccine (LAV) and reassortants with genome segments derived from AHSVtypes 1, 3, and 4 from a LAV used in South Africa. These findings show thatdespite effective protection of vaccinated horses, polyvalent LAV may,paradoxically, place susceptible horses at risk for AHS. Orbivirus, familyReoviridae  is a severe, often fatal disease of equids that is caused byAHS virus (AHSV), a member of the genus AHS is the only equine disease for which the World Organisation for Animal Health (OIE)observes official recognition status, such that OIE member countries are required tohave legally enforceable AHS control measures in place and are required to immediatelynotify OIE of any change to their country’s AHS status  (12). MoSince its creation in 1997, a total of 6 outbreaks of AHS in the AHS controlled area havebeen reported to OIE, in1999, 2004, 2006, 2011, 2013, and 2014  .http://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA271179) andidentified each by a unique virus strain name , 2011 Mamre (7 isolates), 2014 Porterville (14isolates), and 2014 Robertson (2 isolates) outbreaks of AHS in Western CapeProvince of South Africa ; AHSVLAain name . We isolated individual viruses of each type included in the polyvalent AHSV-LAVindependently, as previously described (http://www.geneious.com) (25). We used a combination of de novoassembly followed by mapping to obtain the full-length consensus genomesequences of each virus strain.We prepared sequencing templates by using sequence-independent whole-genomeRT-PCR amplification  (http://www.atgc-montpellier.fr/phyml)  to analyze theconcatenated whole genome sequence maximum-likelihood tree to genetically (andnot evolutionarily) classify the sequences into genotype groups based onpatristic distances (We used RAMI (We used Recombination Detection Program (RDP) version 4.63  and the 39 fieldisolate strains by using MAFFT ; 1 segment (encoding NS3)derived from a virus resembling 4/Lab/ZAF/98/OBP-116 (p = 9.31 ×10−240); and 1 segment (encoding VP4) that could plausiblyhave been derived from either 3/Lab/ZAF/98/OBP-116 or 1/Lab/ZAF/98/OBP-116 (p =7.66 × 10−4) (−216). Genotype groups 3a and 3b viruseshave 9 segments derived from a virus resembling 1/Lab/ZAF/98/OBP-116 and asingle segment (NS3) derived from a virus resembling 4/Lab/ZAF/98/OBP-116 (p =9.31 × 10−240) (Explicitly testing for intrasegment recombination and reassortment by usingRDP4.63  Table 3. × 10−4)  Table 4. 10−240) .>2 synonymous SNVs and>1 nonsynonymous SNV relative to 3/Lab/ZAF/98/OBP-116,which suggests that the NS1 gene of the virus strains in genotype group 3b aremost probably derived from 3/Lab/ZAF/98/OBP-116, whereas those in genotype group3a are more probably derived from 1/Lab/ZAF/98/OBP-116.Several SNVs relative to the AHSV-LAV–derived viruses 1/Lab/ZAF/98/OBP-116and 3/Lab/ZAF/98/OBP-116 are present in the NS1-encoding genes of virusesincluded in genotype groups 3a (2014 Porterville) and 3b (2014 Robertson)  Table 4.Seven nonsynonymous SNVs were identified between the whole genome sequences ofthe AHSV-1-LAV–derived virus, 1/Lab/ZAF/98/OBP-116, and its parentalvirus, 1/E.cab-tc/ZAF/62/OVI-HS29/62 . SNVs arThe 1/E.cab-tc/ZAF/04/Avt-E040061 strain in genotype group 1c has nonsynonymousSNVs at 3 of the 7 loci (K357N in VP2 and V81A and Q169R in VP6) but aVP5-encoding gene apparently derived by reassortment from a virus resembling4/Lab/ZAF/98/OBP-116, such that the SNVs in the VP5 of this strain were also notconsidered to be mutationally derived.The 7 viruses included in genotype group 2 and the 16 viruses included ingenotype groups 3a and 3b all exhibit potential reversion-to-virulence mutationsat 3 of the 7 nonsynonymous SNV sites that differentiate the AHSV-1-LAV virusfrom its virulent parent (I434T in VP5 and V81A and Q169R in VP6). Additionally,a fourth SNV (K201N in NS3) at 1 of the 7 sites differentiating the AHSV-1 LAVfrom its parent  is alsopresent in the genotype group 2 viruses.The epidemiologic parameters of the AHS outbreaks in the controlled area in 2004,2011, and 2014 were inferred by using the current OIE case definition for AHS. AlthougWhole-genome sequences were compared from 55 field, LAV, and laboratory strains ofAHSV. The field viruses were obtained from horses during outbreaks of AHS ofdifferent clinical severity (CFRs ranging from 4.5% to 78.3%) in the AHS controlledarea of South Africa during 2004, 2011, and 2014. Phylogenetic analyses confirmedthat genetically distinct viruses were responsible for each outbreak and that thesewere all closely related to viruses contained in the trivalent AHSV-LAV (combination1) used in South Africa. Evaluation of nonsynonymous SNVs confirmed some outbreakviruses to be revertants of the vaccine AHSV-1 strain toward the virulent parentaltype. Furthermore, some outbreak viruses were clearly reassortants with individualgenome segments derived from multiple different virus types that are present in thetrivalent vaccine preparation.Potgieter et al.  virus type 1 fieldisolates from the Stellenbosch, Mamre, Porterville, and Robertson AHSoutbreaks in the AHS controlled area in Western Cape Province, South Africa,2004–2014.Additional details about relationships between African horse sickness virusesrelated to outbreaks in South Africa and reference viruses.


