Plant roots establish interactions with several beneficial soil microorganisms including arbuscular mycorrhizal fungi (AMF). In addition to promoting plant nutrition and growth, AMF colonization can prime systemic plant defense and enhance tolerance to a wide range of environmental stresses and below-ground pathogens. A protective effect of the AMF against above-ground pathogens has also been described in different plant species, but it seems to largely rely on the type of attacker. Viruses are obligate biotrophic pathogens able to infect a large number of plant species, causing massive losses in crop yield worldwide. Despite their economic importance, information on the effect of the AM symbiosis on viral infection is limited and not conclusive. However, several experimental evidences, obtained under controlled conditions, show that AMF colonization may enhance viral infection, affecting susceptibility, symptomatology and viral replication, possibly related to the improved nutritional status and to the delayed induction of pathogenesis-related proteins in the mycorrhizal plants. In this review, we give an overview of the impact of the AMF colonization on plant infection by pathogenic viruses and summarize the current knowledge of the underlying mechanisms. For the cases where AMF colonization increases the susceptibility of plants to viruses, the term “mycorrhiza-induced susceptibility” (MIS) is proposed. In natural environments, plants interact with pathogenic and beneficial microorganisms that might affect their growth, performance and survival. Arbuscular mycorrhizal fungi (AMF) (subphylum Glomeromycotina)  establisEnhanced resistance of mycorrhizal plants against soilborne pathogens was often observed , while cThe boost of basal defenses in mycorrhizal plants was defined mycorrhiza-induced resistance (MIR) and several studies pointed to priming  as a maiViruses are obligate pathogens able to infect virtually all organisms, including plants. Their infection process depends on the host machinery, allowing the virus to multiply and spread in the host. In plants, virus infections generally induce a disease syndrome, with symptoms such as developmental abnormalities, necrosis and chlorosis. For all major agronomical crops, viral diseases cause huge losses in production and quality, representing a serious threat to global food security . Virus iSustainable and effective approaches to limit viral diseases include the development of viral-resistant/tolerant crop, the integration of crop management strategies to reduce the disease spreading , the intAMF inoculation has been proposed as a cost-effective and sustainable solution for plant virus control. However, despite some information were available from the early 70’s, the studies on the effects of AMF on plant-virus interactions are surprisingly low and contradictory. The aim of this review is to summarize the actual knowledge on the effect of AMF on virus infection and the underlying mechanisms. We propose the term “mycorrhiza-induced susceptibility” (MIS) for the cases where a better performance of the virus, defined by replication efficiency and induced symptomatology, is observed in mycorrhizal plants. Furthermore, the state of the art on the effect of virus infection on mycorrhization will be reported; finally, we suggest different aspects that deserve further investigations.Solanaceae or Cucurbitaceae plant species and positive single stranded RNA viruses, with the exception of Table 1). In Tomato yellow leaf curl Sardinia virus (TYLCSV) although AMF colonization could not contrast the reduction of root biomass induced by the virus. Since, TYLCSV encodes proteins able to interact with the plant hormone pathways : milder symptoms and a significant stimulation of shoot growth were observed in PVY-infected plants inoculated with Rhizophagus irregularis.Differently from R. irregularis and infected by Tobacco mosaic virus (TMV) and Cucumber green mottle mosaic virus (CGMMV), respectively, showed reduced disease symptoms and virus titre if compared to non-mycorrhizal plants . Results indicate that mycorrhizal colonization facilitates or enhances virus multiplication, suggesting a prevailing detrimental effect of AMF on plant virus infection, for which we propose the term “mycorrhiza-induced susceptibility” (MIS). Even if, in the first days after inoculation by Tomato aucuba mosaic virus (now a TMV strain), the virus titre in tomato plants colonized by Funneliformis macrocarpa (formerly Endogone macrocarpa) was lower in respect to the control ones, at 8–12 dpi, it became higher in mycorrhizal plants and increased over time (Potato virus X (PVX)  and experimental design . Several studies report increased virus multiplication and/or symptom severity in infected mycorrhizal plants. They considered plants belonging to the ver time . Similar X (PVX) . No data X (PVX)  and was Glomus sp. The MIS outcome, consisting in viral titre increase and worsening of symptoms, was confirmed by Tomato spotted wilt virus (TSWV) titre in infected mycorrhizal tomato plants at 34 and 56 dpi, but not at 14 dpi, compared to non-mycorrhizal controls. A delay in recovery .More recently, 2, observed that, under elevated CO2 concentration, the titre of both Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV) increases in the grasses Bromus hordeaceus L. and Avena fatua L. colonized by R. irregularis; no differences were observed under normal CO2 concentration.Claroideoglomus etunicatum (formerly Glomus etunicatum) on sour orange and Duncan grapefruit seedlings infected by Citrus tristeza virus and Citrus leaf rugose virus, respectively, observed that AMF colonization did not significantly reduce the pathogenic effect caused by virus infection. A reduction of roots fresh weight and plant growth in the virus-infected mycorrhizal plants in respect to non-infected mycorrhizal plants was found. Similarly to Less investigated is the effect of mycorrhization on fruit trees. F. mosseae intraradical development. This result is consistent with the up-regulation, in both mycorrhizal and TYLCSV-infected mycorrhizal plants, of five selected plant genes previously described as mycorrhiza-responsive and preferentially expressed in arbuscule-containing cells. Similarly, 2 concentration, BYDV and CYDV infection increased the fungal colonization of roots, suggesting that, in this case and condition, AMF and virus interacted to stimulate each other success.Most studies addressing the plants-viruses-AMF interaction mainly focused on the effect of AMF on virus infection; however, viral infection can impact mycorrhization. In this regard, Figure 1). In the early stage of AMF colonization, MIR has been associated with SAR (Systemic Acquired Resistance)-like priming of SA-dependent genes, while in the later stage, MIR coincides with priming of JA- and ethylene-dependent defenses. In addition, ABA has been proposed as a new candidate acting as a complementary long-distance signal controlling MIR, and several reports considered JA and its derivates  as key signals operating in this process . Indeed, the interaction among virus, AMF and plant is a complex system where several factors, including viral pathogen lifestyle, plant nutritional status and timing of interaction, can move the dynamic equilibrium toward the final establishment of a MIS or MIR outcome. A key role is probably played by the hormonal crosstalk that finely tunes both plant-AMF and plant-virus interactions ( process , 2015. I process . Interes process  and regu process . This me process  and addist plant , suggestR. irregularis can improve the survival of the aphid Macrosiphum euphorbiae, vector of Cucumber mosaic virus, thus possibly improving viral spread, but also activate indirect defenses, attracting the aphid parasitoid Aphidius ervi (The complexity of the AMF-plant-virus interaction may further increase if considering that mycorrhiza can impact other trophic levels such as the interaction between plants and insects , includiius ervi .These observations highlight the intricate network of processes that regulate the plant-virus-AMF interaction, and, far to be conclusive, indicate that several factors able to direct the dynamic equilibrium of the system toward a MIS or MIR outcome remain to be evaluated such as the different changes induced by AMF colonization performed before or after viral infection, and the importance of timing in evaluating the interaction outcome. Moreover, since only few AMF have been analyzed so far, future studies should consider different AMF species and isolates  and evenAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Chiroptera (meaning “hand-wings” in Greek), which is the second largest order of mammals in terms of species number. Thus far, there are more than 1300 known species of bats across six continents, constituting 20% of the total number of mammalian species. Indonesia, for example, is home of more than 200 bat species, the largest variety in the world. Bats are monophyletic. Traditionally, they were classified into two suborders: Megachiroptera (megabats) and Microchiroptera (microbats). In general, megabats are larger than microbats, although some large microbats can be larger than some small megabats. Most megabats are frugivorous and eat fruit, nectar or pollen and most microbats are insectivorous. Some microbats also eat small vertebrates or even other bats. Recently, extensive molecular studies and phylogenetic analysis have led to a revision of the classification of bats into two suborders, Yinpterochiroptera and Yangochiroptera, of which Yinpterochiroptera includes the megabats and a number of microbat species. In general, bats are nocturnal animals that are mainly active during night time, dusk or dawn. They roost in caves, trees, mines and water tunnels and most rest and sleep in an upside-down position. They usually live in groups known as colonies, with hundreds to thousands of individuals, which may facilitate intraspecies transmission of viruses. They are capable of echolocation, which help them to locate themselves as well as their food. Bats are a unique but diverse group of mammals in the order Alphacoronavirus and Betacoronavirus in the current model of coronavirus evolution, the only two coronavirus genera with coronaviruses that are known to infect humans [Tylonycteris bat CoV HKU4 and Pipistrellus bat CoV HKU5 in Tylonycteris pachypus and Pipistrellus abramus, respectively, have also greatly facilitated the study of the Middle East Respiratory Syndrome coronavirus when it emerged in the Middle East in 2012 [The high diversity of bats, and hence the highly diversified cell types and receptors, have facilitated this group of animals to harbor a myriad of viruses. Bats are well recognized to be the hosts of a number of highly pathogenic viruses, such as rabies virus, Hendra virus, Nipah virus and Ebola virus, for a long time. Since the discovery of bats as the reservoir of Severe Acute Respiratory Syndrome (SARS) coronavirus ,2, theret humans . The dis in 2012 ,17. In fIn addition to its diversity, the capability to fly also facilitate bats to disseminate the viruses they harbor, and hence, increases the chance of both intraspecies and interspecies transmission, which involves two bat species or one bat species and another kind of animal. With their forelimbs adapted as wings, bats are the only mammals that are capable of sustained flight. Echolocation enables bats to fly in dark places. Bats have long finger-like bones in their wings, and hence are more maneuverable than the wings of birds. However, unlike birds, they do not have a lot of lift. Furthermore, the fact that they are nocturnally active helps them avoid direct competition with birds. The ability to fly makes bats important vehicles for the dissemination of seeds and pollens, as well as viruses. Contacts between human and bats or bat products are not uncommon. Huge bat caves, such as the Batu Caves in Malaysia, are major tourist attractions. Caves explorers are sometimes bitten by bats, and their mucous membranes or wounds may contact the saliva, urine, etc. of bats, resulting in virus transmission. In addition to these direct contacts, bats are used as food in southern China and some parts of Asia such as Indonesia. A variety of bat dishes, minced bat meat, and even hot pot with the whole bat cooked in a pot of soup are available in restaurants in southern China. Dried bat droppings are used as traditional Chinese medicine, for the treatment of diseases such as night blindness. Bat dung that is mined in caves as guano can be used as organic fertilizers. All these uses of bats and their derived products have created countless opportunities of human-bat interaction, and hence, have increased the chance of virus transmission. In the recent few years, numerous novel bat viruses have been discovered and other downstream studies performed. In this special issue, a number of novel bat viruses, including coronavirus, poxvirus, paramyxovirus and orbivirus, were described ,22,23,24


