L. plantarum though were reported to attenuate ALD, their in vivo health benefits are limited by their survival and sustenance in the adverse gut conditions. Therefore, to enhance their in vivo performance, chitosan coated alginate beads entrapping L. plantarum were prepared, characterized, and evaluated for their efficacy against ALD in rats. Following chronic alcohol exposure, rats developed endotoxemia, showed enhanced levels of liver enzyme markers, NF-κB levels, and increased cytokines such as TNF-α and IL12/p40 subunit, and reflected significant histological changes in the intestine and liver. However, cosupplementation with double layered microencapsulated probiotic significantly (P < 0.05) reduced the levels of endotoxemia, serum transaminases, NF-κB, and cytokines complemented with restoration of normal histoarchitecture of the intestine and liver. It is being documented here for the first time that the probiotics have the potential to inhibit IL-12/p40 subunit which is a recently explored potential marker for developing novel therapeutic agents. This study reveals that microencapsulation of probiotics may offer a biopharmacological basis for effective management of ALD.Alcohol-related disorders are one of the challenging current health problems with medical, social, and economic consequences. Endotoxemia, oxidative stress, and release of a variety of inflammatory molecules are established mediators in alcoholic liver injury (ALD). Probiotics like  Sever Lactobacillus GG also ameliorates oxidative stress and intestinal permeability in alcoholic liver injury. Probiotics such as Lactobacillus plantarum, Lactobacillus GG, and Bifidobacterium bifidum have demonstrated a significant reduction of oxidative stress and restoration of normal bowel flora [Use of probiotics to manage the alcohol-induced endotoxin mediated liver injury is attributed to a variety of its health benefits including immunomodulatory and anti-inflammatory effects. A randomized trial carried out by Kirpich et al. in 2008 and 2012 showed an improvement of liver function tests and restoration of normal bowel flora upon administration of probiotics for the treatment of alcoholic liver disease. Further to this,el flora –11.κB which either enhances or suppresses the downstream pathways [Several important mechanisms by which the probiotic provides various health benefits include modification of the gut microbiota, competitive adherence to the mucosa and epithelium, strengthening of the gut epithelial barrier, and modulation of the immune system of the host. Evidence also demonstrates that probiotics communicate with the host by pattern recognition receptors, such as toll-like receptors which modulate key signaling pathways, such as nuclear factor-pathways . in vivo is that they must survive and sustain transit through the extreme conditions of the gut in large quantities to facilitate their colonization in the host to confer these benefits. In this context, encapsulation techniques may ensure greater survival of probiotic bacteria under gastric conditions. This is a method by which bacteria are protected from detrimental factors of environments such as high acidity (low pH), bile salts, molecular oxygen in case of obligatory anaerobic microbes, bacteriophages, and chemical as well as antimicrobial agents [However, major concern for the use of probioticsl agents –15. Howe Alginate is a linear heteropolysaccharide extracted from algae, with two structural units consisting of D-mannuronic and L-guluronic acids and it forms hydrogels in the presence of divalent ions such as Ca2+. However, certain disadvantages are attributed to alginate beads including the susceptibility to acidic environments and deterioration of beads when subjected to monovalent ions or chelating agents which absorb calcium ions. These limitations can be efficiently compensated by coating alginate with suitable compounds so as to impart it with mechanical strength. Chitosan is a linear polysaccharide with negative charge arising from its amine groups which are obtained by deacetylation of chitin [Amongst the encapsulation devices, microencapsulation in calcium alginate microparticles has been widely used owing to its ease of handling, nontoxic nature, and low cost.f chitin . Chitosaf chitin . Anticip Lactobacillus plantarum (MTCC 2621), used as a probiotic, was acquired from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh (India).Absolute ethanol (99.9%) was purchased from Brampton, Ontario. The probiotic microorganismL. plantarum was cultivated in Lactobacillus MRS broth (1% inoculum) at 37°C for 18 hours. The cells were harvested by centrifuging the culture at 8000 rpm for 15 min at room temperature. The cells were washed twice with 0.1% peptone water and were suspended in 1 mL of the same.L. plantarum was encapsulated in calcium alginate by the method of Krasaekoopt et al. 2003, 2004 [ L. plantarum in a solution of sodium alginate was then dropped into 1% sterile calcium chloride with stirring on a magnetic stirrer. The beads were left for hardening by continuous stirring for 2 hours. The formed L. plantarum encapsulated alginate beads (AL beads) were then coated with medium molecular weight chitosan  using the method of Krasaekoopt et al. 2003 [n = 6). The alginate loaded microparticles were named as AL and AL beads coated with chitosan were referred to as AL-CA.03, 2004 , 19 via al. 2003 , in whic(1) Determination of Particle Size. The particle size of the formed beads (AL and AL-CA beads) was determined in triplicate and mean size was recorded using particle size analyzer . (2) Scanning Electron Microscopy (SEM) for Surface Morphology. Freeze dried calcium alginate beads (AL and AL-CA beads) were coated with gold film under vacuum to modify the conducting materials and surface morphology was studied. (3) Drug Entrapment Efficiency (DEE) of Probiotic Beads. For bacterial enumeration, the beads (AL and AL-CA beads) were crushed in 1% sodium citrate solution while stirring for 60 minutes. Serial dilutions were made in 0.1% peptone water and spread plate on MRS Agar and incubated for 48 hours at 37°C (n = 6) and number of colonies forming units (cfu) was counted. Thereafter percentage of entrapment was calculated using the following formula:. (4) Viability of Bacteria Postentrapment. To assess the viability of entrapped bacteria the beads (AL and AL-CA beads) were crushed in 1% sodium citrate solution while stirring for 60 minutes. Serial dilutions were made in 0.1% peptone water and 0.1 mL was spread plated on MRS Agar plates and incubated for 48 hours at 37°C.(5) Determination of Porosity. Beads (AL and AL-CA beads) were filled in a 10 mL graduated measuring cylinder up to the mark. The cylinder was tapped 500 times and the final volume was noted. Initial volume was kept the same in all the cases and the final volume gave the tap volume. The porosity was calculated according to the following equation and mean % porosity and standard deviation were recorded [Vb: bulk volume of the beads = 10 mL; Vp: true/tap volume of the beads;recorded :(2)Poro(6) Viability of Entrapped Probiotic Bacteria in Simulated Gastric Fluid (SGF) and Sequentially in Simulated Intestinal Fluid (SIF). Free probiotic cells and the beads (AL and AL-CA beads) were incubated in SGF without pepsin  for 4 hours and the samples were then sequentially transferred to SIF without pancreatin  for 2 hours and viable count was studied as described earlier [ earlier . (7) In Vitro Release Studies. Release study of probiotic beads (AL and AL-CA beads) was carried out in SIF under aseptic conditions. 100 mg beads were incubated at 37°C in test tubes containing 10 mL of SIF for 3 hours. At intervals of 1 hour, the supernatant from each tube was analyzed for cell count. For bacterial enumeration, serial dilutions of the supernatant were made in 0.1% peptone water and spread plate on MRS agar and incubated for 48 hours at 37°C. (8) Bile Salt Tolerance. The viability in the presence of bile salts was assessed by suspending the free cells and beads (AL and AL-CA beads) in MRS broth supplemented with 0.3% (w/v) bile salts (sodium deoxycholate and sodium taurocholate) for 4 hours and bacterial count was estimated as described earlier.The experiment protocols were approved by the Institutional Animal Ethics Committee  and performed in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, on animal experimentation. All efforts were made to minimize the suffering of animals. ad libitum access to food  and water. It has been reported in the literature that females develop more severe liver injury than males due to smaller amount of body water and lower activity of alcohol dehydrogenase enzyme (ADH) in stomach [Female Wistar rats 200–250 g) were procured from Central Animal House, Panjab University, Panjab University, Chandigarh (India). The animals were housed under standard laboratory conditions, maintained on a 12 : 12 h light : dark cycle, and had0–250 g wAlcohol Dosing. Rats were administered 10 g/kg of body weight/day of 35% (v/v) ethanol by oral gavage in double distilled water for two weeks. Thereafter, the dose was increased to 14 g/kg of body weight/day and was continued for 10 weeks through oral gavage [l gavage .Probiotic Dosing. 1010 cfu/mL of Lactobacillus plantarum was dispersed in 1 mL of PBS (pH 7.2). Chitosan coated alginate beads containing equivalent L. plantarum were dispersed in 1% carboxymethyl cellulose and were administered to rats through oral gavage.After an acclimatizing period, rats were randomly divided into the following four groups each comprising 10–12 rats as depicted in After 10 weeks of alcohol administration, blood was taken from the tail vein 1.5 h and 2.5 h after gavage. Blood alcohol levels (BAL) were measured using the alcohol dehydrogenase kit from Sigma Chemical Co., USA.μL of plasma was incubated with 50 μL of limulus amebocyte lysate (LAL) at 37°C for 45 min. After several subsequent reactions, the samples were read spectrophotometrically at 405 nm. The plasma endotoxin levels were calculated against a standard curve of endotoxin concentrations of 0.1, 0.04, 0.02, 0.01, and 0.005 EU/mL.Endotoxin level in the plasma samples was measured using Toxin Sensor Chromogenic LAL Endotoxin Assay Kit (Hycult Biotech). Briefly, 50 Assessment of Liver Function. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities in serum were determined using ERBA test kits . Alkaline phosphatase (ALP) was estimated using Enzopak Diagnostic kit .Histological Studies. Liver and intestine tissues removed aseptically from the animals were cut into small pieces and fixed in 10% buffered formalin. Samples were dehydrated in different grades of alcohol, washed with xylene, and embedded in paraffin wax, and the sections were stained with hematoxylin-eosin and examined under the light microscope. Histological interpretation was done by Dr. B. N. Datta, Ex-Professor of Pathology, Post Graduate Institute of Medical Education and Research, Chandigarh (India).Mechanistic Studies. Livers removed aseptically from the rats were rinsed in 0.05 M phosphate buffer saline (pH 7.4) (PBS). A 25% (w/v) tissue homogenate was prepared in PBS using a Potter Elvehjen homogenizer. Assay for NF-κB p50 Subunit. Assay for NF-κB/p50 subunit in the nuclear extracts was performed in all the groups by commercially available Transcription Factor Assay kit  according to the manufacturer's instructions. This assay combines the principle of the electrophoretic mobility shift assay (EMSA) with the 96-well based enzyme linked immunosorbent assay (ELISA). Briefly, cellular extracts were prepared from liver tissue using Chemicon's Nuclear Extraction kit. During the assay, the capture probe, a double stranded biotinylated oligonucleotide containing the consensus sequence for NF-κB, was mixed with the nuclear extract in the transcription factor assay buffer provided directly in the streptavidin coated plate and incubated for 2 h at room temperature. Plates were then washed to remove the unbound material. The bound NF-κB transcription factor subunit p50 was detected with rabbit anti-NF-κB p50 (specific primary antibody). HRP conjugated secondary antibody was then used for detection using 3,3,9,5,5,9-tetramethybenzidine (TMB/E) as the substrate and absorbance was read at 450 nm. Positive and negative controls were also run simultaneously [aneously . α (TNF-α)Assay for Tumor Necrosis Factor-. Assay for TNF-α was performed by ELISA in the liver homogenates using commercially available cytokine assay kit  according to the manufacturer's instructions described by us earlier [α. To each of the designated wells, 50 μL of assay diluent was added; the plates were sealed with acetate plate sealer and incubated at room temperature for 2 h. Plates were then washed five times with the wash buffer and 100 μL of rat TNF-α conjugate was dispensed into each well. Plates were again sealed and incubated at room temperature for 2 h, after which they were washed five times with the wash buffer and 100 μL of substrate solution was dispensed into each well. Plates were finally incubated at room temperature (in dark) for 30 min. 100 μL of the stop solution was added into each well to stop the reaction and absorbance was read at 450 nm. The results were expressed as picogram/mL of the TNF-α released. The ELISA was sensitive to 5 picogram/mL of the TNF-α released.  earlier . BrieflyAssay for IL12/p40 Subunit. To check the levels of IL12/p40 subunit a double antibody sandwich ELISA was performed using the commercially available kit  according to the manufacturer's instructions. Briefly, standards (50 μL) and test samples (10 μL of liver homogenate + 40 μL of sample diluent) were added to the 96-well microtiter plates precoated with monoclonal antibody specific for rat IL-12/p40 subunit. To these designated wells, 50 μL of HRP labelled IL12/p40 subunit antibody was added and incubated for 60 minutes at 37°C. Following incubation, the excess liquid was discarded, dried, and washed five times with washing buffer. 50 μL each of chromogens A and B was dispensed and incubated for 10 minutes at 37°C away from light. The assay was stopped by adding 100 μL of stop solution and the absorbance was read at 450 nm.P < 0.05) increased the size of the probiotic loaded beads but also smoothened the surface of alginate microparticles (P < 0.05) lower than the AL-CA beads 80.4 ± 1.5 mm  microparticles  were spharticles . The mea± 1.5 mm .n = 6) and the mean viable count was calculated. In all the cases, the initial cell count was kept in the range of 10.2–10.73 log cfu/mL. Maximum cell entrapping was observed to be 80% in the probiotic loaded beads where the concentration of sodium alginate was kept as 2%  were more porous (95%) as compared to the alginate (AL) probiotic beads (84%).P < 0.05) in cell count for free probiotic than AL beads and AL-CA beads. However, chitosan coated probiotic alginate beads (AL-CA) provided the best protection  than the AL  and free probiotic . The cell count of free probiotic gradually decreased by ≈6 log units with incubation in SGF without pepsin for four hours. A decrease in cell count (≈4 log units) was also observed with AL beads. However, the viability of Lactobacillus plantarum was reduced by only ≈2 log units when double encapsulation was provided with chitosan over alginate beads. In the alkaline environment, the cell viability of the free probiotic further was reduced to ≈2 log units which shows that the free probiotic cannot tolerate the harsh environment of gastrointestinal pathway.To assess the likelihood of microencapsulated and free probiotic bacteria surviving passage through the stomach following oral administration, they were tested in simulated gastric fluid  without pepsin for four hours and sequentially in simulated intestinal fluid  without pancreatin for two hours.  in vitro release studies (P < 0.001) was observed between the AL-CA and AL probiotic beads (~80%). The AL-CA beads could not affect the release of probiotic rather it offered better stability.The studies  showed t L. plantarum were the most effective  in providing protection against bile salts  provided better protection in all the above-mentioned parameters to the probiotic than the alginate beads (AL), therefore, the former were used for furtherAfter 12 weeks of regular alcohol administration, the blood alcohol levels were found to be significantly increased in the alcohol supplemented group when compared to other groups. Blood alcohol levels (BAL) 1.5 h and 2.5 h after ethanol administration by gavage in the alcohol group were 243.2 mg/dL and 198.6 mg/dL, respectively. BAL in the alcohol treated and chitosan coated probiotic alginate beads supplemented group were 214.7 and 176.1 mg/dL after 1.5 and 2.5 h of alcohol administration, respectively.P < 0.01) lower (0.312 EU/mL) than group 2  suffered from significant endotoxemia (0.54 EU/mL) as compared to the control rats (0.16 EU/mL). The plasma endotoxin levels in alcohol administered and AL-CA supplemented group were significantly ( group 2 .P < 0.05) results were obtained with AL-CA beads when coadministered during alcohol abuse   as compared to the control groups . However, the levels were attenuated in the alcohol treated, free probiotic fed group. More pronounced  . The entLiver. The liver sections of alcohol administered rats showed vacuolar degeneration, micro- and macrofollicular fatty changes, and focal collection of lymphocytes. Portal tract inflammation  was also observed  group a Figures . The pro Figures . Slight  Figures .Intestine. The control group showed normal intestine ( per se group of both the free probiotic supplemented group (ntestine . The intntestine . The intntestine  and encantestine ) treateded group  and encaed group  showed nκB p50 subunit (O.D.450 nm—2.601) as compared to control group , whereas the supplementation of encapsulated probiotic significantly lowered the levels of NF-κB (O.D.450 nm—0.890). However, the entrapped probiotic decreased the level of NF-κB significantly as compared to the alcohol abused rats and rats coadministered with free probiotic (O.D.450 nm—1.470)  .α and IL-12/p40 subunit, decreased significantly (P < 0.05) with the cosupplementation of encapsulated probiotic L. plantarum as compared to the alcoholic group. Alcohol consumption caused a 2.9-fold rise in the levels of both the cytokines (35.5 ± 2.4 pg/mg protein of TNF-α and 34.64 ± 1.5 pg/mg protein IL12/p40) as compared to the control value (12.08 ± 0.98 pg/mg protein TNF-α and 11.66 ± 0.56 pg/mg protein IL-12/p40) shown in Figures α and 8.46 ± 0.76 pg/mg protein of IL12/p40, respectively. The encapsulated probiotic caused 2.5-fold in the levels of TNF-α and 4-fold decrease for IL12/p40 subunit as compared to alcoholic rats.The levels of both the proinflammatory cytokines, that is, TNF-Bioencapsulation consists of entrapment of a biologically active material inside a microparticle, providing immobilization, safety, and controlled release as well as physical structure or functions. Therefore, in the present study, probiotic was microencapsulated and evaluated for its efficacy in alcohol-induced endotoxin-mediated liver injury.SEM images revealed that the alginate beads entrapping probiotic have a wrinkled surface. This may be explained by the fact that the particles usually are heterogeneous with a dense surface layer and a loose core which results in their collapse and hence wrinkled shape . The sizAL-CA beads encapsulating the probiotic were found to be more spherical and had a smoother surface than the AL beads. Due to low viscosity, chitosan diffuses rapidly into the microparticles and uniformly distributes itself through the whole shell giving the bead a spherical shape and smoother surface . On coat in vitro to in vivo animal models, the beads were tested for their potential to withstand and grow under the simulating conditions encountered in stomach (extreme acidic pH) and intestine . Hence free and encapsulated L. plantarum beads were exposed to these conditions. The bacterial population was maintained when encapsulated with alginate and further coated with chitosan, whereas a significant reduction in the count of free probiotic was observed. The free probiotic could not resist the harsh extreme dual environment of the gut which is in accordance with the earlier study [ in vivo studies.In order to extrapolate the properties of microencapsulated probiotic fromer study . The reser study , which ser study . Krasaeker study , 25 alsoIn the present study, the observed BAL in the rats confirmed the appropriate alcohol consumption which is broken down in the liver generating potentially dangerous by-products in the presence of alcohol dehydrogenase. Perhaps more so than alcohol itself, these products contribute to alcohol-induced liver damage. Moreover, chronic alcohol consumption mediates endotoxemia which occurs due to alterations in the gut microbiota (dysbiosis) as well as compromised gut barrier function leading to increased intestinal permeability. This indicates that the therapeutic strategies targeting the gut microbiome may be effective in the treatment of ALD .In this context, probiotics are being explored extensively in view of their potential to maintain the composition of normal bowel flora, in addition to their competition for nutrient and adhesion sites, production of inhibitory compounds such as bacteriocins, and lowering of cationic pH by the production of short chain fatty acids . It has κB after alcohol consumption as compared to the free probiotic which might have reduced TNF-α levels. Blocking of NF-κB resulting in the downregulation of TNF-α has been reported by us earlier [ Lactobacillus delbrueckii and Lactobacillus fermentum ameliorated the inflammation by decreasing concentration of IL-6 and expression of TNF-α and NF-κB p65 in ulcerative colitis [κB, TNF-α, IL-6, and p-Akt and increased IL-10 expression in colonic tissues in acute colitis [In this study, the probiotic beads, because of their nature of controlled release as mentioned above, were found to be better in restoring liver and intestine histology, reducing endotoxemia, and attenuating inflammation. The entrapped probiotic significantly lowered the levels of transcription factor NF- earlier . The res colitis , 30. VSL colitis .α therapy is that it lacked the efficacy and loss of response in some patients and led to potential adverse effects [κB, anti-TNF-α, and anti-IL-12/p40 subunit activity of the probiotic correlated well with the functional activity of transaminases resulting in restoration of clinical manifestations of the disease in terms of tissue architecture.The use of antitumor necrosis factor (TNF) therapies has been a huge success in immune-mediated inflammatory diseases (IMIDs) . However effects . Thus, r effects . The encTo the best of our knowledge, this is the first report wherein the improved efficacy of probiotic after being microencapsulated in liver damage has been demonstrated. Further, it may be noticed that microencapsulated probiotic ameliorated ALD by suppressing molecular inflammatory markers particularly IL-12/p40 subunit which remained unexplored earlier.