Aspergillus fumigatus, an opportunistic pathogen causing fungal lung diseases in humans and animals, was recently shown to harbour several different types of mycoviruses. A well-characterised defence against virus infection is RNA silencing. The A. fumigatus genome encodes essential components of the RNA silencing machinery, including Dicer, Argonaute and RNA-dependent RNA polymerase (RdRP) homologues. Active silencing of double-stranded (ds)RNA and the generation of small RNAs (sRNAs) has been shown for several mycoviruses and it is anticipated that a similar mechanism will be activated in A. fumigatus isolates infected with mycoviruses.Mycoviruses are viruses that naturally infect and replicate in fungi. A. fumigatus sRNAs, sRNA-seq libraries of virus-free and virus-infected isolates were created using Scriptminer adapters and compared. Three dsRNA viruses were investigated: Aspergillus fumigatus partitivirus-1 , Aspergillus fumigatus chrysovirus  and Aspergillus fumigatus tetramycovirus-1  which were selected because they induce phenotypic changes such as coloration and sectoring. The dsRNAs of all three viruses, which included two conventionally encapsidated ones PV and CV and one unencapsidated example NK, were silenced and yielded characteristic vsiRNAs together with co-incidental silencing of host fungal genes which shared sequence homology with the viral genomes.To investigate the existence and nature of A. fumigatus microRNA-like (miRNA-like) sRNAs and small interfering RNAs (siRNAs) were detected and validated. Host sRNA loci which were differentially expressed as a result of virus infection were also identified. To our knowledge, this is the first study reporting the sRNA profiles of A. fumigatus isolates.Virus-derived sRNAs were detected and characterised in the presence of virus infection. Differentially expressed The online version of this article (doi:10.1186/s12864-017-3773-8) contains supplementary material, which is available to authorized users. Aspergillus fumigatus can infect humans, causing life-threatening opportunistic infections in immunocompromised individuals including transplant, AIDS and leukaemia patients, as well as being the causative agent of allergic diseases [A. fumigatus virulence is multifactorial and is a consequence of its structure , growth capacity and its ability to damage its host [Aspergillus is a mould fungus which occurs worldwide in decaying vegetation, seeds and grains. diseases , 46. A. its host .A. fumigatus was initially investigated in 61 isolates and revealed that apparently none contained dsRNA elements [A. fumigatus isolates [-1 , Aspergisolates ) and AspfuTmV-1; ) have siisolates  none of RNA silencing is an internal defence mechanism which protects genomes against invasion by mobile genetic elements such as viruses and transposons . Virus-dCryphonectria parasitica where virus infection cannot be eliminated and viral RNA levels increased in mutants of RNA silencing components. It was reported that in the presence of Cryphonectria hypovirus-1 (CHV-1) (an ssRNA virus) a C. parasitica Dicer mutant was debilitated as compared to the wild type [C. parasitica, [Magnaporthe oryzae [Aspergillus nidulans [C. parasitica hypoviruses on RNA silencing using Dicer mutants revealed that silencing responses differed for each virus irrespective of the fact that the proteins encoded by the viruses were 90–99% identical at the amino acid sequence level [Colletotrichum higginsianum [The antiviral role of the RNA silencing machinery has also been described in fungi. It was first observed in the chestnut blight fungus ild type . Furtherasitica, , Magnapoe oryzae  and signnidulans  demonstrce level . Subsequce level , 60, parce level , quadrivinsianum , have alinsianum , 58.A. fumigatus sRNAomes in the presence and absence of three mycoviruses: Aspergillus fumigatus partitivirus-1 , Aspergillus fumigatus chrysovirus  and Aspergillus fumigatus tetramycovirus-1 . Both AfuPV and AfuCV cause visible phenotypic alterations and result in a decreased growth rate as compared to virus-free isolates; however they had no impact on fungal pathogenicity as assessed in the murine model [A. fumigatus mycoviruses on the pathogenicity of as assessed using larvae of the greater wax moth Galleria mellonella and an uncharacterized A. fumigatus mycovirus shown to cause mild hypervirulent effect [A. fumigatus isolates using next generation sRNA sequencing.In this paper we investigated ne model . The efft effect . Since aIllumina sequencing yielded a total number of reads varying between 12–30 million (M) reads. A high proportion of the reads  did not contain unassigned nucleotides; on average >80% of the reads contained the first 6 nt of the 3' adapter indicating a reliable library preparation  which confirmed that sequencing bias was low and there was no favoured or over-amplified sRNA sequences in the sequencing result.A. fumigatus nuclear genome  using PatMaN [Mucor circinelloides [A. fumigatus may also be due to the quality of the genome assembly [Next, the size class distributions  were investigated. The distributions for all (redundant) reads, before genome matching, showed that the lengths of the majority of sRNAs were between 19–23 nt. Two peaks of abundance were observed for 20mers and 21mers; these corresponded to low complexities, suggesting the existence of a small number of unique reads with high abundance  whereas in virus-infected samples the abundance of the virus-matching reads were in the millions.The size class distribution of fungus mapping sRNA population showed that the majority of the reads were in the 20–21 nt range. However low complexity was observed at 24–25 nt even though no obvious peak was observed in this size range  which confirms the quantile normalisation was an appropriate normalization for the current data.For normalisation, the RPM method  was applca. 