African swine fever is one of the most dangerous diseases of swine. We confirmed the 2019 outbreak in Vietnam by real-time reverse transcription PCR. The causative strain belonged to p72 genotype II and was 100% identical with viruses isolated in China (2018) and Georgia (2007). International prevention and control collaboration is needed. Since its first identification in East Africa in the early 1900s, African swine fever (ASF) spread to Kenya in the 1920s; transcontinental outbreaks in Europe and South America in the 1960s and in Georgia (Caucasus) in 2007 led to subsequent transmission to neighboring countries east of Georgia. Along with the outbreaks in the eastern territory of the Russian Federation, acute ASF outbreaks were reported in China in 2018 . http://www.mediandiagnostics.com). We named the detected ASF virus VNUA/HY-ASF1 and deposited the following complete genome sequences into GenBank: p10 (accession no. MK795932), p11.5 (MK795933), p12 (MK795934), p14.5 (MK795935), p17 (MK795936), p22 (MK795937), pE248R (MK795938), p30 (MK757460), p54 (MK554697), p72 (MK554698), and Cd2v (MK757459). We aligned the nucleotide sequences by using BioEdit version 7.2  with ClustalW  and calculated sequence identity. Using MEGA7 (https://www.megasoftware.net)and the neighbor-joining method, we based phylogenetic analysis on the genetic information and calculated bootstrap values with 1,000 replicates. The genotype was determined by p72 gene characterization as reported previously  from dying pigs and submitted them to the diagnostic laboratory at the Vietnam National University of Agriculture for ASF diagnosis. All specimens underwent homogenization, followed by extraction of viral DNA . However, the clinical forms and pathophysiology of ASF varied according to virus virulence, exposure dose, and transmission route.http://www.fao.org/3/i8805en/I8805EN.pdf). Therefore, it is likely that the virus originated in China.Considering the epidemiologic features of the site where ASF has recently occurred, the virus is highly likely to have reached Vietnam via infected wild boar, by movement of pigs and pork products, or by infected fomites  increases the possibility of virus spread to nearby swine-raising Southeast Asia countries, including Laos, Thailand, Cambodia, and Myanmar. Although ASF has occurred in many countries, including Russia and Europe, its outbreak in Asia is far more critical because 60% of the world’s pig population is concentrated in that area and the socioeconomic effects of swine disease would be greater than that in other regions. Therefore, to avoid great economic losses worldwide, we highly recommend that preventive and control measures be developed and implemented through international collaboration.


Following the principle of structure deciding the properties, a reformation in the molecular structure of A2B copolymer is made by appending three segments of a third component C with the same length to the three arms, resulting (AC)2CB 3-miktoarm star terpolymer. A reverse micellar structure in self-assembly is expected by regulating the C length and the pairwise repulsive strength of C to A/B, aiming to enrich its application range. Keeping both A and B lengths unchanged, when the repulsion strength of C to A is much stronger than C to B, from the results of mesoscopic simulations we found, with a progressive increase in C length, (AC)2CB terpolymer undergoes a transition in self-assembled structures, from a cylindrical structure with B component as the core, then to a deformed lamellar structure, and finally to a cylindrical structure with A component as the core. This reverse micellar structure is formed with the assistance of appended C segments, whose length is longer than half of B length, enhancing the flexibility of three arms, and further facilitating the aggregation of A component into the core. These results prove that the addition of a third component is a rational molecular design, in conjunction with some relevant parameters, enables the manufacturing of the desired self-assembly structure while avoiding excessive changes in the involved factors.The most common self-assembly structure for A The thrLx × Ly × Lz spatial lattice of a 50 × 50 × 5 nm3 grid, whose width in the Z axis is 10% of the width in either the X or Y axis, to model thin films. Additionally, the bond length is set as 1.1543 to ensure the isotropy of all grid-restricted operators. To neglect the size influence on self-assembly, all beads are an equal size of 0.01 nm3 for simplicity. In MesoDyn simulations, the bead diffusion coefficient provides the link between the real timescale of the simulation and the dimensionless units, and a default value of 1.0 × 10-7 cm2·s-1 is adopted. Owing to the inverse relationship of the dimensionless noise parameter to the level of thermal noise applied, a maximum value of 100 for the noise-scaling parameter is employed to realize the minimum in noise. Lastly, the compressibility parameter is fixed at 10.0, the dimensionless time step is set as 0.5, and each simulation is carried out at a constant temperature of 400 K for 1.0 × 105 steps .Compared with three dimensions (3D), some structures that result from the simulation in two dimensions (2D) may be missed, such as bi- or tri-continuous gyroid structures. However, in industry, many materials, benefiting from 2D ordered structures fabricated in thin films, manifest great potential, which reveals the structure control in 2D space possesses an essential significance that cannot be replaced. Therefore, all the simulations in this paper are carried out in the 2B topology are also simulated under the interaction situation of C[00], C[55], C[15], and C[51]. The relatively low composition of C beads compared with that of A and B beads, as well as the innermost location of the C bead, both severely restrict the C bead’s influence on the other components, resulting in no apparent difference in the average sizes under various interaction conditions. With the increase in the length of the C segment, the tendencies in the change of the average size under various conditions present evident differences. Only the average sizes of systems with the C[00] interaction show an almost direct proportional change tendency with the increasing length of each C segment. For the same kind of non-preferential interaction of C[55], whose change tendency for the average size is similar to that of C[00], the strong repulsion of C beads to both A and B beads results in a larger average size than that of C[00]. Particularly, the 3C4 system exhibits the highest value of the average size and breaks the monotonically increasing tendency of average size, which will be given a detailed analysis later. With increasing C beads, the change tendency for the average sizes with C[15] interaction approximately rises at first and then decreases. Except for the 3C3 system, whose average size is the highest and higher than the same system with the C[00] interaction, the other systems with the C[15] interaction all possess an average size that is less than that of the corresponding system with the C[00] interaction. The systems with the C[51] interaction are relatively complicated, as only the single addition of a C bead per C segment results in a tremendous increase in the average size of the 3C1 system. Then, two non-cylindrical structures are formed for 3C2 and 3C3 systems. Finally, the 3C4, 3C5 and 3C6 systems are cylindrical in structure, but the constituent components of the core and shell are reversed, and a decreasing trend for their average sizes is displayed.Most of systems assemble into cylindrical structures, whose average sizes are readily calculated and are shown in Taking the 3C4 system as an example, from a track of average size with time shown in The C[00] and C[55] interactions are two typical representatives of non-preferential interactions, but their distinctly different repulsive strengths for the interaction between C beads and A and B beads cause their different self-assembly structures, as shown in As shown in For comparison, the same kind of density profiles for the 3C4 system affected by the C[15] interaction are shown in Different from the rising trend (or roughly rising trend) in the average size changing with the increase in the number of inserted C beads present in non-preferential interaction systems , a decreasing trend is present in the systems with four or more C beads in each inserted C segment under a preferential interaction , which results from their distinguishing structures. At first, leaving aside the position exchange of the A and B components under two preferential interactions, an outstanding feature lies in the overlapped distribution of the C beads and the A or B beads also making up the shell part and is distinct from peaks associated with the aggregation of C beads between the aggregation of A beads and the aggregation of B beads under a non-preferential interaction. Furthermore, the wide distribution of C beads and the sharing scope of A or B beads reinforce the repulsive strength interacting with the components making up the core. Thus, the increasing repulsion from more C beads ‘squeezes’ the components of cores inward to become more condensed, resulting in the production of smaller-sized cylinders; this result is presented as the decreasing average sizes in 3 steps, an initial partition forms via the aggregation of the same component, with four peaks of A beads found following the selected route. Additionally, not only are the aggregates in various patterns, but also the value and scope of the four peaks are not distinct. After 0.1 × 103 steps, further aggregating of the same component causes the growth in densities of the four peaks. In the following 1.2 × 103 steps, along with the fusion of small aggregates and the splitting of large aggregates happening simultaneously, the irregularity of aggregating patterns is reduced, and the surrounding C-shells are gradually constructed. The completion of normal-shaped cylinders along the selected route is first gained in 2.5 × 103 steps, but the acquirement of a uniform density distribution among the four cylinders is achieved in the subsequent 1.2 × 103 steps. Finally, including the four cylinders mentioned above, other cylinders also achieve the normal shape, even layout of position and uniform density distribution. Three long-chained C segments situated between A and B segments are helpful for inverting the core-forming component, i.e., A beads aggregating in the core region, in conjunction with the concurrent fusion and splitting among aggregates, brings about the least time needed to reach the equilibrium state of average size, whereby the amplitude of variation in the changing curve of the average size is also the smallest, as shown in Due to the particular structure formed by the 3C4 system under the