The seasonality of influenza virus infections in temperate climates and the role of environmental conditions like temperature and humidity in the transmission of influenza virus through the air are not well understood. Using ferrets housed at four different environmental conditions, we evaluated the respiratory droplet transmission of two influenza viruses  and concurrently characterized the aerosol shedding profiles of infected animals. Comparisons were made among the different temperature and humidity conditions and between the two viruses to determine if the H3N2 variant virus exhibited enhanced capabilities that may have contributed to the infections occurring in the summer. We report here that although increased levels of H3N2 variant virus were found in ferret nasal wash and exhaled aerosol samples compared to the seasonal H3N2 virus, enhanced respiratory droplet transmission was not observed under any of the environmental settings. However, overall environmental conditions were shown to modulate the frequency of influenza virus transmission through the air. Transmission occurred most frequently at 23°C/30%RH, while the levels of infectious virus in aerosols exhaled by infected ferrets agree with these results. Improving our understanding of how environmental conditions affect influenza virus infectivity and transmission may reveal ways to better protect the public against influenza virus infections. Influenza viruses display distinct seasonal patterns in temperate climates with peak infection rates occurring in the winter months and little to no influenza virus activity detected at other times of the year . HoweverThere are three generally accepted modes of influenza virus transmission . Aerosolin vitro studies have demonstrated that influenza viruses in droplets and aerosols maintain viability better at low humidity [Temperature and humidity are inextricably linked when considering their effects on evaporation rates and on infectious particle dynamics . Althoughumidity ,17. Animhumidity . The ferhumidity . To moreAll ferret procedures were approved by Institutional Animal Care and Use Committee (IACUC) of the Centers for Disease Control and Prevention and in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility. Animal studies were performed in accordance with the IACUC guidelines under protocol #2234MAIFERC: "Transmissibility of influenza viruses with pandemic potential".Virus stocks of A/Panama/2007/1999 (H3N2) were propagated in the allantoic cavity of 10 day old embryonated hens’ eggs while A/Indiana/8/2011 (H3N2v) was grown in Madin-Darby canine kidney cells (MDCK) as previously described [3 AH); 23°C/30%RH (6.2 g/m3 AH); 23°C/50%RH (10.3 g/m3 AH) and 23°C/70%RH (14.4 g/m3 AH). Prior to inoculation, ferrets were allowed to acclimatize inside the chamber for at least four days. Baseline temperatures were measured using an implantable subcutaneous temperature transponder  and baseline serum, weight and minute volume (MV) of respiration measurements were collected as described [Male Fitch ferrets 5–11 months of age (Triple F Farms), serologically negative by hemagglutination-inhibition (HI) assay for currently circulating influenza viruses, were housed during each experiment in cages placed inside a custom environmental chamber  with HEPA filtration operating at 20 air changes per hour. Experiments were conducted at four environmental settings with consideration for the comfort of the animals: 5°C/70%RH  and was found to be negligible (<0.05 m/s) for all of the cages. One day post inoculation (dpi) a naïve ferret was placed in a cage adjacent to each inoculated animal. Nasal washes (NW) were collected every other day for up to 11 dpi or days post contact (dpc)  for viruThree inoculated ferrets from each experiment were anesthetized and exhaled aerosol samples were collected and analyzed for size distribution using an aerodynamic particle sizer . Aerosol samples were collected from ferrets immediately after exhalation on 2, 4 and 6 dpi for 15 minutes of closed-mouth, normal breathing and for 5 minutes of induced sneezing as described . Size diExhaled aerosols from inoculated animals were analyzed for infectious virus using a viable two-stage cascade impactor  on 1, 3, and 5 dpi as described with modifications . Aerosol2–103 pfu) directly on prepared impactor plates and placing the impactor assembly inside the environmental chamber at one of the four environmental conditions [The effects of the aerosol collection procedure on influenza virus recovery was evaluated in duplicate by applying a known amount of virus .3.8–105.5 pfu of PN99 or IN11 virus   and allo11 virus . One dpiRegardless of the inoculum, changes in activity levels in infected animals were minor (relative inactivity indexes ranged from 1.0–1.2) while anorexia and nasal discharge were the most common clinical signs observed in ferrets housed at any of the controlled environmental settings. Peak temperatures were detected 2–3 dpi in inoculated animals and ranged from 1.2–2.4°C above baseline for PN99 virus and 1.9–3.3°C above baseline for IN11 virus . MV of r4.2 to 104.8 pfu/mL in ferrets inoculated with PN99 virus and were 3–24 times higher (105.2 to 105.7 pfu/mL) in ferrets inoculated with IN11 virus housed under the respective environmental condition; the greatest difference was observed at the 23°C/70%RH condition  on 1, 3 and 5 dpi and then quantified for influenza virus by plaque assay as well as real-time RT-PCR. Infectious virus detected at any time point and in either size range in normal breathing samples was ≤10 pfu  and in sThe impact of the aerosol collection procedure on virus recovery was tested for each controlled environmental condition by spiking the collection medium on each impactor stage with known quantities of virus. After air was pulled through the impactor for 15 or 5 minutes, to represent normal breathing and sneezing sampling times, respectively, the collection medium was harvested and processed as described for the ferret samples. PN99 virus RNA recovery from either stage and for the 15 and 5 minute collection times ranged from 54 to 79%, similar to IN11 virus RNA recovery which was 47 to 81% . InfectiThe influence that temperature and humidity have on aerosolized influenza virus and the consequence for transmission remain poorly understood despite the potential impact on the seasonality of influenza epidemics in the temperate regions of the world. Climatic conditions in tropical regions of the world represent a different set of parameters affecting transmission, and furthermore, routes of transmission not involving airborne virus (e.g. direct or indirect contact) demonstrate the complexity of global influenza virus transmission and the multitude of potential mechanisms at play each impacted differently by environmental conditions. Here, we evaluated the transmissibility of two H3N2 influenza viruses (PN99 and IN11) using ferrets housed under diverse environmental conditions and concurrently evaluated the aerosol shedding profiles of infected animals. We compared the two virus groups to each other and then combined them to identify common differences at each environmental condition. Higher levels of IN11 virus were measured in ferret nasal washes and in exhaled aerosol samples compared to PN99 virus but overall, we found little difference in the transmission capabilities of the two viruses at any of the environmental conditions with transmission most frequently observed at 23°C/30%RH (83%) and least frequently observed at 23°C/50%RH and 5°C/70%RH (17%).The viruses chosen for this study were selected because one represents a seasonal H3N2 strain PN99 virus), the other one is a swine-origin H3N2 variant strain (IN11 virus) responsible for a summertime infection ,25 and b9 virus, Any successful transmission event through the air requires that virus pass from an infected host to a susceptible one while maintaining infectivity. As this occurs, aerosols are released from the warm, humid environment of a respiratory tract, through the ambient conditions outside of the host and eventually reach the warm, humid environment of a recipient’s respiratory tract. Depending on the ambient conditions and the solute composition of the aerosol, evaporation and condensation processes may have deleterious effects on any virus present in the aerosols . Using cDetection of influenza virus in exhaled aerosols has been largely limited to the detection of viral genetic material via RT-PCR with few reporting infectious virus detection in humans ,13 or laIn the temperate regions of the world, people spend the vast majority of their time indoors, especially during the winter months where the environment is typically maintained at a warm temperature and low RH . This sc


Here, we report the 10,807-nucleotide-long consensus RNA genome sequences of three spatiotemporally distinct and genetically divergent Zika virus strains, with the functionality of their genomic sequences substantiated by reverse genetics: MR-766 , P6-740 , and PRVABC-59 . Flaviviridae, genus Flavivirus (Aedes species mosquitoes (–––19–––––Zika virus (ZIKV) is a positive-stranded RNA virus of the family avivirus . ZIKV issquitoes , but it squitoes –7 or by quitoes –, 9 or blquitoes –. In natuquitoes ––16. Althuitoes ––, 18, it uitoes –––5, 19–21es –––19––33 to ths –––19–––40, and  –––19––––46; it i–––19–––––50.Aedes aegypti mosquitoes in 1966 ; however, little is known about the genetic variation among these geographically and temporally distinct ZIKVs because of the limited number of fully sequenced ZIKV genomes , 51–61. 51– in 1966 ; and  flanked by a 106- or 107-nt 5′ noncoding region (NCR) and a 428- or 429-nt 3′ NCR . The ORFThe accession numbers deposited at the GenBank sequence database are listed in


Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research. Drosophila melanogaster, and large-scale surveys show that many are common in the lab and in the field.Sequencing of metagenomic RNA and small RNA identifies more than 20 new viruses associated with the fruit fly  Drosophila melanogaster is extensively used as a model species for molecular biology and genetics. It is also widely studied for its evolutionary history, helping us understand how natural selection has shaped the genome. Drosophila research has been particularly valuable in determining how the insect immune system interacts with viruses and how co-evolution between hosts and viruses can shape the host immune system. Understanding insect–virus coevolution is important because some viruses—such as those which cause dengue and yellow fever in humans—also infect their insect vectors, and because the viruses of bees and other pollinators are implicated in pollinator decline. Although we have an increasingly good idea of how flies recognise and combat viral pathogens, we still have much to learn about the viruses they encounter and interact with in the wild. In this paper, we sequence all of the genetic material from a large collection of wild fruit flies and use it to identify more than 20 new viruses. We then survey individual wild flies and laboratory stocks to find out which viruses are common, which are rare, and which species of fruit fly they infect. Our results provide valuable tools and an evolutionary and ecological perspective that will help to improve Drosophila as a model for host–virus biology in the future.The fruit fly  Drosophila melanogaster, provides a valuable tool to understand the biology of viral infection  for known extant lineages) of approximately 100–200 years ago  and 50–300 years ago , respectively. This rapid movement of viruses is consistent with the rapid global movement that has been seen in transposable elements , Wolbacholbachia , and eve genomes . Neverth genomes . For theD. melanogaster and D. simulans might either be reciprocally monophyletic, with substantial divergence between host-specific lineages, or the hosts may be distributed across the virus phylogeny, if between-host movement is common. By incorporating information on host species (D. melanogaster versus D. simulans) in the phylogenetic analysis, we were able detect past movement of RNA viruses between these host species  and non-synonymous (dN) substitution [dN-dS or dN/dS; dN/dS ratios , although only Nora Virus displayed codons with strong evidence of positive selection. There were no clear patterns of dN/dS variation across genes within DAV or Nora virus genomes .D. melanogaster in the wild and shown that some of them are common in the laboratory and the field. A substantial fraction of the virus lineages described here are newly , negative sense RNA genomes , and double-stranded RNA genomes , but few with DNA genomes  repoiviruses  to date)oviruses ; retro-eDrosophila viruses will prove valuable for the use of D. melanogaster as a model for viral infection and antiviral immunity. First, these data will facilitate the curation and management of laboratory stocks and cell cultures. Second, they will facilitate the isolation and culture of viruses for future experimental work, including natural pathogens of Drosophila that are closely related to medically or economically important viruses such as Flaviviruses and Bunyaviruses, or Chronic Bee Paralysis Virus. Third, our analyses of virus prevalence, distribution, and dynamics will provide an informed evolutionary and ecological context to develop D. melanogaster and its relatives as a model system for viral epidemiology and host-switching. In combination, we hope this work will provide a key reference point for future studies of Drosophila-virus interaction.This newly discovered diversity of Drosophila melanogaster and other sympatric drosophilid species were made in 2010 by netting flies from fruit bait. For United Kingdom samples (denoted S and T), flies were morphologically screened to exclude species other than D. melanogaster. For non-UK samples , flies were screened to bear a superficial similarity  to D. melanogaster. Samples E and K, each of ca. 700 adult pale-bodied Drosophilidae, were collected in Kilifi (Kenya) by JA in July 2010. Sample I of ca. 650 adult pale-bodied Drosophilidae were collected in Ithaca  by BPL in August 2010. Sample S of ca. 1,250 adult D. melanogaster were collected in Sussex (UK) by DJO and EHB in August 2010. Sample T of ca. 1,700 adult D. melanogaster were collected in Twyford (UK) by DJO and Alasdair Hood in July 2010. In each case, flies were maintained in groups of up to 200 for 5–10 d on a hard-agar/sugar medium before maceration under liquid nitrogen. RNA extraction was performed separately on each of the five samples.For metagenomic viral discovery, five large collections of D. melanogaster and D. simulans were made at the following locations between November 2008 and October 2012: Thika (Kenya) by John Pool in January 2009; San Diego  by DJO in November 2008; Edinburgh (UK) by DJO in August 2009; Athens (Greece) by Natasa Fytrou in June 2009; Marseille (France) by Nicolas Gompel in July 2009; Ithaca  by BPL in September 2009; Athens  by PRH in September 2009; Sussex (UK) by DJO in August 2009; Two locations in Accra (Ghana) by CLW and JA in January 2010; Plettenburg (South Africa) by Francis Jiggins in January 2010; Edinburgh (UK) by CLW in September 2009; Marrakech (Morocco) by CLW in September 2010; Montpellier (France) by PRH in September 2010; Torquay  by BL in February 2011; Lisbon  by DJO in October 2012; Porto  by DJO in October 2012  and DNAse treated (Life Technologies) according to the manufacturer’s instructions. For metagenomic RNAseq and small-RNA sequencing, aliquots of the samples E, I, K, S, and T were initially mixed into two pools: Dmel/UK (denoted ST) and mixed-species/non-UK (EIK). RNAseq was performed by Edinburgh Genomics (Edinburgh). Following ribosome-depletion using two cycles of RiboMinus (Life Technologies), around 40 million high-quality paired-end 100 nt Illumina reads were generated from a single sequencing lane of each library (accessions SRR1914484 and SRR1914527). Small-RNA sequencing from the same two pools was performed with a periodate oxidation step to reduce the relative ligation efficiency of miRNAs, which do not carry 3′-Ribose 2′O-methylation , and seqD. melanogaster reference mtDNA) drawn from multiple species. This identified the EIK RNA sample to have been a mixture of D. simulans (0.8%), D. hamatofila (1.2%), D. ananassae (12.4%), Scaptodrosophila latifasciaeformis (16%), D. melanogaster (24.1%), and D. malerkotliana (45.5%). It also identified a small amount of cross-species contamination in the D. melanogaster ST sample . To estimate the contribution of each of several potential sources of RNA, reads were also mapped to known Drosophila viruses, the D. melanogaster, D simulans, and D ananassae genomes, Wolbachia from D. melanogaster, D. simulans, D. willistoni, D. suzukii, and D. ananassae, and the bacteria Pseudomonas entomophila, Providencia sneebia, P. rettgeri, P. burhodogranariea, P. alcalifaciens, Gluconobacter morbifer, Enterococcus faecalis, Commensalibacter intestini, Acetobacter pomorum, A. tropicalis, and A. malorum (To estimate species-composition of the UK (ST) and non-UK (EIK) pools, RNAseq datasets were quality trimmed using ConDeTri2.0 and mapped using Bowtie2  to a 359 malorum . The per−10 to identify the top 10 hits. Contigs with no BLAST hit at this threshold were translated to identify the longest open reading frame in the contig, and the resulting amino acid sequence compared to Genbank protein reference sequence using BLASTp with default parameters and an e-value threshold of 1 × 10−5 to identify the top 10 hits. For each contig with BLAST hits, the phylogenetic hierarchy of the best hits was traversed upward to identify the lowest taxonomic classification displaying a 75% majority taxonomic agreement, and this was used to guide subsequent cross-assembly and manual curation using the SeqManPro assembler (DNAstar). The resulting BLAST-candidate virus sequences were then used as targets for (RT-)PCR and Sanger sequencing confirmation , with primer design guided by assuming synteny with close relatives. Final sequences for phylogenetic analysis and submission to NCBI were generated either by PCR and Sanger sequencing, or from a majority consensus derived by re-mapping RNA-seq reads to the combined de novo/PCR contig (sequences have been submitted to Genbank as KP714070-KP714108 and KP757922-KP757936).All paired-end RNAseq datasets were combined and de novo assembled using three different approaches. First, all reads were assembled using Trinity . SecondDrosophila Dcr2-Ago2 RNAi pathway processes double-stranded RNA derived from viruses and transposable elements into 21 nt siRNAs [Drosophila OSS cell culture expressing Piwi [Drosophila immune system.The t siRNAs ,8,102. It siRNAs ), howeveing Piwi , these hDrosophila viruses DCV, DAV, Nora virus, and DMelSV as threshold a guide, we then used a high ratio of siRNA:RNAseq reads and a high ratio of siRNA:piRNA reads to corroborate the BLAST-candidate viruses outlined above, and to propose novel siRNA-candidate viruses amongst contigs lacking BLAST hits  with Ribo-Zero rRNA depletion. For each contig this resulted in a count per unit length of the number of 21–23 nt siRNAs, the number of 25–29 nt putative piRNAs, and the number of RNAseq reads. Using the well-studied AST hits . Final sWe used translations from the novel viruses to perform a BLASTp similarity search of the Genbank protein reference database to identify relatives and suitably conserved genomic fragments for phylogenetic inference. In addition, we searched the Genbank transcriptome shotgun assemblies (TSA: database tsa_nt) using tBLASTn  to identD. melanogaster reference genome (Flybase). The 14 siRNA datasets comprised: each of E, I, K, S, and T ligated using the NEBnext protocol according to manufacturer’s instructions; each ligated using the NEBnext protocol replacing oligos with “High Definition” equivalents that incorporate four additional random bases [To quantify the relative number of small RNAs produced from each virus, RNAseq reads and small RNAs from the EIK and ST pools were re-mapped using Bowtie2 . To quanom bases ; and theom bases . To idenom bases .D. melanogaster [Several viruses are known to be endemic in laboratory stocks and cell cultures of nogaster ,21, and nogaster  , and unmapped reads digitally normalised (Khmer) and de novo assembled using Trinity [To detect additional novel RNA viruses present in these public datasets, but absent from the literature or from our metagenomic sequencing of wild flies, we further selected 2,188 Illumina RNAseq datasets for viral discovery. The first 20 million read pairs  from each dataset were mapped to the  Trinity  as aboveD. melanogaster genomes can provide evidence that these sequences do not represent “fossilised” endogenous viral elements (EVEs). However, if only a minority of individuals carry a particular EVE, and if genomes are derived primarily by mapping to a reference, the EVEs may not appear in assembled datasets. We therefore mapped all reads (forward and reverse reads mapped separately) from 779 Drosophila NEXUS genomic read datasets .We reasoned that the absence of virus-like sequences from  genomes ) to knowDrosophila viruses , ten novel BLAST-candidate viruses , and two novel siRNA-candidate viruses . In addition, we surveyed the same RNA extractions by RT-PCR for the presence (but not titre) of Wolbachia pipientis, using primers for the Wolbachia surface protein (wsp_F1 GTCCAATARSTGATGARGAAAC and wsp_R1 CYGCACCAAYAGYRCTRTAAA).We used RT-PCR to survey wild populations for the presence of four previously published D. melanogaster individuals and 658 D. simulans individuals sampled across 17 locations (see “(median n = 63). RNA extractions and random hexamer reverse transcription was