22% , 50%  and 25%  as compared to 0.001% , 0.0003%  and 0.002%  for the virus-free counterpart samples.Comparisons of the total reads for two biological replicates which map to the three virus genome sequences in infected samples for AfuCV, AfuTmV-1 and AfuPV-1 were respectively Size-separated presence plots of PV are shown in Fig. For each virus, four virus-derived sRNAs were confirmed following northern hybridisation. Probes for the northern blots were designed to hybridise with the most abundant virus-derived sRNAs as identified on the presence plots  transposon sequences; their differential expression between the virus-free and the virus-infected samples was confirmed by northern hybridisation. The low throughput validation proved that all of the proposed siRNAs were detectable only in corresponding virus-infected samples and that the sizes were as anticipated  and AfuTmV-1  were respectively 93.8% and 100% similar to two different regions in ted Fig. . Conversted Fig.  and AfuTted Fig.  presenceA. fumigatus rRNAs  was present in both AfuCV-infected and virus-free isolates and differentially expressed during the virus infection; the sequencing-based expression levels were confirmed by quantitative northern hybridisation Fig. .Fig. 5QuA. fumigatus LINE-like transposons; based on sequencing data it was predicted to be differentially expressed in all three virus-infected isolates. Northern hybridisation confirmed the presence of this miRNA-like candidate only in the AfuPV-1-infected isolate and its absence from AfuCV- and AfuTmV-1 infected isolates  between each virus-free and virus-infected isogenic line were annotated in order to determine the loci which may play a regulatory role in the presence or absence of virus infection  genome were predominantly to the negative-strand despite the fact that it possessed a dsRNA genome [Botrytis genus . Upon further investigations, this information could be linked to the replication properties of dsRNA viruses as it may be the indicator that encapsidated dsRNA would be exposed to the cytoplasm in another form such as a replication intermediate which could be targeted by host RNA silencing.Another interesting point observed in sRNAome of o common , 39, 42.A. fumigatus. Such alterations following virus-infection may be the result of simultaneous silencing of mycovirus dsRNAs and sequence-specific degradation of host mRNAs. Initial checks, conducted using qPCR, revealed that the expression levels of some genes potentially targeted by virus-derived sRNAs were reduced following virus infection . Another gene assayed using qPCR assays was the pyrG gene, important for filamentous growth and pathogenicity .One hypothesis emerging from this study is that vsiRNAs may influence host mRNA expression and lead to phenotypic changes in A. fumigatus genome and the viruses, it is hard to distinguish which location was the source and/or potential target. The second important observation was that pathogenicity and secondary metabolite process-related genes were incident with sRNA loci on the A. fumigatus genome. Although, for sRNA loci predictions, the source and target cannot be unequivocally distinguished using sequencing data, it is accepted that a region predicted based on perfect alignments of the incident reads  has a high probability of being the source locus [A. fumigatus [The analysis of sRNA loci allowed us to identify putative sRNA producing regions  in the hce locus , 43). Inumigatus . In addiumigatus . The sRNA. fumigatus. In addition, putative sRNA loci in the host genome during virus infection were identified. As differences in phenotype such as pigmentation and growth rate were noted between virus-free and virus-infected isogenic lines, it was considered that A. fumigatus dsRNA mycoviruses can affect gene expression via RNA silencing and can also affect pathogenicity. The work presented here inputs a wide range of information on sRNA profiles of A. fumigatus mycoviruses while offering preliminary data about milRNAs and possible targets of the sRNAs. Taken together, our data provide insights into RNA silencing against mycoviruses in an important human pathogenic fungus A. fumigatus.Here we report for the first time virus-derived sRNA in the presence of AfuCV, AfuPV-1 and AfuTmV-1 and also first miRNA-like candidates in A. fumigatus strains .All ns Table  were sprTotal RNA was extracted from 100 mg of mycelium using Trizol according to the manufacturer’s protocol. From the total RNA preparations sRNAs were enriched using the PEG-8000 precipitation method with slight modifications . BrieflyThe sRNA libraries from 2 μg sRNAs were generated using the ScriptMiner Small RNA-Seq Library preparation kit  according to the manufacturer’s protocol. The 5’ adapter was 5’GUUCAGAGUUCUACAGUCCGACGAUC3’ and the 3’ adapter was 5’AGATCGGAAGAGCACACGTCT3’. The cDNA libraries were amplified using ScriptMiner index primers including different barcodes to tag different samples. PCR amplification was performed using Phusion High-Fidelity DNA polymerase (NEB) and different cycles were set to determine the optimal cycle number of each sample. At least 2 PCR amplification reactions per sample were performed in order to maximise library yields. Following PCR amplification, libraries were size fractioned by electrophoresing the products twice through 10% native polyacrylamide gels and excising the bands of interest. Sequencing was done by the Earlham Institute  previously known as The Genome Analysis Center  using Illumina HiSeq2500 sequencing.The sRNA sequencing output files were delivered in FASTQ format. The sequencing was directional, single ended and the length of the reads was 50 nt. The data was analysed on a Linux server, using Perl (version Strawberry Perl 5.18.2.1) and R (version 3.0.3) custom made scripts. The UEA sRNA Workbench was used for the prediction of sRNA loci , 54.χ2 test.Sets of 6 samples, corresponding to each biological replicate, were loaded on a sequencing lane. To evaluate the equal loading of each library in each lane, we compared the distribution of total number of reads per library to a random uniform distribution (on the same lane sequencing depth), using the A. fumigatus, (ii) AfuCV, (iii) AfuPV-1 and (iv) AfuTmV-1. The A. fumigatus genome  has a length of 29.4 Mb and is divided into 8 chromosomes together with a 32 kb mitochondrial genome [Aspergillus Genome Database  to the fungal genome and three different viral genomes using PatMaN  allowingl genome . The ful . The se  between the treatments - virus-free versus virus-infected samples. A first measure to assess the replicate versus treatment sample similarity and localise the differences between the samples was the Jaccard similarity index which was calculated on the top 1000 and 5000 most abundant reads . Next, tlisation  adapted lisation  for eachlisation , (ii) dilisation . The sRNmiRNA identification was performed using three approaches; (i) similarity search against the miRBase database and known fungal miRNA-like entries, using a maximum of 3 mis-matches and no gaps (ii) miRNA prediction using miRCat , (iii) aTo detect conserved miRNAs, reads incident to either genome  were mapped to all mature miRNA sequences in the miRBase database  described in Mohorianu et al. . DiffereAdditional analyses were done to identify any putative new classes of vsiRNAs. With this aim, the viral presence plots were created. Next, the