C[51] interaction, the formation process of the reverse cylindrical micellar structure is necessarily studied, as shown in 2CB 3-miktoarm star terpolymer in a thin film have been studied via MesoDyn simulations. To minimize the number of factors influencing the self-assembly, only the length of C segment and the interaction between the C component and A/B component are changed.The self-assembled structures and mechanism of an (AC)Owing to the extraordinary high value of the average size of the 3C4 system affected by C[55], as well as the 3C4 system being also on two decreasing lines for the average size with the increase in C segment affected by C[15] and C[51], the 3C4 system is selected as a representative system for further analysis. Different forming processes can be speculated upon from the trajectory of the average size with time under different interaction conditions, that is, a splitting process (under the C[00] interaction), multistep coalescence process (under the C[55] interaction), three-step coalescence process (under the C[15] interaction), and speedy uncertain process with no clear change (under the C[51] interaction).After a detailed analysis of the densities, the associated mechanisms can be revealed definitely. For the C[00] interaction, where the repulsions between C beads and A and B beads are both zero, leading to a nonselective distribution of C beads in A- and B-aggregating regions, the interaction between A and B beads weakens, and then, the unstable cylindrical structure shows a wide-amplitude of change in the average size starting from the splitting of several bulk aggregates. For the C[55] interaction, where the repulsions between C beads and A and B beads are equal and both strong, giving rise to a C shell forming between A and B aggregations, the coalescence of pairwise smaller aggregates is accompanied by the change in the surrounding type of C shell and is presented as a multistep coalescence mechanism for the self-assembly process. For the C[15] or C[51] interaction, where the C beads preferentially repulse B beads or A beads, causing the covering of C-aggregating regions on A-aggregating regions or B-aggregating regions, respectively, to compose an outer shell. This ‘squeezing effect’ from such a heterogeneous outer shell strengthens with the C-segment length, so a decreasing trend in average size can be interpreted. The most delightful thing is the appearance of the reverse cylindrical micellar structure, and it is also a result of rational molecular design, which happens in the 3C4 system under the C[51] interaction. This result fills in the shortage of such reverse micellar structures by changing only two influential factors and offers a significant opportunity in the design of materials with novel structure, which enables the fabrication of a wide range of useful devices.


Beads of different sizes were formed using a compression-molding method, and their morphology was evaluated via scanning electron microscopy. Intrabead incorporation of antifungal agents was evaluated via Fourier-transform infrared spectroscopy, and in vitro fluconazole liberation curves of PLGA beads were inspected via high-performance liquid chromatography. When we implanted the drug-incorporated beads into the bone cavity of rabbits, we found that a high level of fluconazole (beyond the minimum therapeutic concentration [MTC]) was released for more than 49 d in vivo. Our results indicate that compression-molded PLGA/fluconazole beads have potential applications in treating bone infections.Fungal osteomyelitis has been difficult to treat, with first-line treatments consisting of implant excision, radical debridement, and local release of high-dose antifungal agents. Locally impregnated antifungal beads are another popular treatment option. This study aimed to develop biodegradable antifungal-agent-loaded Poly( Candida, Aspergillus, and other common fungi are characterized by the formation of a biofilm that resists antifungal treatment, thus further strengthening the infection [Fungal osteomyelitis is an uncommon disease presenting significant challenges to orthopedic surgeons. The severity of a fungal infection is associated with the immune status of individuals and the fungal species. A large number of fungal infections have been reported in both immunocompromised and immunocompetent individuals . Fungal nfection . Certainnfection . Howevernfection ,7.Candida spp., and Candida albicans in particular [Candida albicans infections [Candida infections [Candida osteomyelitis involves surgical debridement and long-term administration of antifungal agents. The guidelines of the Infectious Diseases Society of America (IDSA) for the treatment of Candida osteomyelitis suggest the administration of fluconazole for 6–12 months [The most common fungi causing bone and joint infections are rticular . Fluconafections . Furtherfections . Treatme2 months . Current2 months . The bea2 months . Further2 months . An ideaBiodegradable antifungal-agent-loaded beads possess advantages over conventional PMMA beads in four ways. First, biodegradable beads provide high concentrations of antifungal agents for the extended time needed to completely treat the particular orthopedic infection. Second, variable biodegradability from weeks to months permits various types of infections to be treated. Third, the biodegradable vehicles degrade eventually, and surgical removal of the beads is not required. Fourth, the biodegradable beads dissolve gradually and the soft tissue or bone defect slowly fills with tissue, it is thus not necessary for bone/tissue reconstruction .d,l-lactide-co-glycolide) (PLGA) beads and assessed drug release dynamics in vitro and in vivo. Among the various polymeric materials available to develop local drug release systems, PLGA is promising, owing to its degradable polymer that facilitates long-term drug delivery at high doses to the target region [This current study developed biodegradable antifungal-agent-loaded vehicles for a long-term drug release. We utilized a compression-molding method to fabricate fluconazole-incorporated Poly, and intrabead antifungal drug incorporation was assessed via Fourier-transform infrared (FTIR) spectroscopy. In vitro fluconazole liberation curves of PLGA beads were inspected via high-performance liquid chromatography (HPLC), and in vivo fluconazole release was investigated by implanting the drug-incorporated beads into the bone cavity of rabbits. We found that a level of fluconazole greater than the minimum therapeutic concentration (MTC) was released for more than 49 d in vivo, indicating that compression-molded PLGA/fluconazole beads are a promising candidate for the treatment of bone infections.All materials utilized in this study, including PLGA (50:50), amphotericin B, and fluconazole, were acquired from Sigma-Aldrich .PLGA/amphotericin B and PLGA/fluconazole beads were prepared using a laboratory-scale compression-molding system equipped with an isothermal oven with a temperature range of 25 °C to 300 °C. Beads of two different polymer:drug ratios (6:1 and 4:1) and three sizes  were fabricated. PLGA, amphotericin B, and fluconazole at predetermined weights were first mixed using a dry mixer and placed in molds  customizN = 3) containing 1 mL buffered solution at 37 °C. The tubes were deposed in an isothermal oven for 24 h, and the eluent was harvested and substituted with fresh solution (1 mL). The process was carried out in duplicate for 40 d. Amphotericin B and fluconazole levels in the harvested eluents were quantified via HPLC, carried out using a Hitachi L-2200 System . All experiments were performed in triplicate (N = 3).In vitro release of amphotericin B and fluconazole from the biodegradable beads was assessed via an elution method. PLGA/amphotericin B and PLGA/fluconazole beads were placed in test tubes (−1 (32 scans). The drugs were compressed as KBr discs, and spectra were recorded over 400–4000 cm−1. Fluconazole was considered stable if no obvious structural changes occurred due to temperature. The FTIR spectra of pure PLGA beads were compared with those of fluconazole-loaded PLGA beads.The thermal stability of fluconazole was determined via FTIR spectrometry to evaluate whether the chemistry and orientation of material structures varied with temperature. FTIR analysis was conducted using a Nicolet iS5 spectrometer  at a resolution of 4 cmN = 3).The water contact angles of the beads were measured on a contact angle measurement device  (4 cells/well) in Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 °C and 5% CO2/95% for 48 h. Cell viability was monitored via the CCK-8 assay and quantified using an ELISA reader.Cytotoxicity of fluconazole-loaded PLGA beads was examined via a Cell Counting Kit-8 (CCK-8) assay  for cell viability, in accordance with the manufacturer’s instructions. Eluents harvested at 1, 2, 3, 7, and 14 d were placed in 96-well culture plates. Human fibroblasts obtained from foreskins of patients (1–3 years of age) undergoing surgery were seeded , and all experimental animals were provided care in accordance with the regulations of the Ministry of Health and Welfare of Taiwan under the supervision of a licensed veterinarian.Four adult New Zealand white rabbits  weighing approximately 3.5 ± 0.3 kg were enrolled in the experiment. The rabbits were housed in individual cages in a temperature- and light-controlled room, with ad libitum access to standard rabbit chow and sterilized drinking water. All animals were administered general anesthesia via inhalation of isoflurane through a vaporizer  in a plastic box (40 cm × 20 cm × 28 cm). Anesthesia was maintained during the entire surgical procedure via mask inhalation of isoflurane.2) was induced at the right femoral middle site, and a polymethylmethacrylate (PMMA) spacer was initially inserted. The wound was closed with 3-0 Vicryl sutures . After 2 weeks, the PMMA spacer was surgically excised and fluconazole-impregnated PLGA cylindrical beads (5.0 × 6.0 mm2) were placed into the right femoral bone cavity  in a designated fluconazole mid-resistant infection was 64–128 μg/mL. In a fluconazole-susceptible strain of Candida, the median MIC was 0.5 μg/mL [50) for more than 49 d (Sustained local release of high levels of antifungals contributes to infection control. Louie et al. reported that the minimum inhibitory concentration median (MIC.5 μg/mL , and thehan 49 d . FurtherAlthough the current study has generated promising preliminary data, some limitations should be noted. First, we used a non-infected animal model, and therefore it is unclear whether the antifungal beads might perform differently in infected tissue. Further evaluation of the antifungal agent-embedded PLGA copolymer beads in an animal model of fungal infection is necessary to address this limitation. Second, despite the experimental data showing that 80% of the drug was released at the end of the study, it would have been faster given larger amounts of liquid. The sink condition analysis is needed to ensure the drug was released freely. Third, although no obvious sign of inflammation was observed in the in vivo test, the influence of controlled release of drugs and carriers on the local irritation should be further examined. Finally, the relevance of the present findings to patients with bone infections remains unclear and warrants further investigation. We intend to further explore these topics in future studies.This study is the first to perform in vitro and in vivo analyses of drug release dynamics in the bone cavity from compression-molded antifungal-incorporated PLGA beads prepared without organic solvents. Biodegradable PLGA/fluconazole beads released high concentrations of antifungals for over 49 d at the target site, while the antifungal agent blood concentration remained low. In summary, the compression-molded PLGA/fluconazole beads that we describe here have potential applications for treating bone infections.


Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant’s antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants.H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed.In the present study, we investigated the virome of leaf and sapwood samples from native Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host.Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a ‘very difficult to manage’ sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.The online version of this article (10.1186/s12985-018-1095-3) contains supplementary material, which is available to authorized users. Hevea brasiliensis (the rubber tree) is described as source of higher quality latex and rubber among all known plant species. The latex produced by this plant has a set of features such as elasticity, abrasion and impact resistance, heat dispersion and malleability, which makes it the ideal feedstock for building products in many different areas, such as engineering, medical and pharmaceutical industries [H. brasiliensis latex irreplaceable [dustries , 2. To dlaceable . Naturallaceable .Pseudocercospora ulei [H. brasiliensis is also known to be susceptible to many other diseases caused by fungi [Colletotrichum gloeosporioides [Oidium hevea [Carlavirus, which is associated with the Leaf Disease of Viral Origin [Currently, more than 90% of natural rubber production occurs in Asia, mainly in Malaysia, Thailand and Indonesia, even though the rubber tree originated in the Brazilian Amazon . Commercora ulei . Apart fby fungi , such asum hevea . Virus hl Origin .In response to viral infections, eukaryotic organisms have developed different strategies to protect their genomes. RNA interference (RNAi) is an important regulatory mechanism to induce the silencing of self and non-self RNA through sequence-specific homologous interactions . In thisTherefore, the sequencing of sRNAs is a useful tool for detecting phytoviruses, also in asymptomatic and infected plants , 18. TheH. brasiliensis trees using sRNA deep sequencing, bioinformatics tools and PCR techniques. This strategy allowed to identify viral sequences, which after their assembly, were found to be phylogenetically related to Grapevine fleck virus (GFkV), genus Maculavirus. Our findings expand the knowledge about the H. brasiliensis virome and suggest that other studies are necessary to explore the viral biodiversity in this plant.Here, we investigated the virome of asymptomatic native H. brasiliensis species prevail in these areas, which are characterized by a short dry season and excessive precipitation, with a mean annual temperature of 25.9 °C and 82% humidity. The permissions for the study were obtained from the Sistema de Autorização e Informação em Biodiversidade (SISBIO), authentication code 31162617.This study was carried out in two different areas in the state of Pará, Brazil: Caxiuanã National Forest - CNF  and Tapajós National Forest - TNF  Fig. . Native Leaf and sapwood samples were randomly collected from five adult individuals in CNF (C1-C5) and from 10 individuals in TNF (T1 – T10), from which five were adults and five were plantlets. All collected samples were originated from asymptomatic plants with the leaves showing homogenous green coloration without any kind of discoloration, wilting, or necrotic lesions. Five fragments from each leaf Fig.  and fiveLeaf fragments from CNF were ground with liquid nitrogen and 500 mg aliquots were used for RNA extraction using TRIzol Reagent®  following the manufacturer’s recommendations. The quality and quantity of RNA were evaluated using spectrophotometry  and automated electrophoresis systems .RNA samples were stored with 30 μL of RNA protection reagent . The samples were prepared for sequencing with the NEXTflex Small RNA – Seq Kit V3 and sRNAs were selected by size (15–35 nt) in denaturing SDS-PAGE electrophoresis  and sequenced using Illumina HiSeq .H. brasiliensis reference sequences available on the National Center for Biotechnology Information - NCBI (https://www.ncbi.nlm.nih.gov/) using Bowtie, allowing one mismatch [H. brasiliensis genome was downloaded from the Genome Online Database – GOLD (https://gold.jgi.doe.gov).Pre-processing of sRNA libraries and virome analyses were performed as described by . Brieflymismatch . The H. in-house Perl scripts and plotted using the R program with the package ggplot2 [Sequences that did not present similarities with bacteria, fungi or the host were used for contig assembly and subsequent analyses. Assembled contigs greater than 50 nt were characterized based on sequence similarity and pattern-based strategies. The identification of conserved domains was performed using HMMER . The sRN ggplot2 .e-value of 1e− 5) to select the closest references. Once the genome viral references were selected  using Blast , quantitative PCR (qPCR) and digital droplet PCR (ddPCR) amplification techniques were performed using oligonucleotides designed to amplify four larger fragments  with random primers (500 ng/uL)  and M-MLV Reverse Transcriptase , following the protocols described by . Conventficiency .The fragment amplification by qPCR was performed using TaqMan Universal Master Mix II  in a solution containing 2.0 μL (100 ng) of cDNA in a reaction volume of 8.5 μL containing 5.0 μL of TaqMan Universal Master Mix II  and 1.5 μL of primers (10 pmol/μL) with probes (5 pmol/μL), and 2.0 μL of DNase/RNase-free water. Amplification reactions were performed according to the TaqMan recommendations  with an annealing temperature of 60 °C. After amplification, the qPCR products were cleaned up with a GenElute PCR Clean-up Kit  and sequenced using traditional Sanger technology. The ddPCR was carried out using ddPCR Supermix for Probes , 2.0 μL (100 ng) of cDNA in a reaction volume of 18.0 μL containing 12.5 μL of 2Xand ddPCR Supermix for Probes , 1.5 μL of primers (10 pmol/ μL) with probes (5 pmol/ μL), and 4 μL of DNase/RNase-free water. The droplet was transferred to a semi-skirted 96-well PCR plate , which was sealed and subjected to amplification in a Px2 Thermal Cycler . Following PCR amplification, the plate was placed in a QX200 droplet reader . The amplicon quantity was evaluated using QuantaSoft version 1.7  by determining the threshold value and the number of positive copies of the target in a 20 μL reaction.Tymoviridae, Alphaflexiviridae, Gammaflexiviridae and Betaflexiviridae families using MAFFT version 7 using the G-INS–i criteria. Viral sequences of the referred families were obtained from NCBI protein databases under the following accessions: Tymoviridae family: Grapevine fleck virus (NP_542612); Citrus sudden death-associated virus (YP_224218); Mayze rayado fino virus (AAK52838); Oat blue dwarf virus (NP_044447); Anagyris vein yellowing virus (YP_002308578); Diascia yellow mottle virus (YP_002048673), Eggplant mosaic virus (NP_040968); Nemesia ring necrosis virus (YP_002308442); Ononis yellow mosaic tymovirus (NP_041257); Plantago mottle virus (YP_002308445); Turnip yellow mosaic virus (AAB2649); Alphaflexiviridae family: Shallot virus X (NP_620648); Botrytis virus X (AAL17722); Lolium latent virus (YP_001718499); Indian citrus ringspot virus (NP_203553); Potato virus X (YP_002332929); Sclerotinia sclerotiorum debilitation-associated RNA virus (YP_325662); Betaflexiviridae family: Apple stem grooving virus (NP_044335); Cherry virus A (NP_620106); Aconitum latent virus (NP_116487); Citrus leaf blotch virus (NP_624333); Apple stem pitting virus (NP_604464); Grapevine rupestris stem pitting-associated virus (NP_047281); Peach chlorotic mottle virus (YP_001497153); Apple chlorotic leaf spot virus (NP_040551); Peach mosaic virus (YP_002308565); Grapevine virus A (NP_619662) and Gammaflexiviridae family: Botrytis virus F (NP_068549).The viral contigs were translated into amino acids, concatenated and aligned with reference sequences of the http://tree.bio.ed.ac.uk/software/figtree/) and midterm rooted.Phylogenetic analyses were carried out in the Geneious 9 and MEGA 7 software using distance-based (distance matrix) and character-based (maximum likelihood) methods, respectively. Mean distances and the neighbor-joining algorithm were used for distance analyses, and the best-fit model of protein evolution, previously selected in ProtTest 3.2 with Akaike Information Criterion (AIC), was used for maximum likelihood analyses , 30. ClaH. brasiliensis, one leaf sample obtained from one tree was selected to construct two sRNA libraries based on RNA quality. The two libraries were pooled, totalizing 21,446,061 reads. Reads with low quality or containing ambiguous bases were excluded, which resulted in 18,591,249 reads left. The pre-processing of the data resulted in a decrease of only ~ 13% of the reads, highlighting the representativeness and quality of our libraries. We enriched reminiscent reads for viral sequences by filtering out reads matching bacterial, fungal and host genomes. The filter step reduced the number of reads to 1,737,014, which were then used for contigs assemblage and characterization of the putative viral genome  viruses of The investigation of the molecular characteristics of the sRNAs mapped into assembled contigs showed that most of the sequences with similarity to Plantae, Bacteria and Fungi have a broad size distribution and originated mostly from positive sense strand RNAs. On the other hand, small RNAs mapped into contigs exhibiting similarity to viral sequences showed a size distribution between 20 and 22 nt derived from both strands, which was consistent with the signature of sRNAs derived from vsiRNA pathway , qPCR and ddPCR techniques. However, apart from the leaf sample from CNF, no signal of