performed on single flies  or small bulks of 2–20 flies . Fly species was confirmed using a competitive 3-primer PCR for the single-copy nuclear gene Ago2, which gives a 300 bp product in D. simulans and a 400 bp product in D. melanogaster .This survey comprised a total of 1,635 ons see “” above, For each of these 16 viruses we designed up to four PCR assays based on conserved positions in the metagenomic RNAseq datasets, and these were tested on the E, I, K, S, and T metagenomic discovery RNA pools and on a selected subset of wild-collected individual flies. Those primer sets that consistently gave repeatable bands in agreement with other assays for the same virus, and which could be sequenced to confirm band identity, were selected for use as assays for the wider geographic sampling , and only two were “significant” using the Benjamini-Hochberg approach to limit the false discovery rate to 5% .As only one fly needs to carry a virus for the bulk to test positive for that virus, the proportion of “positive” single-fly and/or small-bulk assays does not directly estimate the frequency of virus carriers. We therefore used a maximum likelihood approach to infer the underlying prevalence . Briefly, we assumed that for each species/location combination there was a single underlying prevalence  and that the number of flies testing positive or negative by PCR assay would be binomially distributed given this prevalence. Then, given the number of flies included in each small bulk, and the number of small bulks or single flies testing positive, we searched across the range of possible prevalence values (zero to one) to identify the prevalence which maximised the likelihood of the observed set of positive/negative PCR assays. It is possible that differences in the sex ratio between individual-fly assays  and small-bulk assays  could make this approach misleading if prevalence differs between males and females. However, based on likelihood ratio tests  we were unable to identify any widespread significant differences between the sampling regimes. Specifically 4.4% of tests were nominally “significant” at Eleven of the viruses with high prevalence  were selected for further sequence analyses. PCR products amplified during the prevalence survey were supplemented with additional amplicons and sequenced in both directions using BigDye V3.1 (Life Technologies). Reads were assembled into partial or near-complete genomes using SeqManPro (DNAstar) and all variable sites within or between chromatograms were examined by eye. Virus sequences that displayed substantial within-sample  heterozygosity for any amplicon, and therefore deemed likely to represent mixed infections, were discarded. Sequences were submitted to Genbank under accessions KP969454-KP970120.Sequence alignments were divided into blocks, minimising missing data within blocks, and these alignment blocks were tested for evidence of recombination using GARD  from the−4 site-1 year-1 and 95% of the prior density between 1 × 10−4 and 11 × 10−4. When greater time-sampling depth was available (DAV and Nora Virus) we ran models using a hierarchical prior for global nucleotide clock rate, in which clock rates for individual segments are drawn from a shared log-normal distribution. To infer rates of host-switching between D. melanogaster and D simulans, and rates of movement among broadly-defined sampling locations , tips were labelled by location and host species and treated as discrete traits [8 steps sampling every 50,000 steps, and convergence was inferred for all parameter values both visually from stationary distributions, and from effective sample sizes of >1,000. In one case (Thika virus), time and date parameters mixed poorly, and the analyses were re-run using a hard upper limit to the nucleotide clock rate for one alignment block (2.5 × 10−3 site-1 yr-1). Posterior parameter estimates and maximum clade credibility trees were inferred from the two independent parallel runs combined after discarding 10% of steps of each as burn-in.Alignment blocks (containing no detectable recombination) were subjected to phylogenetic analysis using BEAST  under a e traits ,109. AlldS) and nonsynonymous (dN) substitution, and evidence for positive selection (dN-dS > 0) were inferred using the FUBAR [dN-dS and sites under selection by pre-computing a grid of conditional likelihoods for dS and dN (where the expectation of dS is 1) and reusing the precomputed values to infer the posterior distribution of dN-dS without recalculating likelihoods. The analysis used a 20 × 20 grid with ten independent MCMC chains each providing 1,000 subsamples from the posterior (each 5 × 108 steps after 5 × 108 burn-in steps). Sites were considered to display evidence of positive selection if more than 95% of the posterior sample for that site supported dN>dS.Patterns of synonymous Click here for additional data file.S2 DataDrosophila and associated microbiota. The contigs are uncurated and are likely to include chimeric assemblies. As such, they are not suitable for submission to Genbank and should be treated with caution.Raw metagenomic contigs derived from Trinity , Trinity(ZIP)Click here for additional data file.S3 DataProtein sequence alignments used to generate the trees presented in Figs (ZIP)Click here for additional data file.S4 DataMaximum clade credibility trees with Bayesian posterior support are provided in BEAST nexus format, suitable for display with FigTree.(ZIP)Click here for additional data file.S5 DataThe tab-separated text file contains counts of virus-derived small RNA and RNA-seq reads from 9,656 publicly available sequencing “run” datasets (downloaded from the European Nucleotide Archive on 9 May 2015). Up to 2 million reads  were mapped for each dataset. Datasets with no mappable reads were excluded.(ZIP)Click here for additional data file.S6 DataD. melanogaster RNAseq datasets (which cannot be submitted to Genbank), including Brandeis Virus, Berkeley Virus, a Totivirus-like sequence, and several reovirus-like sequences.Virus-like contigs assembled de novo from publicly available (ZIP)Click here for additional data file.S7 DataAlignments and BEAST models as BEAST xml for Figs (ZIP)Click here for additional data file.S8 DataPosterior summaries of BEAST output for Figs (ZIP)Click here for additional data file.S9 DataFUBAR (HyPhy) command batch files and output. Alignments were those provided in (ZIP)Click here for additional data file.S1 FigD. melanogaster transposable elements, Drosophila miRNAs, the remainder of the D. melanogaster genome, Wolbachia and other bacteria, the four “classical” viruses , the new named viruses, other BLAST-candidate viruses, and siRNA-candidate viruses. Counts exclude unmapped reads, and reads mapping to Drosophila ribosomal sequences. EIKST refers to the metagenomic pools or mixtures thereof, “BGI” and “EG” indicate sequencing was performed by the Beijing Genomics Institute or Edinburgh Genomics (respectively), “HD” indicates the use of “High Definition” ligation adapters for small RNA sequencing, “RM” and “RZ” indicate the use of rRNA depletion by RiboMinus or Ribo-Zero, and “DSN” indicates double-stranded nuclease normalisation. Raw counts data are provided in The proportion of RNAseq and small (17-29nt) reads that map to characterised (TIF)Click here for additional data file.S2 FigDrosophila ribosomal protein gene RpL32. Note that DCV and Twyford Virus were each detectable in a single pool. Error bars represent 80% credibility intervals for means, based on two or three replicates per sample and assuming the efficiencies inferred from dilution series Click here for additional data file.S3 FigDrosophila–associated viruses (red), previously published viruses from other taxa (black) and sequences from the Transcriptome Shotgun Assemblies (blue). Bayesian posterior support is shown for second-order nodes above and where space permits, and the scale is given in amino-acid substitutions per site. Genbank nucleic acid or protein identifiers are given after each sequence. (A) Craigie’s Hill Virus and Nodaviruses; (B) Kilifi Virus, Thika Virus, DCV, and Dicistroviruses; (C) Bloomfield Virus, Torrey Pines Virus, and Reoviruses; (D) Newfield Virus, DAV, and Permutotetraviruses; (E) Galbut Virus and TSA sequences; (F) La Jolla Virus, Twyford Virus, and a closely related Iflaviruses; (G) Motts Mill Virus, Sobemoviruses, and Poleroviruses; (H) Charvil Virus and Flaviviruses; (I) Dansoman Virus and viruses related to Chronic Bee Paralysis Virus; (J) Chaq Virus and TSA sequences; (K) Brandeis Virus and related Negeviruses; (L) Berkeley Virus, DCV, and a selection of Picornavirales sequences; (M) Partitiviruses; (N) Bunyaviruses; (O) Kallithea Virus and Nudiviruses (Note that D. innubila Nudivirus [Mid-point rooted maximum clade-credibility trees showing the inferred relationship between new and previously known udivirus  is exclu(PDF)Click here for additional data file.S4 Figx-axis represent reads mapping to the positive strand and those below represent reads mapping to the negative strand. Bars are coloured according to the proportion of reads with each 5′-base . The approximate number of reads for each virus in each pool is shown inset, and only viruses that had >100 small RNA reads in that pool are shown. Pairs of rows show technical replicates, and are labelled by metagenomic pool  or pool-mixture. For mixed pools (blue background) the reads in the lower rows were sequenced by Edinburgh Genomics following an oxidation step (to reduce miRNA representation), and the upper rows by BGI without an oxidation step. For unmixed pools (white background) the reads in the lower rows were sequenced using a “High Definition” ligation protocol (both without oxidation). Note that the relative number of longer (23–27 nt) reads seen in DCV, Nora Virus, Kilifi Virus, and Thika Virus appears to be reduced in the presence of an oxidation step, as is the number of Kallithea miRNA reads. Raw count data for this figure are provided in The bar plots show the size distribution of small RNAs (17–29 nt) for selected viruses (columns) separated across the 14 different sequencing libraries (rows). Bars plotted above the (TIF)Click here for additional data file.S5 FigPart A “sequence logo” plots show biased base composition along the length of virus-derived small RNAs, where the relative letter sizes indicates the base frequency among reads at that position, and the total height represents the information content . For each virus, only the most common read length is shown, and reads are combined across all sequencing libraries. Note that most viruses show a small bias toward A and U (plotted as T) at the 5′ base, while Twyford Virus is biased toward U at both 5′ and 3′ positions. Kallithea DNA virus 22 nt reads are dominated by the miRNA ATAGTTGTAGTGGCATTAATTG. Part B “sequence logo” plots show biased base composition amongst viRNA-genome mismatches in 22 nt