most abundant sequences mapping to the viral genomes were checked for differential expression using the normalized expression levels obtained from the analysis described in Section 2.6.2. Validation was done by northern blot analysis and potential targets were searched on the host genome using both plant and animal targeting rules , 8.A. fumigatus isolates were fractionated by electrophoresis through 7 M urea 16% denaturing polyacrylamide gels. Following electrophoresis the RNAs were transferred onto Hybond NX membrane (Amersham) using the semi-dry transfer system (Bio-Rad). Cross-linking was performed using 1-ethyl-3-(dimethylaminopropyl) carbodiimide at 60 °C for 2 h as described previously [32 labelled probes which were reverse complement oligonucleotides to the sRNAs of interest. To assess the relative expression levels, quantifications were done three times using ImageQuant software  and averages were plotted. The sequences of the probes used and validated sRNAs are listed in Additional file Northern blotting was used to validate the presence of vsiRNAs. Briefly, 2 μg of sRNA from virus-free and virus-infected eviously . HybridiP values were estimated using unpaired t-test with Welch’s correction. Error bars were calculated using the standard error values. P values less than 0.05 were accepted as statistically significantly different and are indicated with an asterisk.GraphPad Prism 6.0 software was used to analyse the qPCR results and Additional file 1:Size class distribution  and complexity (C) of the samples before and after genome matching to the fungal and viral genomes, respectively. Biological replicates were represented as two shades of same colour. Blue: all reads, green: reads matching to the fungal nuclear genome, red: reads matching to viral genomes. The CV, NK and PV correspond to Aspergillus fumigatus chrysovirus (AfuCV), a strain of Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) and Aspergillus fumigatus partitivirus-1 (AfuPV-1), respectively. (PDF 632 kb)Additional file 2:Quantile normalisation. MA plots and distributions of DE  separated per size class. In the MA plots, the average abundance (in log 2) is shown on the axis and the OFC (with offset = 20) is shown on the y axis. The CV, NK and PV correspond to Aspergillus fumigatus chrysovirus (AfuCV), a strain of Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) and Aspergillus fumigatus partitivirus-1 (AfuPV-1), respectively. (PDF 5547 kb)Additional file 3Presence plots indicating the distribution and abundance of (A) Aspergillus fumigatus chrysovirus (AfuCV) and (B) Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1)-derived sRNAs along the 4 segments of the related genomes. Double-stranded RNA segments are shown as 1, 2, 3 and 4 respectively. Colour code for the lines are red, green, orange and blue for 19 nt, 20 nt, 21 nt and 22 nt, respectively. Genomic coordinates were represented on the x axis. On the y axis, the point cumulative abundance of all incident reads  was represented. (PDF 8465 kb)Additional file 4:List of probes designed to validate selected virus-derived sRNAs. The CV, NK and PV correspond to Aspergillus fumigatus chrysovirus (AfuCV), a strain of Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) and Aspergillus fumigatus partitivirus-1 (AfuPV-1), respectively. (PDF 37 kb)Additional file 5:List of most abundant variants (with abundance greater than 100) which can hybridize with the PV probes. PV correspond to Aspergillus fumigatus partitivirus-1 (AfuPV-1). For each PV probe we present shorter and longer variant sequences, with up to 2 mis-matches relative to the probe. For each sequence we show the normalized abundance in the two infected replicates and the variant length. (PDF 38 kb)Additional file 6A. fumigatus. For each miRNA-like candidate we present the hairpin sequence, the mature sequence, incident annotation based on version Af293, version s03-m04-r22 of the A. fumigatus genome, the samples for which the candidate is differentially expressed and the corresponding secondary structure. (PDF 217 kb)Characteristics of miRNA-like candidates in Additional file 7:Characteristics of Folded-1 (PDF 787 kb)Additional file 8:Histograms showing the differentially expressed (−2 ≥ DE or DE ≥ 2) sRNA loci and annotations. The sRNA loci of all three viruses were identified and expressions plotted for CV (A), NK (B) and PV (C). The CV, NK and PV correspond to Aspergillus fumigatus chrysovirus (AfuCV), a strain of Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) and Aspergillus fumigatus partitivirus-1 (AfuPV-1), respectively. Annotations of the regions of putative sources of sRNAs were presented in red to indicate those differentially expressed in virus-infected cases and in blue to indicate the ones differentially expressed in virus-free examples. Comparisons were made for all three combinations . (PDF 1446 kb)Additional file 9:qPCR validations of RNAi genes and genes responding to the virus infection. (PDF 501 kb)


Simultaneous detection of multiple pathogens and samples (multiplexing) is one of the key requirements for diagnostic tests in order to enable fast, accurate and differentiated diagnoses. Here, we introduce a novel, highly scalable, photonic approach to multiplex analysis with single virus sensitivity. A solid-core multimode interference (MMI) waveguide crosses multiple fluidic waveguide channels on an optofluidic chip to create multi-spot excitation patterns that depend on both the wavelength and location of the channel along the length of the MMI waveguide. In this way, joint spectral and spatial multiplexing is implemented that encodes both spatial and spectral information in the time dependent fluorescence signal. We demonstrate this principle by using two excitation wavelengths and three fluidic channels to implement a 6x multiplex assay with single virus sensitivity. High fidelity detection and identification of six different viruses from a standard influenza panel is reported. This multimodal multiplexing strategy scales favorably to large numbers of targets or large numbers of clinical samples. Further, since single particles are detected unbound in flow, the technique can be broadly applied to direct detection of any fluorescent target, including nucleic acids and proteins. Early diagnosis and treatment are essential for preventing the transmission of the infectious agent and development of long-term complications2. Therefore, advancement of sensitive and accurate diagnostics systems with low complexity for use in low-resource settings is crucial. However, it is a challenge to accurately diagnose infectious diseases due to low natural