viral contigs was detected in the samples.The presence of viral sequences was evaluated in all the Maculavirus with strong statistical support (bootstrap = 100%). In addition, the putative virus was also phylogenetically close to Marafivirus and Tymovirus (bootstrap = 77.9%) . This analysis also revealed that the contig was related to Marafivirus and Tymovirus (bootstrap = 82%)  in reference to its host.Based on the contig ordering strategy, we generated a 1913-aa long concatenated sequence that was used to infer the phylogenetic relationships of the putative virus. Using the distance-based method with the Neighbor-joining algorithm, it consistently clustered with the Grapevine fleck virus (GFkV) 9%) Fig. . Maximum2%) Fig. . AltogetH. brasiliensis leaf, since the predominant size of reads entering in the viral contigs assembly was  20-22 nt in both strands.Viral dsRNA is used by the plant’s immune system to produce virus small interfering RNAs (vsiRNAs) 20–22 nucleotides in length , 13. TheTymoviridae family: methyltransferase (Mtr), RNA helicase (Hel), papain-like cysteine protease (P-Pro), RNA-dependent RNA polymerase (RdRp), and the coat gene.Metagenomic studies usually generate fragmented sequences, which hamper the characterization of genome structure and phylogenetic analyses , 32. To The ordering and characterization of the viral contigs allowed the design of specific oligonucleotides to experimentally confirm the presence of the viral RNA sequences in our sample via PCR amplifications. Conventional PCR was not suitable to detect their presence, while using qPCR three out of four contigs were amplified. Small amounts of target molecules in a qPCR reaction could decrease amplification efficiency due to the reduced probability of binding oligonucleotides in the template . DigitalMaculavirus (Tymoviridae family).The use of in silico similarity searches associated with experimental confirmation of the RNA viral sequences allowed us to generate a 1913 aa long fragment, which was used to estimate the phylogenetic relationships of the putative virus. Distance and character-based phylogeny methods indicate that HBrV clusters with the Grapevine fleck virus, genus Tymoviridae family differ in the number of ORFs (from 1 to 4) and in the distribution of genes, but they all encode a large polyprotein essential for viral replication [Maculavirus contains four ORFs, the longest (ORF1) encoding a replication polyprotein constituted by four domains: a methyltransferase (Mtr); a papain-like cysteine protease (P-Pro); an RNA helicase (Hel); and an RNA-dependent RNA polymerase (RdRp), which are conserved among all the ssRNA viruses [Genomes of viruses belonging to the lication . The gen viruses , 37. In Carlavirus, order Tymovirales) was found to infect native H. brasiliensis trees in Amazonia [Maculavirus, together with Marafivirus and Tymovirus, compose the Tymoviridae family . Members of the Tymoviridae family are usually associated with eudicotyledonous plants, which include H. brasiliensis, except for some Marafivirus that infect monocots (Poaceae). Infections by maculaviruses are known to be phloem-limited [Although hundreds of plant viruses have been described over the past 120 years, only one species . As far as we know, this is the second time that viral sequences were identified in this plant; the first, Carlavirus, was described more than 30 years ago. Our results highlight the necessity of new studies to identify viruses associated with this economically important, but poorly studied plant.Small RNA deep sequencing combined with bioinformatic tools and in vitro amplifications proved to be a robust strategy for the accurate identification of viral sequences in Additional file 1:Figure S1. Overview of the strategy used for the in silico contig ordering. Contigs were anchored in the genome of related viruses based on sequence similarity searches. The position of contigs and the score associated to each position was stored for each reference genome assessed . After the evaluation of all references, the final contig position was defined by the highest cumulative score obtained through sum of partial scores. (PDF 422 kb)Additional file 2:Table S1. Oligonucleotides designed for the amplification of viral contigs. (DOCX 59 kb)Additional file 3:Table S2. Summary metrics of the sequencing and from the contigs assembled in sample C2. (DOCX 49 kb)Additional file 4:Figure S3. Schematic representation of the Grapevine fleck virus (GFkV) organization. ORF1 (upper box) codes for the replication associated polyprotein (RP) containing the domains of methyltransferase (MTR); papain-like protease (PRO); helicase (HEL); RNA-dependent RNA polymerase (POL); and the coat protein (CP), ORF2 (lower box) encodes the putative movement protein (MP). Bars represent the contigs assembled according to the sequence similarity searches. Red arrows represent the oligonucleotides designed to amplify four fragments covering contigs regions across the genome. (PDF 399 kb)Additional file 5:Table S2. DNA sequences corresponding to the viral contigs identified in the Hevea brasiliensis sample. (DOCX 119 kb)Additional file 6:Table S4. Identification and characterization of the assembled contigs based on NCBI database searches. Similarity and coverage percentages, accession numbers, contig position and sizes are listed. (DOCX 92 kb)


The responses toward the antigen beads were both antigen specific and sensitive, with a detection threshold of 1 IFNγ producing T-cell per 18,000 PBMCs.Reliable and sensitive detection of antigen specific cells is essential in several fields of research, whether it concerns monitoring responses to infectious agents or exploring the auto-antigen repertoire in autoimmune diseases. Identification of these cells is however difficult, especially when the cells often are rare and methods not sensitive, specific or practical enough. We propose a novel method of processing antigens before stimulation of cells which consists of covalently binding protein antigen to superparamagnetic micro-beads and using denaturing washes to remove contaminants. Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated using both cytomegalovirus and tetanus-diphtheria antigen-beads as well as non-antigenic protein-beads as negative control in an IFNγ FluoroSpot assay in order to detect Th1 and CD8•Covalently binding antigen to paramagnetic beads allows for harsh denaturing washes without loss of antigen.•Microbeads are phagocytosed by antigen presenting cells, resulting in efficient uptake, processing and presentation of the antigens.•The method allows the usage of relatively impure starting antigen material and whole PBMC samples without high background levels in follow up cellular assays. Specifications TableE. coli expression of recombinant proteins include high amounts of bacterial contaminants like lipopolysaccharide (LPS), which can be difficult to remove and will interfere in follow-up cellular assays [Identification of antigen-specific T-cells is an important aspect of several different areas of immunological research, from infections to autoimmune diseases and cancer immunology. There are several different methods available for detecting antigen-specific T-cell responses, such as cell-proliferation assays, ELISpot/FluoroSpot or flow-cytometry based assays. There are advantages and disadvantages with each method, but problems often arise due to assays having a high background noise owing to an abundance of non-specific responses while the cells specific for the antigen in question are rare . The char assays . This isr assays ,5. Usingr assays . These p6 cells/ml. The PBMCs were then frozen at −1 °C/min using a CoolCell  and subsequently stored at −150 °C. All donors gave their written informed consent before participating. The study was approved by the regional ethics board, Regionala Etikprövningsnämnded in Stockholm and was conducted in accordance with the Declaration of Helsinki principles.Venous blood samples were drawn from healthy donors (HDs) (n = 28) using EDTA tubes (BD diagnostics) and peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation  in the SepMate system (Stemcell Technologies) as per manufacturers protocol. Remaining red blood cells were lysed by ACK-lysing buffer (Sigma-Aldrich) for 5 min before the cells were washed and resuspended in freezing medium (45% RPMI 1640 (Sigma-Aldrich), 45% heat inactivated FCS and 10% DMSO) at RT at a concentration of 10–15 × 10E. coli BL21-AI (ThermoFisher Scientific).A combination of epitopes from tetanus toxin (T) and diphtheria toxin (D), as previously described , with anE. coli were grown in 500 ml medium supplemented with kanamycin, glycerol, Mg2S04, arabinose, lactose and glucose for auto-induction at 25 °C overnight. Cells were centrifu ged at 9000 g and lysed using a 6 M Guanidinium-HCl, 10 mM Tris-HCl, 50 mM NaHPO4, 100 mM NaCl and 20 mM β-mercaptoethanol, pH 8.0 buffer followed by freezing at −80 °C. Supernatants were collected after centrifugation at 23,500 g. The antigens were purified using His Mag Sepharose Ni Beads  and eluted with a 6 M Urea, 0.05% Tween 20, 10 mM β-mercaptoethanol, 10 mM MES, pH 3.0 buffer. The antigens were adjusted to pH 5.0 by addition of 0.82 M MES buffer pH 6.0.Transformed N-hydroxysuccinimide (NHS) (Sigma-Aldrich) and 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fisher Scientific) in MES-buffer. After washing twice in MES-buffer, the antigen was added at a concentration of 1 mg/ml in MES-buffer, 67 μg protein/mg (n = 109) beads, and incubated for 30 min at room temperature. After antigen coupling, still reactive carboxylic groups were blocked by incubating with 50 mM