and 23 nt viRNAs from Twyford Virus (KP714075), indicating that many of the 3′U residues are non-templated. Raw count data for this figure are provided in (TIF)Click here for additional data file.S6 Figx-axis represent reads mapping to the positive strand, and bars below the x-axis represent reads mapping to the negative strand. Bars are coloured according to the proportion of reads with each 5′-base . All peak at 21 nt and show the expected 5′-base composition (a slight bias against G), except for siRNA Candidate 14 (KP757950) which shows the 22–23 nt 5′ U-rich peak seen in Twyford Virus (KP714075). Count data are provided in The bar plots show the size distribution of small RNAs (17–29 nt) for those unnamed siRNA-candidate loci that have >80 small RNA reads. Inset is the total number of small RNA reads summed across all libraries. Bars plotted above the (TIF)Click here for additional data file.S7 FigDrosophila miRNA miR-34-5p), and the number of virus RNA-seq reads . Ratios were then normalised to the lowest rate (Kilifi Virus in sample EIK) to give relative rates, such that 104-fold more viRNAs are derived from DMelSV than from Kilifi Virus. Where viruses were present in both the EIK and ST pools, the correlation between the two datasets is high (rank correlation coefficient >0.99), suggesting that this variation is repeatable. Normalised data are provided in To quantify differences in the rate at which small RNAs are generated from different viruses, we calculated the relative viRNA production for each of 16 different viruses in the ST and EIK metagenomic sequencing pools. The bars show the ratio between the relative number of 21–23 nt small RNAs mapping to each virus Click here for additional data file.S8 FigDrosophila and Drosophila cell culture  that map to viruses . Only datasets that have at least one virus present at ≥100 viral reads per million total reads were included, and only viruses present in at least one dataset at ≥100 viral reads per million total reads were included. Note that different parts of the segmented viruses generally co-occur within datasets, which allowed us to provisionally associate siRNA-candidate sequences with BLAST-candidate sequences . The viruses from DAV to Drosophila melanogaster Birnavirus were reported previously, the others are newly described here. Counts are provided in The grid shows the proportion of reads from each of 3,144 publicly available RNAseq and small RNA datasets from (TIFF)Click here for additional data file.S9 FigDrosophila cell culture lines, and all forward reads were mapped to viruses. Twenty-four of the datasets were drawn from ModEncode cell culture sequencing [A single exemplar RNAseq dataset was selected for each of 26 widely-used quencing , and two(TIF)Click here for additional data file.S10 FigWolbachia prevalence in D. melanogaster and D. simulans for each of the 17 locations at which flies were collected. Values are maximum likelihood estimates with 2 log-likelihood intervals, and are plotted on a log scale. Where no bar or confidence interval is provided, no flies of that species were sampled. Location details and population prevalence are provided in Charts show virus and (TIF)Click here for additional data file.S11 FigD. melanogaster and D. simulans carrying at least one of the surveyed viruses. Bars are maximum-likelihood estimates with 2 log-likelihood intervals. Coloured bars represent carriers of DAV, DCV, Nora, DMelSV, Twyford, La Jolla, Kallithea, Dansoman, Torrey Pines, Craigie’s Hill, Thika, Newfield, Bloomfield, or Motts Mill viruses ; grey bars show the large increase in overall prevalence if the siRNA-candidate viruses  are included. Missing bars indicate that the host species was absent from the collection location  or included (E), while (D) and (F) show the equivalent plots for D. simulans. Panels (C-F) are calculated using single-fly assays only (not bulks), and are averages across populations.Panels (A) and (B) show the percentage of (TIF)Click here for additional data file.S12 FigWolbachia in prevalence, across the sampled populations. Inset are rank correlation coefficients, and a simple linear regression (virus ~ Wolbachia) for illustration. The analysis does not account for any spatial autocorrelation in prevalence, and does not correct for multiple testing. Points are coloured by location Click here for additional data file.S13 FigD. melanogaster and D. simulans, and (E) the relative rates of synonymous (dS) and nonsynonymous (dN) substitution (dN-dS > 0 implies positive selection). For panels A–D points are the median of the posterior sample and 95% credibility intervals, panel E shows the median and range across codons. For all viruses except DAV and Nora Virus, a strong prior was placed on mutation rate , and mutation rates and MRCA dates were inferred separately for each alignment block. The underlying BEAST XML files  are provided, along with the resulting mcc tree files and summaries of posterior distributions, in Each panel shows parameter estimates of viral evolution for the eleven RNA viruses for which sufficient sequence data were available. Panels are (A) mutation rate, (B) date for the most recent common ancestor, (C) the range of movement between geographic regions (defined as continents and laboratory), (D) the rate of switching between (TIF)Click here for additional data file.S14 FigdN/dS are shown for each codon in open reading frames of DAV and Nora Virus . Bar colours illustrate posterior support that the site is constrained (blue for strong support that dN < dS) or positively selected (red for strong support that dN > dS). Positions coloured grey have little support for either positive selection or constraint. The dashed horizontal line indicates neutrality (dN = dS), so that bars higher than this are candidate sites for positive selection. The solid horizontal line shows the mean of the dN/dS estimates for that open reading frame. dN/dS estimates greater than 3 have been truncated to 3 for clarity. FUBAR batch files and parameter estimates are provided in Point estimates of (TIF)Click here for additional data file.S1 TableDrosophila, bacteria, and viruses.Counts of raw metagenomic reads mapping to (XLSX)Click here for additional data file.S2 TableDetailed descriptions of BLAST-candidate virus fragments and named siRNA-candidate viruses.(XLSX)Click here for additional data file.S3 TableD. melanogaster 3′ UTRs predicted by miRanda, and Gene Ontology enrichment analyses for genes with a miRanda score >150.Potential miRNA binding sites in (XLSX)Click here for additional data file.S4 TableCollection data  and virus prevalence for each species at each location . (XLSX)Click here for additional data file.S1 TextOutput from the software package mirDeep  showing (PDF)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


Cydia pomonella) populations against the Mexican isolate of its granulovirus (CpGV-M), raised questions on the sustainability of the use of this biological insecticide. In resistant host cells, CpGV-M is not able to complete its replication cycle because replication is blocked at an early step. Virus isolates able to overcome this resistance have been characterized—among them, the CpGV-R5 isolate. In mixed infections on resistant insects, both CpGV-M and CpGV-R5 viruses replicate, while CpGV-M alone does not induce mortality. Genetically heterogeneous virus populations, containing 50% of each CpGV-M and CpGV-R5 appear to control resistant host populations as well as CpGV-R5 alone at the same final concentration, even if the concentration of CpGV-R5 is only half in the former. The use of mixed genotype virus preparations instead of genotypically homogeneous populations may constitute a better approach than traditional methods for the development of baculovirus-based biological insecticides.The detection of resistance in codling moth ( Cydia pomonella granulovirus; genus Betabaculovirus; family Baculoviridae) [Cydia pomonella) and some related moth species. The CpGV-M genome comprises double stranded circular DNA of 123.5 kb in length [The Cydia pomonella granulovirus Mexican isolate (CpGV-M) (species viridae)  was discviridae) . This vin length , which hRW, while resistant males can be ZRZR or ZRZS [Resistance to CpGV-M was first reported in Germany in 2005 , and sub or ZRZS . The mec or ZRZS .pe38 gene, the function of which remains unclear [Research was conducted to obtain new viral variants able to overcome the resistance mechanisms of host cells. Extensive collections of CpGV isolates were carried out in the original apple distribution area . New iso unclear .Both resistant and susceptible individuals are usually present simultaneously in field host populations and this raises questions on the ability of a single viral genotype to provide adequate control of the pest population, and the risk of the development of resistance to other genotypes in the virus population.Genetic diversity in parasite populations is an important driver for the evolution of host defense capabilities ,16. Natuet al. [As pathogens and their hosts are continuously coevolving, it was thought that pest populations were unlikely to develop resistance to a pathogen-based product , contraret al.  noted thA major question arising is why resistance to CpGV spread recently in apple and pear agrosystems and has not been detected in natural populations of codling moth. Fuxa pointed out the importance of the ability of the virus to adapt and therefore overcome resistance of the host in natural conditions . PossiblBaculoviridae, genus Alphabaculovirus) [i.e., the possibility of transmission of more than one genotype in a single OB, has been demonstrated for Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) [Genetic variability has been described both in nucleopolyhedroviruses  ,25,26 anlovirus) ,28. In rlovirus) . Co-occl(SfMNPV) ,31. GeneSpodoptera frugiperda, Simon et al. have shown that genetic heterogeneity contributes to the efficacy of the viral population by increasing the transmissibility of virus OBs [In the virus-host system SfMNPV and irus OBs , and thaTwo laboratory colonies were used in this study: the susceptible colony CpNPP, used for the industrial production of CpGV by Natural Plant Protection SA , is susceptible to both CpGV-M and CpGV-R5 virus isolates. CpNPP has been reared in the laboratory for more than 25 years and originally comes from northern France.GV of Cydia pomonella was derived from a natural resistant population (St-A) found in the region of Saint-Andiol  as previously described [GV colony harbors the major genetic determinant for resistance that is present in resistant European populations [RZS) are resistant to CpGV-M infection. Accordingly, the RGV colony is periodically subjected to artificial selection by rearing the survivors of a CpGV-M treatment in order to maintain a high level of resistance [The resistant colony Rescribed . The RGVulations . As thisulations , and usisistance .Both colonies are mass reared on artificial diets as previously described .GV colony and produces a productive lethal infection in resistant insects. No genetic polymorphism has been detected in this virus isolate [The virus isolates used in this work, CpGV-M (laboratory stock 2020-s1) and CpGV-R5 (laboratory stock 2016-r16) have been described previously . CpGV-R5 isolate .Five mixed virus populations were constructed by mixing OBs resulting from infections of pure isolates CpGV-M and CpGV-R5 propagated on permissive CpNPP larvae. The proportions of each isolate in the mixed virus populations were 99% CpGV-M + 1% CpGV-R5; 95% CpGV-M + 5% CpGV-R5; 90% CpGV-M + 10% CpGV-R5; 50% CpGV-M + 50% CpGV-R5; 10% CpGV-M + 90% CpGV-R5. Pure CpGV-M and CpGV-R5 were used as control populations. These OBs are referred as passage zero OBs (P0).Heliothis Diet, Ward’s Science, Rochester, NY, USA) in 24-well plates. One L3 larva was then placed in each well, the plates sealed and incubated at 25 °C (±1 °C) with a 16:8 h (light/dark) photoperiod and a relative humidity of 60% (±10%). After 4 days, larvae showing clear signs of infection were removed from the wells and kept without diet at 25 °C for one more day. Infected larvae were then crushed in distilled water and the resulting mixture was filtered through nylon to eliminate debris. The suspension was then centrifuged 5 min at 8000× g, and the resulting pellet was resuspended in distilled water. The final volume of the virus stocks was of 100 µL per infected larva, which corresponds to 1011 OB/mL on average. This suspension constituted the first amplification (P1) of each viral mixture.Amplification of the different P0 populations was performed as previously described ,34. BrieHeliothis Diet, Ward’s Science, Rochester, NY, USA) were inoculated by spreading 6 µL of an OB suspension over the surface of each piece of diet by pipetting. Diet in control wells was treated with the same volume of distilled water. One larva, aged less than 12 h, was placed in each well. Six CpGV concentrations from 2 to 6250 OBs/µL on fivefold serial dilutions were used in all but CpGV-M bioassays on resistant populations, in which concentrations between 10 and 3,000,000 OBs/µL were employed. Early mortality due to handling was excluded from the test. Virus induced mortality was recorded at 7 days post-infection. Mortality data were subjected to probit analysis [2 statistic was used following Polo Mix Software (LeOra Software) [Bioassays were performed on virus populations, (P0), using established protocols . Brieflyanalysis  using thanalysis . To testoftware) . Each bi2CO3, 0.34 M NaCl, 0.02 M EDTA; pH = 10.5) was added to 100 µL of OB suspension (~1 × 107 OBs/µL) and incubated 30 min at 37 °C. The suspension was neutralized with 20 µL 1 M HCl and 11 µL 10% (wt/vol) sodium dodecyl sulfate (SDS) was added. A 1 min centrifugation step at 13,000 rpm was performed and the resulting supernatant was diluted (1/100) in sterile distilled water before (polymerase chain reaction) PCR amplification.OB stocks were obtained as detailed in 2.4. Lysis of OBs was performed in order to extract viral DNA from the OB suspensions obtained previously (P0–P1). A volume of 100 µL of extraction buffer , 20 µL of 2.5X PCR Hotmastermix  and 24 µL water. The amplification conditions were as follows: a 3 min predenaturation at 94 °C, followed by 35 cycles of 15 s, 94 °C; 40 s, 65 °C and 1 min 30 s, 72 °C.PCR was performed in a final volume of 50 µL, containing 2 µL of each primer (10 pmol/µL MWG-Eurofins), 2 µL of viral DNA  in tris-borate-ethylenediaminetetraacetic acid (TBE) buffer. Fragments were visualized on a UV transiluminator after ethidium bromide staining. .The concentration-mortality response of each viral population was estimated by probit analysis in our previous works ,34 Tabl.GV resistant colony was very low. The LC50 value of CpGV-M OBs in RGV larvae was 2.22 × 106 OBs/µL, , as observed previously.CpGV-M was highly pathogenic towards the susceptible insect colony, CpNPP , line 1, OBs/µL, , line 5.50 of CpGV-R5 alone ranged between 6.76 OBs/µL for the CpNPP colony .For CpNPP susceptible insects, the LCGV insect colony, the presence of CpGV-R5 genotypes, even at a low proportion (10%), results in a marked increase in pathogenicity compared to CpGV-M alone (measured both at the LC50 and LC90 levels) (For the R levels) , line 6.For the mixed virus population, 50% CpGV-M + 50% CpGV-R5, the Independent Joint Action hypothesis was rejected both on susceptible and resistant insects, , suggestThe suitability of the primers was tested both on the pure virus isolates CpGV-M and CpGV-R5 and on the laboratory mixed virus populations (P0). Single amplification fragments of the expected length were obtained for CpGV-M and CpGV-R5 pure isolates, confirming the specificity of the primers. For mixed P0 virus populations, both fragments were detected. a. FragmeGV hosts with CpGV-R5, no CpGV-M markers were detected in the progeny OBs  larvae that subsequently developed obvious signs of infection.OBs produced in both susceptible and resistant host colonies after infection with the experimental virus mixed populations were analyzed using this approach. Following infection of CpNPP and Rgeny OBs b. SimilaGV indicating that CpGV-M was able, in the presence of CpGV-R5, to infect resistant host cells and to replicate. , CpGV does not induce mortality in resistant RGV larvae. Although only RGV larvae showing clear symptoms of infection were used for PCR, the amplification obtained using high virus concentration could be due to the inoculum OB that contaminated the larvae. Even in these conditions, no CpGV-R5 specific band was observed, as it would be expected if a latent infection was ongoing.Using the Rf CpGV-M . Howevernjection . The difnjection  carried pe38 viral gene [p38 should not be able to productively infect a resistant insect. Given this scenario, we expected that no “M” type pe38 would be present in the progeny OBs that replicated in RGV insects. The presence of markers originating from both genotypes in the progeny of all mixed virus populations indicates that both viruses infected the larvae and replicated productively. The mechanism involved in the blocking of CpGV-M replication in resistant insects can be overcome, at least partially, by the presence of CpGV-R5.As CpGV-M does not induce mortality in resistant insects at the concentration of 800 OB/µL, using artificial mixed genotype preparations, we would expect a positive correlation between the relative proportion of CpGV-M in the inoculum and the dose required to kill larvae. This was clearly not the case. The presence of CpGV-M in the inoculum contributed to the overall pathogenicity of the mixed virus population. The markers we selected are linked to the ability to replicate in resistant hosts, recently demonstrated to reside in the ral gene . This metrans and those acting in cis. Tanada and coworkers [trans; that is, one particle (an OB of a granulovirus) could help another particle (a virion of a nucleopolyhedrovirus) to initiate the infection of insect midgut cells. This action is due to the presence of a perithrophic membrane (PM) degradating enzyme present in the granulovirus OBs [Per Os Infectivity proteins (PIFs) [cis. The helper protein needs to be in the same particle [Helper mechanisms can be classified in two main classes, those acting in oworkers  first deirus OBs . A diffes (PIFs) . For theparticle .trans-acting effect that acts both at the level of the midgut and other larval tissues in resistant insects.A detailed analysis of the blocking point for CpGV-M infection of the CpRR1 colony of resistant insects has been published . These aA major difference between multicapsid nucleopolyhedroviruses (MNPVs) and GVs should be considered. In MNPVs, OBs contain Multiple Occlusion Derived Virions (ODV), and each ODV contain a variable number of genomes. Defective virus genomes can be cooccluded in the same OB and probably co-enveloped in the same virion with replication-autonomous genomes, and can thus be transmitted together to a new host, the complete genome acting as a helper . In GVs,50 for CpGV-M in susceptible insects has been estimated at 1.2–5.0 OBs/larva [The LDBs/larva . In our 50 on susceptible insects) randomly selected from a mixed population containing 50% CpGV-R5 and 50% CpGV-M; the probability of “had eaten both CpGV-M and CpGV-R5” is 0.84. In a population containing 90% CpGV-M and 10% CpGV-R5, for the same doses, only 38% of the insects would have eaten both virus genotypes. This probability increases when the number of OBs ingested increases. In our conditions, only when challenging hosts with a virus population containing 50% CpGV-R5 and 50% CpGV-M, almost all larvae will have consumed both virus genotypes (apart from insects treated with the lowest virus concentrations).Accordingly, at low doses, most larvae that died from granulosis disease would have consumed only one or a few OBs. The highest probability for obtaining a mixed infection is when both viruses are present at the same frequency. For a larva eating 5 OBs (the upper estimation for LDIn addition, as in GVs, occlusion derived virions carry a single genome, and this helper effect for replication cannot occur unless a particular host cell is infected by at least two virions, one of which is a helper-genotype. Given these conditions, multiple infection of a midgut cell is probably a very rare event. Moreover, each infecting virus particle must be able to accomplish the whole cycle of infection, replication and cell-to-cell transmission within the infected insect. Accordingly, as the presence of CpGV-R5 in the host helps CpGV-M virions to infect, a possible explanation would involve a diffusible factor secreted by CpGV-R5-infected cells that renders non-infected cells permissive to CpGV-M infection. Alternatively, some cells could host a latent blocked infection of CpGV-M that becomes released if a CpGV-R5 superinfects.Surprisingly, both virus genotypes were detected after replication of all experimental virus populations on RGV larvae, raising questions concerning the possible interactions between the viruses when infecting the larvae and the individual cells that should be explored further.The use of mixed genotype virus isolates as the basis for biological insecticides allows efficient pest control while preserving virus diversity and probably reducing the impact of selective pressures on the pest population that favors resistance development. This would likely reduce the speed or the probability of generation of insect populations showing resistance to these virus isolates. Combining this approach with co-evolution of virus populations with the natural pest populations they are targeting will likely prevent development of new cases of resistance, making the biological control of this insect sustainable.