concentration levels of the pathogens and their similar early symptoms. Current diagnostic tools, such as real-time polymerase chain reaction (RT-PCR), amplify the small amounts of target nucleic acids of the pathogens to be able to detect them5. Even though this technique is highly sensitive and specific, it is limited to one analyte type (nucleic acids) and not ideal for rapid point-of-care detection, since it requires complex procedures. On the other hand, eliminating the amplification step requires highly sensitive devices that are capable of reliable sensing of low concentration targets.Infectious diseases are responsible for a large fraction of fatalities around the world, accounting for more than 8% of all deaths7.In addition to a low limit of detection and large dynamic range, multiplex analysis is another key element of many diagnostic panels as different diseases can present with similar symptoms. Standard tests for influenza or other respiratory infections, for example, simultaneously screen for 4–8 pathogen types10. In the past couple of years, several optofluidic approaches have shown the capability to optically detect single biological nanoparticles such as viral particles (virions). These include resonance shifts in microspheres upon virus binding11, interferometric imaging of viruses bound to a functionalized surface12, and fluorescence imaging of virions using a smartphone camera13. However, despite their single particle sensitivity, implementing the other requirements of a diagnostic device  is not as straightforward in these approaches.Optofluidic approaches that seek to apply integrated photonic principles to biosensing, bioanalysis, and other applications have produced a number of promising devices15. Intersecting liquid-core and solid-core ARROWs allow for detection of single biomolecules in flow down to individual nucleic acids at clinically relevant concentrations18. Moreover, these silicon-based chips can be integrated with advanced sample preparation steps on dedicated microfluidic chips, enabling full sample-to-answer analysis of molecular biomarkers on a chip-based system20. In order to provide multiplexing capabilities, we recently introduced a new paradigm – optofluidic wavelength division multiplexing21. By replacing the single-mode solid-core excitation waveguide with an integrated multi-mode interference (MMI) waveguide22, well-defined multi-spot excitation patterns were created in a single fluidic channel over the entire visible optical bandwidth21. With proper design, different patterns can be generated by different excitation wavelengths that are matched to the absorption spectrum of fluorescent labels. This spatio-temporal encoding of spectral information allowed for direct identification of three different influenza strains labeled with different fluorescent tags with extremely high sensitivity and fidelity at clinically relevant concentrations. As an added benefit, the multi-spot excitation approach increases the signal-to-noise ratio and sensitivity of these biosensors, with a SNR enhancement of up to over 50,000 reported in experiments for 8 spot excitation23. However, this spectral multiplexing capability is ultimately limited by the finite absorption bandwidth of the dye labels which can lead to channel cross-talk if the absorption spectra overlap. While a further increase of the multiplexing capacity was demonstrated using combinatorial multi-dye labeling21, this approach still faces challenges for scaling to large target numbers.We have developed a biophotonic analysis platform based on Antiresonant Reflecting Optical Waveguides (ARROWs)single MMI waveguide that crosses multiple fluidic channels  waveguide is excited by a narrow single-mode waveguide to create location dependent multi-spot patterns for three orthogonally intersecting liquid-core waveguides Fig. . This al22. At certain propagation distances where the relative phases of these modes match up constructively, well-defined spot patterns are created. Here, we design the MMI and the intersection locations of the 3 liquid channels such that well-defined, but distinct, integer numbers of spots are generated at a certain wavelength in each channel. The dependence of the spot number (N) on distance from the beginning of the MMI (L) and the excitation wavelength λ, can be derived from standard MMI theory22 and is given by:w is the effective MMI-WG width (here: 75 µm) and nc the effective refractive index of the MMI-WG (here: 1.46). When λ = 633 nm; 8, 6, and 4 well-defined spots are produced at the MMI lengths of L1 = 1676µm, L2 = 2243 µm, and L3 = 3350 µm respectively. This provides a way for spatial multiplexing. In addition, we designed the chip such that we can choose another wavelength that generates a second set of distinct, integer spot numbers in each channel, adding the option for spectral multiplexing. For the exact same device, using Eqn.  was used to minimize propagation loss in the curved waveguide sections. Based on these simulations, the fan-out angle of the outermost collection arms is kept small (~8°) while keeping the waveguide short. Moreover, it is important to balance the signal strength from the first and third channels to increase our detection sensitivity. The MMI waveguide has a finite propagation loss which results in a smaller excitation power in the third channel, as compared to first and second channels. However, the emitted signal intensity from a fluorescent particle depends on the excitation power of each individual spot, which is given by the total excitation power at that channel, divided by In order to characterize the excitation spot patterns, 3 intersecting fluidic channels were filled with solutions containing a mixture of quantum dots which have a broad excitation spectrum. The MMI section was excited with the tunable laser source, and the wavelength is tuned to optimize the fidelity of the spot patterns. The most well-defined patterns were found at λ = 520 nm and λ = 633 nm. Their fluorescence when imaged with an overhead CCD camera shows the desired well defined excitation spots for all three channels and 2 separate wavelengths Fig. .For the first demonstration of a spatially multiplexed virus detection assay, red (Dylight 633) fluorescently labeled deactivated H2N2 virus solution was pipetted into the three input reservoirs. The solutions were then pulled through the 3 different liquid-core analyte channels and past the MMI excitation areas via negative pressure applied at the output reservoir. The laser source was tuned to 633 nm to excite the labeled viruses. As the targets traveled down the liquid core waveguides, they