Tris pH 7.4 for 15 min.The protein antigens  were covalently linked to 1 μm paramagnetic polystyrene beads  using amine coupling. A magnet  was used to facilitate recovery of the beads between each following steps. To keep the beads in suspension all incubation steps were performed in 1.5 ml tubes on a shaker block at ≥900 RPM. In order to minimize bead losses when handling the beads, low retention tubes and pipette tips were used throughout the whole procedure. The beads were first washed twice in 25 mM MES buffer, pH 6, followed by incubation for 30 min at room temperature with 0.1 M 6 beads were incubated with 5 μl FITC-HSA (Abcam) for 30 min at RT and subsequently washed twice in PBS before acquisition in a FACSVerse . Using non-antigen beads as a negative control, the number of beads containing a significant amount of the antigen was gated using flow cytometry software   and in the CMV- and T-D- antigen constructs. This allowed for staining the bead-bound antigens using a fluorophore-conjugated human serum albumin (HSA). In this method, ˜10CC, USA) . While tAs an integral part of the antigen preparation, the beads were washed with an NaOH solution to remove endotoxin. As different proteins can be expected to contain different amounts of endotoxin, and trace amounts of LPS can facilitate antigen-specific responses , the opt9 beads/ml and incubated at 95 °C for 5 min before testing. The FBS used in the cell culture media in the FluoroSpot assay was similarly tested. All tests were done in duplicates in a 96-well plate and LPS-concentration was determined by OD at 405 nm. The rationale for briefly incubating the beads at 95 °C prior to testing was to release as much endotoxin as possible from inside folded proteins. Testing the beads together with the supernatant increased the sensitivity of the assay, while the beads themselves did not interfere with the OD-reading.The remaining endotoxin on the beads was tested using a limulus-amebocyte-lysate-assay  according to the manufacturer’s protocol. The beads were diluted to 0.4–1 × 10l-Glutamine , 100U/ml penicillin and 100 μg/ml streptomycin ). PBMCs were plated in a 96 well FluoroSpot plate at 2.5 × 105 PBMCs/well and antigen beads were added in duplicates to a concentration of 10 beads per PBMC. The plates were incubated at 37 °C, 5% CO2 for 44 h before development according to manufacturer’s protocol. The plates were read and spot forming units (SFUs) counted using an iSPOT Spectrum . As certain antigen beads can be sticky, and small amounts can be lost due to contact with plastic, we found that keeping them in high concentration  minimizes the loss. In order to get a manageable concentration for pipetting during the FluoroSpot assay, the beads are preferentially diluted in cRPMI before adding to the wells, as cRPMI reduces the stickiness. Our titration trials indicate that ˜10 beads per PBMC give the best separation between antigen responsive individuals and background in the FluoroSpot assay, however differences have been seen in the range of 2.5–20 beads per PBMC.To assess the T-cell responses towards the antigen-beads an IFNγ FluoroSpot assay  was used. PBMCs were thawed in a water bath at 37 °C before being washed twice in cRPMI (RPMI 1640 (Sigma-Aldrich) with 10% FBS , 2 mM U test was used to compare the responses to the antigen-beads and negative control beads. A Spearman correlation was used to correlate the individual CMV and T-D responses. Before this correlation, each individual’s background response was subtracted from the antigen response (ΔSFU). P < 0.05 was set as significance threshold for all analyses.Statistical analyses were performed using GraphPad Prism 7 . A Mann-Whitney 5 PBMCs, indicating a very low background noise level. Both T-D-beads and CMV-beads gave rise to a significantly higher response than the background , indicating a targeted response towards these antigens. To see whether there was a discrepancy in individuals’ responses to T-D and CMV-beads, indicating antigen specificity, the individual HD responses towards CMV and T-D were correlated , T-D+CMV− , T-D−CMV+  and T-D−CMV−  populations. Using a mean+3SD above the NC-response as the limit for detection, the assay was able to detect antigen specific cell responses as rare as 1 IFNγ producing T-cell per 18,000 PBMCs. The mean SFUs of antigen-responsive and antigen-non-responsive individuals were 61.2 vs 6.4 for the T-D beads and 165.0 vs 5.6 for the CMV-beads, giving a signal to noise ratio of 9.6 and 29.5 respectively. The validation was based on FluoroSpot as the follow-up assay. While the FluoroSpot/ELISpot assay is very sensitive as it detects responses in single cells and might thus be optimal when looking for rare antigen-specific responses, the use of antigen-beads instead of soluble antigen as stimuli can likely be used in other cellular methods as well. Additionally, this validation used IFNγ production as a measurement of response, meaning it mainly detected Th1 and CD8+ cells. In order to detect other types of antigen-specific responses which could be more relevant to the study in question, one must combine this antigen-processing method with other readouts, e.g. IL-10, TNFα or IL-17 production or cell-proliferation.After the NaOH wash, the levels of endotoxin contamination of the beads were very low . All antrrelated B, showinCBD solutions, Neuroförbundet, Swedish Research Council (2017-00777), Swedish Brain Foundation, Margareta af Ugglas Foundation and Stratneuro. None of the funding sources have had a role in the study design, collection, analysis or interpretation of the data.The study has been partly funded by grants from We wish to draw the attention of the editor to the following facts which may be considered as potential conflicts of interest. There are pending patents regarding parts of the methods presented in this manuscript, more precisely the usage of antigen-beads for detection of antigen-specific T-cells. Mattias Bronge and Hans Grönlund are the inventors of said patents which are held by the private company TCER AB. Hans Grönlund is a co-founder of TCER AB and Claudia Carvalho-Queiroz, Ola Nilsson and Andreas Kaiser hold positions at TCER AB.CBD solutions, Neuroförbundet, Swedish Research Council (2017-00777), Swedish Brain Foundation, Margareta af Ugglas Foundation and Stratneuro. None of the funding sources have had a role in the study design, collection, analysis or interpretation of the data.The study has been partly funded by grants from


We note the reemergence of human monkeypox in Sierra Leone following a 44-year absence of reported disease. The persons affected were an 11-month-old boy and, several years later, a 35-year-old man. The reappearance of monkeypox in this country suggests a need for renewed vigilance and awareness of the disease and its manifestations. Monkeypox, a tropical zoonosis with an estimated death rate of 15% in children, is a resurgent presence in several countries in West and Central Africa  in Koribondo. At this time, the child remained febrile and was exhibiting a nascent-stage rash. At the Koribondo CHC the child was given a presumptive diagnosis of early stage chickenpox and sent home.°C) and disseminated vesiculopustular rash. Notably, lymphadenopathy was absent. On hospital day 4 (day 8 after fever onset), physicians from Médicins sans Frontières working in a pediatric ward at the facility alerted national authorities that they suspected the child might have monkeypox. After consultation with the Directorate of Disease Prevention and Control of the Sierra Leone Ministry of Health and Sanitation, Kenema scientists contacted the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA.Within 2 days of the child’s evaluation at the Koribondo CHC, pustular, umbilicated lesions had spread to cover his body, including his face, mouth, oral mucosa, trunk, back, palms, and genital area. In addition, the child was experiencing sweats, chills, vomiting, loss of appetite, cough, and pruritus. On March 21, the child was taken to the Médicins sans Frontières Gondama Referral Center and was admitted with fever (body temperature 38.9Orthopoxvirus and for West African variant monkeypox virus (Orthopoxvirus IgG and IgM (Diagnostic specimens were collected from the patient on April 1. At that time his rash was desquamating. Lesion crusts and serum samples were obtained, and specimens were shipped to CDC on April 4. On April 8, real-time PCR testing for nonvariola No viable virus was detected in the specimens. Further genetic characterization of the virus was unsuccessful owing to the limited quality and quantity of specimens.Upon confirmation of monkeypox etiology, investigators from the Bo District Surveillance Office and the Lassa Fever Outreach Program traveled to the child’s town Kpetema;  to interOn March 25, 2017, a 35-year-old male farmer in Pujehun district came to Pujehun Government Hospital in Sierra Leone with fever, generalized body pain, enlarged cervical lymph nodes, dysphagia, malaise, and macular rash with cropping of papules and macules. Various samples were taken and shipped out of the country for laboratory investigations. Monkeypox was confirmed, and community sensitization was instituted. The detection of monkeypox virus infection in an 11-month-old child and a 35-year-old man signals the possible reemergence of this disease in Sierra Leone (Multiple factors are probably driving the resurgence of monkeypox in West and Central Africa. Increasing population susceptibility is likely to be one (