passed through 4, 6, or 8 different excitation spots, depending on the channel they were in. The resulting fluorescence signals were captured by the liquid-core waveguides, collected by the solid-core collection waveguides and combined into a single output waveguide by using the 3 × 1 Y-coupler see Fig. . The exc23. It should be noted that the fluctuation in the peak height can arise due to various sources – variation due to changes in a particle’s cross sectional position as it flows across the MMI channel, or optical variations introduced due to imperfect fabrication in the MMI section, or imperfect fiber alignment. However, particle identification only depends on the number of peaks in the pattern, or the temporal spacing ∆T between the peaks, which are very robust against the factors mentioned above.Figure This multi-channel multiplexing ability can be applied in different ways. For example, a test can be run for samples from three different patients to determine which ones contain the target virus and therefore used to screen for a disease in the three different patients simultaneously. Alternatively, different types of viruses can be placed in the three different reservoirs in order to do simultaneous detection of different targets with a single color. Finally, a single sample can be split and analyzed three times faster. Clearly, the multiplexing ability of the triple-core chip allows for versatile sample analysis and can therefore be adapted for different clinical needs.Next, we demonstrated the capability for joint  multiplexing, which significantly increases the multiplexing capacity of the device in a highly scalable manner. For the demonstration of a joint multiplexed virus detection assay, H2N2 deactivated virus type was labeled with red (Dylight 633) fluorescent dye and H1N1 deactivated virus type was labeled with green (Dylight 550) fluorescent dye. They were mixed and pipetted into the three input reservoirs and the flow was initiated via negative pressure applied at the output reservoir as before.Figure 5 particles/mL, which is in good agreement with previous assays at clinically relevant concentrations21. Combining multiple wavelengths with multiple channels enables detection and differentiation of many different target samples based on the number of spots, in the same experiment, without the need for a spectral filter to separate signals.Automated spot counting with a custom-written program identified with 97% accuracy as compared to manual inspection during identification, mostly due to distorted signals caused by flow variations. Furthermore, the velocities and the concentrations of the sample can be extracted from the dimensions of the excitation volume and the time difference between the peaks, respectively. The average velocity is found to be 0.87 cm/sec and the concentration of all viruses is estimated to be 6 × 106 in parallel by using the spatial-spectral capability described above. For these experiments, deactivated virus solutions of H1N1, H2N2 and H3N2 virus strains were labeled with green (Cy3) fluorescent dye, while those of Adenovirus (AV), and Parainfluenza PIV1 and PIV3 viruses were labeled with red (Cy5) fluorescent dye. Three separate virus mixtures were then prepared from the above fluorescently labeled solutions as follows: Solution A with virus strains H1N1 and PIV3; Solution B with virus strains H2N2 and PIV1, and Solution C with virus strains H3N2 and AV. Therefore, each solution A, B and C contained one virus strain labeled with the red fluorescent dye, and one virus strain with green fluorescent dye. Solutions A, B and C were then pipetted into input reservoirs for liquid channels 1, 2 and 3 respectively, with a negative pressure applied at the output reservoir to drive flow in the three liquid channels. The MMI was then excited by laser light at wavelengths of λ = 525 nm and λ = 633 nm to generate the desired six spot patterns in the three intersecting liquid channels.Finally, we demonstrate simultaneous screening for six different strains from a standard influenza panel24. For this assay, the peak number was determined using two independent methods, described in more detail in Supplementary Information (SI). Only if both methods produced the same result for the peak number, was the virus considered as identified. While this results in a somewhat lower identification rate than the single-step identification used in Fig. Figure In summary, scalable joint  multiplexed detection of influenza viruses was achieved on an optofluidic chip consisting of triple liquid-core waveguides intersecting a single solid-core MMI waveguide. We successfully detected and identified single viruses from 6 different strains simultaneously and distinguished among them based on the number of fluorescence peaks they generated. This approach enables scaling up MMI-based multiplexing to large target numbers. It facilitates simultaneous analysis of multiple bioparticles of different types on a single, fully integrated platform while taking advantage of established fluorescence technologies. It should be noted that at high concentrations where multiple particles are excited too close together to be temporally separated, it might not be always be possible to distinguish each particle individually. However this is not a concern at clinically relevant concentrations which are low enough to allow for single bioparticles to be probed individually by the excitation volume at a given time. Further, if the concentration of the sample was higher than the single molecular regime for our device, the sample can be diluted accordingly to suitable concentration ranges.21 we can further increase the capacity of multiplexing while using minimal number of different fluorescent dyes and color channels. For the present example, adding co-labeling with two dyes translates to nine possible targets for only two excitation colors. The optofluidic platform demonstrated here can also be integrated with a dedicated microfluidic layer for sample processing and distribution as shown in previous work25. This platform therefore serves as a powerful diagnostic tool by allowing the single-molecule detection and identification for variety of pathogens and biomarkers.By incorporating the multi-color labeling approach detailed inThe experimental setup implemented for multiplexed virus detection can be seen in Fig. Fluorescently labeled viruses passing through the MMI excitation volume are excited by the spatially distributed MMI spot pattern and thus generate multiple fluorescent peaks per virus in the collected time domain signal. These signals are captured by the