Rousettus aegyptiacus) captured in Zambia in September 2018. The virus was closely related phylogenetically to the viruses that previously caused Marburg outbreaks in the Democratic Republic of the Congo. This finding demonstrates that Zambia is at risk for Marburg virus disease.We detected Marburg virus genome in Egyptian fruit bats ( Marburgvirus, like Ebolavirus, belongs to the family Filoviridae and consists of virus species that cause severe hemorrhagic fever in humans and nonhuman primates. Marburgvirus contains 1 species, Marburg Marburgvirus, and 2 viruses, Marburg virus (MARV) and Ravn virus (RAVV) (hereafter referred to as Marburgviruses) (The genus Rousettus aegyptiacus) are the primary natural reservoir of Marburgviruses. Entry into caves and mines inhabited by Egyptian fruit bats has frequently been linked to MVD outbreaks , approved by the Department of National Parks and Wildlife, Ministry of Tourism and Arts of the Republic of Zambia (act no. 14 of 2015). We sampled lung, liver, kidney, spleen, and colon tissues from 22 bats in February and 25 bats in September . In Novehttps://blast.ncbi.nlm.nih.gov/Blast.cgi), we confirmed detection of Marburgvirus NP, VP35, and L genes (GenBank accession nos. LC465155–7). We also detected the NP gene in the pooled tissue RNA of another bat (ZB18-55) (GenBank accession no. LC465158)  captured in September . By SangC465158) . Nucleothttps://www.ebi.ac.uk/Tools/msa/muscle) and constructed phylogenetic trees by the maximum-likelihood method with 1,000 bootstrap replicates in MEGA7 (,Subsequently, we phylogenetically compared the NP, VP35, and L genes with representative Marburgvirus sequences available in GenBank. We aligned nucleotide sequences by MUSCLE (in MEGA7 . The virMiniopterus inflatus and Rhinolophus eloquens bats, as well as Egyptian fruit bats in Gabon (Recently, the Marburgvirus genome was detected in cave-dwelling Egyptian fruit bats in South Africa (,,,Experimental infection of Egyptian fruit bats with MARV demonstrated that induced virus-specific IgG rapidly declined by 3 months postinfection (We report a potential risk for MVD in Zambia. It is important to clarify whether unrecognized human cases of MVD, including asymptomatic Marburgvirus infection, are present in Zambia. Moreover, further research is needed to elucidate the ecology of Marburgviruses in the entire African region and to estimate potential risks of MVD outbreaks in previously nonendemic countries. In particular, more extensive information is needed on Marburgviruses in the Egyptian fruit bat population, including the genetic diversity of the viruses, the distribution and migratory behavior of the bats, and the seasonal pattern of virus infection prevalence.


Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens.Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg’s disease virus, and 3000 clinical samples were tested for practicability.Specific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 μl). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, The optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits.This optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies.The online version of this article (10.1186/s13071-018-2996-0) contains supplementary material, which is available to authorized users. Insect-borne diseases are infectious diseases spread among animals and human through hemophagia by blood-feeding arthropods, such as mosquitoes, ticks and midges, which are widespread in the environment . These iTo date, multiple molecular detection methods have been established to detect insect-borne pathogens, including reverse transcriptase-polymerase chain reaction (PCR) , real-tiCoxiella burnetii (CB), African swine fever virus (ASFV), West Nile fever virus (WNV), Borrelia burgdorferi (BB), vesicular stomatitis virus (VSV), Rift Valley fever virus (RVFV), Ebola virus (EBV) and Schmallenberg virus (SBV). This optimized liquid array detection system may contribute to the rapid and effective detection of multiple insect-borne diseases at border ports in China.In this study, we established a method that was able to simultaneously detect multiple insect-borne pathogens rapidly and effectively, including bluetongue virus (BTV), epizootic hemorrhagic disease virus of deer (EHDV), Q-fever pathogen Armored RNA technique was utilized in this study. The exogenous RNA fragments packaged by the virus-like particles (VLPs) expression vector pTMSCA2C were used as positive control of BTV, EHDV, WNV, VSV, RVFV, EBV and SBV.NS1 gene of BTV was most closely related to the virus isolate CAR1982/04 (GenBank: KP 822059.1) from the Pirbright Institute, with 97% identity. The segment of the NS3 gene of EHDV was most closely related to the virus isolate EHD1/USA2006/LA/CC332-06 (GenBank: KU140939.1), with 99% identity. The segment of E gene of WNV was most closely related to the West Nile virus isolate 876 (GenBank: KY229074.1), with 99% identity. The segment of N gene of VSV was most closely related to the virus isolate Colorado/21793/2014 (GenBank: KP 202364.1) from the National Veterinary Services Laboratories of USA, with 96% identity. The segment of S gene of RVFV was most closely related to the Rift Valley fever virus strain ZH-501 (GenBank: DQ380149), with 98% identity. The segment of NP gene of EBV was most closely related to the Sudan Ebola virus isolate EboSud-682 2012 (GenBank: KC545392.1), with 99% identity. The segment of N gene of SBV was most closely related to the virus isolate BH80/11-4 (GenBank: KC545392.1) from FLI of Germany, with 99% identity.The segment of the Target gene segments of ASFV, CB and BB were synthesized by Beijing Genomics Institute (BGI), Beijing, China. Then, 3 plasmids, containing above 3 target segments, were constructed as a positive control for further works.P72 gene of ASFV was most closely related to the African swine fever virus E75 (GenBank: FN557520), with 99% identity. The segment of IS1111a gene of CB was most closely related to the Coxiella burnetii NMII RSA439 (GenBank: M80806), with 98% identity. The segment of OspA gene of BB was most closely related to the Borrelia burgdorferi SZ (GenBank: AY502600), with 99% identity.The segment of Other insect-borne pathogens, including dengue virus (DENV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), Xinjiang hemorrhagic fever virus (XHFV), spotted fever group rickettsiae (SFGR), and ehrlichiae and chikungunya virus (CHIKV) used for specificity test, were cultured and conserved by Chinese Academy of Inspection and Quarantine.Based on the specific sequences of each pathogen in the GenBank database, primers and probes (capture probes and reverse-complement (RC) probes) were designed using different software packages, such as DNAMAN, MEGA, BioEdit, DNAStar and Primer Premier 5.0 Table . Before In order to avoid interference by different primers and templates, we designed more than 3 pairs of primers for each pathogen. Primers were designed to meet the following conditions: (i) amplification segments were between 100–300 bp to insure the intensity of fluorescence signal; (ii) melting point is between 50–60°C; and (iii) there are no long dimers and complimentary stem to insure the compatibility in a multiplex-PCR system.Total RNA or DNA of pathogens was extracted and purified using the QIAamp Viral RNA Mini kit or the QIAamp DNA Blood Mini kit  according to the manufacturer’s protocol. The quality and concentration of RNA and DNA were evaluated by the ratios of A260/A280 and A260/A230 using a NanoDrop2000 spectrophotometer . Subsequently, eligible RNA was reverse-transcribed into cDNA using the PrimeScript RT-PCR Kit .2O up to 50 μl. The thermal cycle conditions were as follows: 1 cycle at 50 °C for 30 min and 95 °C for 15 min, followed by 35 cycles at 95 °C for 40 s, 54 °C for 40 s and 72 °C for 1 min, and a final extension at 72 °C for 10 min. Each reaction was repeated in triplicate. PCR products were confirmed by 2% agarose gel electrophoresis.Multiplex asymmetric PCR was applied to DNA amplification. The PCR system was determined as follows: 23 μl buffer mix, 0.5 μl forward primers (10 μmol/ml each), 0.5 μl reverse primers (10 μmol/ml each), 5 μl nucleic acid templates, added with double-distilled H7 beads/ml beads were re-suspended in 0.1 mol/l MES buffer , with the probe , and treated twice with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride  at room temperature away from light for 30 min. The mixture was then rinsed in Tween 20 (1 ml of a 0.02% aqueous solution), followed by sodium dodecylsulfate solution , and re-suspended in Tris-EDTA buffer (pH 8.0) to the final volume of 100 μl. The final concentration of the bead suspension was calculated using a haemocytometer.According to the manufacturer’s protocol, capture probes modified with an amino-C12 linker at the 5'-end were coupled with 10 types of Luminex MicroPlex fluorescent carboxylated micro-spheres (“beads”) . Briefly, for each coupling of probe and bead set, 200 μl of 1.25 × 10According to the manufacturer’s  protocol, the prepared suspension of beads coupled with capture probes was diluted to a final concentration of ≥ 100 beads/μl using 1× tetramethylammonium chloride (TMAC) hybridization solution. Then, 33 μl of diluted bead suspension was mixed with 15 μl Tris-EDTA buffer (pH 8.0) and 1 μl of different concentrations  of RC-probes modified with biotin at the 5'-end. Then, the mixtures were subjiected to PCR amplification  and the PCR products were transferred into a 96-well ELISA plate. The beads were re-suspended in 100 μl of streptavidin-R-phycoerythrin (SAPE) diluted in 1× TMAC hybridization solution , and incubated in a metal bath at 58 °C for 10 min. Then, the reaction systems were analyzed using a Bio-Plex multiplexed liquid array detection system . Each reaction was repeated in