liquid-core waveguide orthogonal to the excitation, transmitted into the collection solid-core waveguides, which then combine into a single solid-core waveguide via the Y-coupler. An avalanche photodiode (Excelitas) is used to detect the fluorescence signal after removing the excitation wavelengths with a set of filters (Edmund Optics 633 nm Notch Filter and Semrock 532 nm Long Pass). No spectral filters are required to separate the emission colors.For the first set of experiments to demonstrate spatial multiplexing and joint multiplexing with two strains of viruses, purified, UV-inactivated A/2/Japan/305/57 H2N2 Human Influenza Virus (Advanced Biotechnology Inc.) was labeled with Dylight 633 and β-Propiolactone-inactivated A/PR/8/34 H1N1 Human Influenza Virus (Advanced Biotechnology Inc.) was labeled with Dylight 550 NHS ester-activated dyes according to manufacturer specifications (Thermo Scientific). Unbound dye was removed by column chromatography using a PD MiniTrap G-25 column and 1 x PBS elution buffer , and efficient labeling was verified by TIRF microscopy.6, were purchased for testing - β-Propiolactone-inactivated A/PR/8/34 (H1N1), UV-inactivated A/2/Japan/305/57 (H2N2) (Advanced Biotechnologies Inc.), UV-inactivated purified Parainfluenza Viruses 1 and 3 and Adenovirus (ViraSource), as well as Formalin-inactivated A/Aichi/68 (H3N2) (Charles River). Viruses were labeled using NHS-activated Sulfo-Cyanine fluorophores (Cy3 and Cy5) according to manufacturer instructions (Lumiprobe). Briefly, virus samples were added to aliquots of dried Cyanine dyes and allowed to incubate at room temperature for 2 hours. Samples were then separated from free dye using PD MiniTrap G-25 gel filtration columns . Fractions were collected dropwise and tested for fluorescent virus and free-dye background on a Leica SP5 confocal microscope to determine if there was efficient labeling of the virus particles. Once identified, the viral fractions were aliquoted and flash-frozen via liquid nitrogen for later use.For our final set of experiments with six-fold multiplexing, six inactivated virus samples from the influenza panel2 (n = 1.47) and Ta2O5 (n = 2.107) were then sputtered over the whole wafer to thicknesses of 265 nm and 102 nm respectively, forming the ARROW layer stack, which acts as the substrate in subsequent fabrication steps. The three 6 × 12 µm hollow-core microchannels, which carry the biological sample in the final device, were defined using standard lithography procedures for SU-826, and then hard baked at a maximum temperature of 250 °C to withstand further processing. A self-aligned pedestal was defined as described in27 to improve the structural integrity of the hollow cores. This step protects the SU-8 core while etching the ARROW dielectric stack. Once the pedestal was defined, a 6 µm thick PECVD oxide layer was deposited on top using a low stress recipe to reduce potential core cracking28. The MMI and collection waveguides were then patterned by photolithography and etched with an ICP/RIE etcher to create 3 µm tall rib waveguides. The SU-8 cores were exposed at the corners by removing the oxide with buffered hydrofluoric acid and then placed in a strong acid to remove the SU-8, hollowing out the liquid-core channel29.The devices were created on top of a < 100 > oriented Si substrate. The mask alignments were designed to cleave across the (100) plane in order to produce clean unpolished facets. Six alternating dielectric layers of SiORequests for materials and data should be addressed to the corresponding author (Prof. Holger Schmidt)Supplementary information for Scalable Spatial-Spectral Multiplexing of Single-Virus Detection Using Multimode Interference Waveguides


We studied late convalescent serum samples from persons with prior DENV or Zika virus exposure. Despite extensive cross-reactivity in IgG binding, Zika virus neutralization was not observed among primary DENV infections. We observed low-frequency (23%) Zika virus cross-neutralization in repeat DENV infections. DENV-immune persons who had Zika virus as a secondary infection had distinct populations of antibodies that neutralized DENVs and Zika virus, as shown by DENV-reactive antibody depletion experiments. These data suggest that most DENV infections do not induce durable, high-level Zika virus cross-neutralizing antibodies. Zika virus–specific antibody populations develop after Zika virus infection irrespective of prior DENV immunity.Cross-reactive antibodies elicited by dengue virus (DENV) infection might affect Zika virus infection and confound serologic tests. Recent data demonstrate neutralization of Zika virus by monoclonal antibodies or human serum collected early after DENV infection. Whether this finding is true in late DENV convalescence ( Zika virus is a member of the flavivirus family of arthropodborne viruses, which includes West Nile virus, yellow fever virus, tick-borne encephalitis virus, and dengue virus (DENV) .50] for a single DENV serotype or Zika virus was >4-fold higher than for any other virus tested). In most cases, the person’s travel history corroborated the primary immune status. Serum that had high levels of neutralizing antibody to >2 flaviviruses were defined as secondary (repeat) flavivirus infections. Most secondary infection samples were from persons who had resided in DENV- or Zika virus–endemic countries for >5 years.Serum that had neutralizing antibodies to 1 DENV serotype or to Zika virus with minimal cross-neutralizing antibodies were defined as primary flavivirus infections   or Vero Cercopithecus aethiops monkey cells (ATCC no. CCL-81). C6/36 cells were grown at 32°C with 5% CO2 in minimum essential medium supplemented with 10% fetal bovine serum (FBS), L-glutamine, nonessential amino acids, and HEPES (2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer. Vero cells were grown at 37°C with 5% CO2 in Dulbecco modified Eagle medium supplemented with 5% FBS and L-glutamine. Virus stocks were titrated on Vero cells by plaque assay or focus-forming assay . Virus stocks were prepared in C6/36 p-nitrophenyl phosphate substrate (Sigma). Absorbance at 405 nm was measured on an Epoch plate reader . ELISAs used to confirm depletion were performed as given earlier, with the exception that 50 ng purified DENV was coated directly to the plate at 37°C for 1 h at 1:50 dilution before serum was tested. ELISA data are reported as optical density values that are the average of technical replicates, unless otherwise indicated. The average optical density for technical replicates using naive human serum at the same dilution factor as test samples serves as the negative control in ELISAs. In depletion experiments, the OD of depleted sample is expressed as percentage of control from same serum for some graphs as indicated.We measured binding of human serum IgG to DENV or Zika virus by ELISA as previously described  equation of Prism 6 . One set of DENV-1–4 neutralization values (DT003) was determined previously by U937 flow-based assay   with DENV and then concentrating DENV-containing supernatants at 4°C by tangential flow ultrafiltration using the Pellicon mini system with a 100-kD cutoff membrane . The flow rate was 400 mL/min, and filtration rate was ≈100 mL/min; pressure was 20–30 psi. Concentrated virus was then purified on a 15%–65% sucrose gradient by ultracentrifugation  at 21,583 relative centrifugal force for 18 h at 4°C. The fractions with maximal content of virus was determined by resolving fractions by SDS-PAGE and protein concentration was measured by Micro BCA Protein Assay Kit .As previously described  in accordance with the manufacturer’s instructions (100 μg/250 μL beads) by incubating overnight at room temperature. Control beads were incubated with equal amount of bovine serum albumin. Beads were blocked with 10 mg/mL bovine serum albumin, washed 3 times with 0.1 M borate buffer (pH 8.5), followed by 3 times with phosphate buffered saline. For depletion, serum was diluted 1:10 in phosphate buffered saline and incubated with 100 μg DENV-1 + 100 μg DENV-2 divided over 3 rounds at 37°C for 1 h each. After incubation, tubes were centrifuged at 20,800 relative centrifugal force to pellet beads with bound antibodies, and serum was pipetted off the undisturbed pellet and transferred to new vials. We confirmed depletion efficacy with direct binding ELISA. Serum with higher titers of binding antibodies was subjected to additional rounds of depletion until IgG binding was reduced to background levels.>2 flavivirus infections, including 2 persons exposed to both DENV and Zika virus.To study human antibody interactions between DENV and Zika virus, we assembled 36 late convalescent serum samples from persons exposed to DENV, Zika virus, or both . The panWe measured total IgG binding to DENV and Zika virus using a virus capture ELISA. We observed extensive cross-reactivity between DENV serotypes and between DENV and Zika virus, confirming that cross-reactive binding antibodies are maintained for many years after infection . Althoug50 >100) against at least 3 DENV serotypes, and 7 were highly neutralizing against all 4 serotypes . In general, IC50 titers were similar for primary Zika virus serum and for secondary Zika virus serum, consistent with the idea that cross-reactive antibodies from prior DENV infection do not contribute to Zika virus neutralization.Zika virus strains are divided into 2 genotypes, Asian and African; contemporary Zika virus isolates from Latin America are uniformly of the Asian genotype, consistent with the model that Zika virus spread from Southeast Asia to Oceania and from there to Brazil . All 4 sTo test the hypothesis that Zika virus infection elicits type-specific antibody, even in the presence of DENV immunity, we incubated serum with polystyrene beads coated with purified DENV antigen to remove DENV-specific and flavivirus cross-reactive antibody , Table 3Because Zika virus is emerging in areas with high rates of dengue prevalence, the extent of antibody cross-reactivity between these 2 viruses needs to be thoroughly evaluated. Recent studies have reported that DENV infection results in antibodies that cross-neutralize Zika virus  of 13 showed Zika virus neutralizing antibodies. The remaining 10 persons had no detectable Zika virus neutralizing antibodies despite having high levels of neutralizing antibodies to multiple DENV serotypes. DT165 and 166 are classified as secondary Zika virus strains on the basis of neutralization profile, epidemiologic context (these donors had fever and rash illness while residing in northeastern Brazil in 2015 during peak Zika virus transmission), and the presence of Zika virus type–specific antibodies in their serum. Accordingly, these persons have high titers of neutralizing antibodies to Zika virus in addition to neutralization activity to >1 DENV serotypes, with little to no cross-neutralization of viruses belonging to other flavivirus serocomplexes (>6 months after infection when the neutralizing antibody response has become more specific to the viruses responsible for infection.A key feature of acute DENV infection is the transient induction of high levels of flavivirus cross-neutralizing and cross-protective antibody (In essence, the question is whether Zika virus will behave as a “fifth serotype” of DENV or as a flavivirus outside the DENV serocomplex. Extensive cross-reactivity in IgG binding between these viruses clearly occurs, consistent with sequence homology and structural similarity among different flavivirus serocomplexes (–In the field, undifferentiated fever in the tropics can have a multitude of causes and often presents a diagnostic challenge (Interest and investigation are ongoing regarding whether Zika virus strain–dependent factors might explain phenomena observed during the current epidemic. Although a more comprehensive and in-depth analysis of Zika virus genetic variation might reveal viral determinants of pathogenesis, our experiments suggest that epitopes conferring susceptibility to neutralization by human serum have not changed substantially over time. We do find that the prototype Zika virus strain MR766 is more readily neutralized than other Zika virus strains. MR766 might provide the most sensitive screen for cross-neutralizing antibody, but relevance of such antibody should be confirmed by an isolate more representative of contemporary circulating viruses.–In conclusion, the current Zika virus epidemic presents many urgent challenges but also great opportunities to dramatically expand our knowledge of humoral immunity to flaviviruses. Although our results argue for infrequent cross-neutralization of Zika virus by DENV-immune serum and for development of independent populations of neutralizing antibody to these 2 viruses, additional and larger studies are needed to determine whether the rate of Zika virus cross-neutralization varies in different populations, particularly in DENV-endemic areas where ongoing subclinical exposure to DENV could further broaden the range of cross-neutralization in polyclonal serum. That effective vaccines have been developed against several flaviviruses is encouraging (Limited cross-neutralization between Zika virus; dengue virus and different Zika virus strains with similar neutralization profiles.