triplicate. The coupling efficiency of beads and probes was evaluated based on the median fluorescent intensity (MFI).2O, the negative control tube by 1 μl of negative PCR product, and the positive control tube by 1 μl of RC-probes (0.01 μmol/l). Each reaction was repeated in triplicate. The PCR amplification and subsequent steps were the same as those described for coupling efficiency detection. The MFI was computed to assess the results according to the manufacturer’s protocol. The Luminex qualitative ratio result (LQRR) was equal to the MFI of the sample (MFIS) divided by the MFI of the blank control (MFIB), that is LQRR = MFIS/MFIB. If LQRR ≥ 3, the result was positive; if LQRR < 2, the result was negative; and if 2 ≤ LQRR < 3, the result was inconclusive and needed to be reanalyzed. The procedures used for the suspension array method are outlined in Additional file The PCR products of pathogens were hybridized with probe-coupled beads using the liquid array. Briefly, the working solution was then prepared by mixing 1 μl of the 10 types of probe-coupled bead suspensions and 330 μl of 1× TMAC hybridization solution. Then, 1 μl of the positive PCR product of each virus was mixed with 33 μl of working solution and 15 μl of Tris-EDTA buffer (pH 8.0) in a tube. The PCR product in the blank control tube was replaced by 1 μl of double-distilled HTo optimize the ratio of forward/reverse primers in the asymmetric PCR, five ratios of forward/reverse primers  were tested. All other conditions remained constant.To optimize the quantity of PCR product for hybridization efficiency, six concentrations of PCR product quantity  were tested. The optimized ratio of forward/reverse primers, determined above, was used in the PCR, and all other conditions remained constant.To optimize the melting temperature (Tm) for hybridization efficiency, six different Tm values  were tested. The optimized ratio of forward/reverse primers and the optimized quantity of PCR product, determined above, were used in the PCR, and all conditions remained constant.To optimize the hybridization duration, six different durations  were tested in the hybridization assay. The optimized ratio of forward/reverse primers, PCR product quantity and Tm, as determined above, were used in the PCR, and all other conditions remained constant.After the detection method had been optimized, 10 standard curves were established for the MFI values obtained from the experiments. A 10-fold dilution of pathogens was added to the system to create correlative mathematical curves with MFI values.Each of the 10 pathogens and other insect-borne pathogens including DENV, TBEV, JEV, XHFV, SFGR and CHIKV were detected to determine the specificity of the liquid array detection system.10 copy number), then serially diluted with different folds (10–1010-fold ). The serially diluted nucleic acids were detected using liquid array method.To determine the sensitivity of the liquid array detection system, nucleic acid of the pathogens was extracted using a Qiagen kit . After detection of both the concentration and purity of the nucleic acid, the nucleic acid concentration of each pathogens was adjusted to the same magnitude  was calculated as CV = (standard deviation (SD)/mean) × 100%.A total of 3000 samples of mosquitoes, ticks and midges provided by the Entry-Exit Inspection and Quarantine departments at five border areas in China Table  were useThe capture probes on the breads and the corresponding RC-probes of the 10 pathogens showed a high hybridization signal. With the concentration of RC-probes gradually decreasing, the MFI also gradually declined  when the ratio of forward/reverse primers was 1:2, 1:3 or 1:4. The MFI for EHDV, VSV and CB was the highest when the ratio of forward/reverse primers was 1:2, whereas the MFI for BB and ASFV was relatively stable regardless of the forward/reverse primer ratio Fig. . As a reThe MFI for BTV, EHDV, VSV, EBV and SBV PCR product was highest when the volume of PCR product was 2 μl, then gradually decreased with increasing volume of PCR product (> 2 μl). Similarly, the MFI for WNV, BB, ASFV, CB and RVFV PCR product was also highest when the volume of PCR product was 2 μl, but showed no obvious decrease with increasing volume of PCR product (> 2 μl) Fig. . TherefoThe MFI for BTV PCR product was higher at 50 °C and 56 °C. The MFI for BB and EBV PCR product was highest at 58 °C, and for the other seven pathogens was highest at 50 °C Fig. . TherefoThe MFI for BB and EHDV PCR product was higher when the hybridization duration was 30 min or 35 min, whereas the MFI for the other eight pathogens was highest when the hybridization duration was 30 min Fig. . Therefo2O up to 50 μl. The thermal cycle conditions were as follows: 1 cycle at 50 °C for 30 min and 95 °C for 15 min, followed by 35 cycles at 95 °C for 40 s, 54 °C for 40 s and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The hybridization system was set up as follows: 33 μl of diluted bead suspension (1 μl of probe-coupled bead suspension added into 330 μl 1× TMAC hybridization solution), 15 μl Tris-EDTA buffer (pH 8.0), and 2 μl PCR products. The hybridization reaction was performed as follows: denaturation at 95 °C for 5 min, and hybridization at 50 °C for 30 min. After hybridization, PCR products were transferred into a 96-well ELISA plate. Beads were re-suspended in 100 μl of SAPE diluted in 1× TMAC hybridization solution (3:1000), and incubated in a metal bath at 58 °C for 10 min. Then the reaction systems were analyzed using a liquid array detection system.Above all, an optimized liquid array detection system was described as below. The asymmetric PCR system was set up as follows: 23 μl buffer mix, 0.5 μl forward primers, 1 μl reverse primers, 5 μl nucleic acid templates, and double-distilled HR2) were greater than 0.95, indicating good relationships between the concentration of the target and fluorescence.After the detection method had been optimized, 10 standard curves were established for the MFI values obtained from the experiments. A 10-fold dilution of pathogens was added to the system to create correlative mathematical curves with MFI values Fig. . All of To confirm the effectiveness of the optimized liquid array detection system, equal proportion of nucleic acid from the 10 pathogens were mixed and then analyzed using the detection system. The LQRR value was greater than 3, indicating a positive result for the mixed viral nucleic acid  infection rate, which was consistent with the results of the detection kit. Furthermore, 53 midge samples from Yunnan Province and Guangdong Province were detected to be positive for BTV, showed as 12.62% (53/420) infection rate, which was also consistent with the results of the detection kit. In addition, 17 tick samples from Inner Mongolia Autonomous Region and Xinjiang Uighur Autonomous Region were positive for CB, showed as 3.62% (17/470) infection rate. Co-infection with BB and CB was found in 5 ticks. All of these samples were reanalyzed using the corresponding gold-standard assays or commercial nucleic acid detection kits, and all of the results were consistent with the findings of the liquid array detection system , which can be used for further analysis, such as the detection of hybridization . The ratFurthermore, the conditions of the hybridization, such as hybridization temperature and duration, and the quantity of PCR product, are critical items that ensure the adequacy of a contact reaction , 20. In High specificity, sensitivity and reproducibility are the desired characteristics of any detection system  StrikingCoxiella burnetii and Borrelia burgdorferi among Dermacentor nuttalli and Ixodes persulcatus in the Inner Mongolia Autonomous Region. The result indicated that the system could also detect the mixture of different targets.This liquid bead array method was used to analyze 3000 clinical samples. The assay detected Lyme disease  and Q fever  among the samples from the Inner Mongolia Autonomous Region and the Xinjiang Uighur Autonomous Region. These two provinces are recognized as the main epidemic areas of LB and QF in China. The results of analysis of these clinical samples indicted that the liquid beads array has promising potential applications in the monitoring of important infectious diseases. Interestingly, it was found the co-infection of Notably, the liquid array detection system was able to simultaneously detect 10 different types of pathogens. This number of species of tick-borne pathogens is higher than that detected by any other established method, such as the xMAP liquid array reported to detect nine tick⁃borne pathogens , the micThis study established a multiplexed PCR-coupled liquid array to simultaneously detect 10 types of insect-borne pathogens. The optimized ratio of forward/reverse primers (1:2) and the conditions of hybridization, including the quantity of PCR product (2 μl), temperature (50 °C) and duration (30 min), were optimized. This improved detection method may be applied for the rapid and effective detection of multiple insect-borne diseases at border ports in China.Additional file 1:Figure S1. Outline of the suspension array method used for the simultaneous and rapid detection of 10 insect-borne pathogens. The hybridization signal of biotinylated PCR product and microsphere beads labeled with specific probes for 10 insect-borne pathogens was detected by instrument. (TIF 220 kb)Additional file 2:Figure S2. Coupling efficiency results for the 10 viral probes and corresponding breads. The error bars indicate the standard deviations. Abbreviations: RC-probes, reverse-complement probes; MFI, median fluorescent intensity. (TIF 1380 kb)