To the Editor: Vaccinia virus (VACV) causes exanthematous disease (bovine vaccinia) in Brazil. Outbreaks of this disease in humans have been reported since the late 1990s and have spread throughout Brazil . In September 2012, during a serologic survey in a rural area of Serro City , Minas Gerais, Brazil  Table 1.Bovine vaccinia lesions were observed on the hand of the father . Serum samples were used for detection of virus-neutralizing antibodies  and molecular diagnostic studies . All family members had positive results by molecular diagnostic test for rus gene  Table 1.ha gene from 3 virus-positive samples were sequenced in both directions in triplicate . Sequences were aligned by using ClustalW  and MEGA4.1 (http://www.megasoftware.net/) and showed 100% identity with each other  for VACV infection of humans should be considered. These alternative routes can include person-to-person or environmental transmission because the 2 daughters did not report any exposure activities related to milking or contact with cattle (In conclusion, person-to-person transmission of VACV in these cases might have been caused by direct contact between the father and family members, contact with virus in the home, or consumption of unpasteurized milk and cheese. Additional studies are necessary to elucidate the role of these transmission pathways in spread of VACV in Brazil.Technical Appendix. Additional information on alternative routes of zoonotic vaccinia virus transmission, Brazil.


Zika virus (ZIKV) is a globally emerging mosquito-transmitted flavivirus that can cause severe fetal abnormalities, including microcephaly. As such, highly sensitive, specific, and cost-effective diagnostic methods are urgently needed. Here, we report a novel electrogenerated chemiluminescence (ECL)-based immunoassay for ultrasensitive and specific detection of ZIKV in human biological fluids. We loaded polystyrene beads (PSB) with a large number of ECL labels and conjugated them with anti-ZIKV monoclonal antibodies to generate anti-ZIKV-PSBs. These anti-ZIKV-PSBs efficiently captured ZIKV in solution forming ZIKV-anti-ZIKV-PSB complexes, which were subjected to measurement of ECL intensity after further magnetic beads separation. Our results show that the anti-ZIKV-PSBs can capture as little as 1 PFU of ZIKV in 100 μl of saline, human plasma, or human urine. This platform has the potential for development as a cost-effective, rapid and ultrasensitive assay for the detection of ZIKV and possibly other viruses in clinical diagnosis, epidemiologic and vector surveillance, and laboratory research. Flavivirus genus of the Flaviviridae family, which includes other globally important pathogens, such as West Nile (WNV), dengue (DENV), and yellow fever viruses14567Aedes species of mosquitoes that transmit ZIKV circulate globally, there is a significant risk of ZIKV spread worldwide911Zika virus (ZIKV) is a member of the Similar to DENV, WNV and chikungunya virus (CHIKV), ZIKV infection in humans can result in a range of clinical signs and symptoms including fever, rash, joint pain, and conjunctivitis, which poses a challenge for differential diagnosis of these viral infections as they often are co-transmitted in the same areas1416171920222325273[B(C6F5)4]2  ECL labels. This step increased the ratio of ECL label to bioanalyte and amplified ECL signals by several order of magnitudes30Electrogenerated chemiluminescence (ECL) is a phenomenon of light emission by an excited state of species generated at electrodes that undergo high-energetic electron-transfer reactions293031333437394142In this paper, we report a highly sensitive and specific ECL-based immunoassay for detection and quantification of ZIKV in biological fluids with a limit of detection of 1 plaque-forming unit (PFU) in 100 μl of samples. This study provides an essential proof-of-concept that ECL-based immunoassays can be developed for the detection and quantification of viruses.3[B(C6F5)4]2, 9,10-diphenyl anthracene (DPA), and 5,6,11,12 tetraphenyl trtracene  were selected for initial testing. Because experimental conditions for ion annihilation ECL generation are strict , and detection of viruses generally involve aqueous solutions, coreactant ECL systemsn-propylamine (TPrA) for anodic ECL, and benzyl peroxide (BPO) for cathodic ECL, were coupled with the three ECL emitters to investigate their ECL responses. As shown in −•, 3R* (R = Ru(bpy)32+, DPA, or RUB), and R+•, which is favorable to ECL generation, BPO can react with the electrogenerated R−•, which reduces the chance of the ion annihilation reaction of R+• + R−• → R*, and diminishes ECL generation. Smaller ECL intensities were observed across the entire concentration range of BPO when DBAE and TPrA were used as coreactants. This could be ascribed to the relative instability of R+• cation radicals in the reaction media as well as the insufficient reducing capacity of TPrA• and DBAE• free radicals. Although the RUB/BPO and the Ru(bpy)32+/BPO systems show similar ECL intensities, we chose the RUB/BPO system for virus detection, because RUB is insoluble in water and commercially available, whereas the synthesis of Ru(bpy)3[B(C6F5)4]2 was nontrivial and expensiveAlthough a variety of ECL emitters are available, only those with extremely low water solubility and high ECL efficiencies are candidates when PSBs are used as carriers of ECL emitters. This is because leached ECL emitters from PSBs during a series of chemical and biochemical interactions or conjugations as well as washing processes could contaminate the reaction system, resulting in an undesirable background. ECL emitters with a constant and maximum loading capacity in PSBs will ensure that, in subsequent processes, strong ECL signals are produced reproducibly. On the basis of the above criteria, Ru(bpy)9 RUB molecules per bead, which was estimated on the basis of the ECL data obtained from the PSB(RUB) dissolved in MeCN and a set of standard RUB solutions using BPO as a coreactant. ECL label loading of PSBs with RUB was confirmed with a fluorescence microscope, which shows that PSB(RUB) beads glow as intense yellow color when exposed to UV light  in 0.1 M PBS  containing 2% BSA were incubated with anti-ZIKV-PSB (40 μl) for 45 min in a Dynal mixer to form ZIKV-anti-ZIKV-PSB complexes. These complexes were washed twice  with PBS, re-suspended in 100 μl PBS containing 2% BSA, then reacted with 10 μl of capture element (anti-ZIKV-MBs) for 45 min in a Dynal mixer form PSB < ZIKV > MB aggregates, which were separated by using Dynal magnetic separator. After centrifugation, some of the PSBs in the control samples (without virus) attach on the wall of the microfuge tube. In comparison, after addition of viruses followed by centrifugation, the virus-PSB complexes settled down at the bottom of microfuge tube (4 PFU) to form aggregates, which were separated magnetically and subjected to extraction and RT-qPCR analysis of ZIKV RNA. The PCR results confirmed that ZIKV bound to PSB < ZIKV > MB aggregates validating the efficiency of ECL-based assay to capture ZIKV  diluted in PBS with 2% BSA. The resulting virus-PSB conjugates were washed to remove unbound viruses and re-suspended. Virus-PSB conjugates then were allowed to react with the capture element  to form sandwich-type PSB < ZIKV > MB aggregates, which were magnetically separated from the reaction medium. PSBs that did not have bound viruses do not form aggregates and remain in solution during magnetic separation. Thus the ECL-intensity obtained from the PSB < ZIKV > MB aggregate is proportional to the concentration of virus in a sample. To detect ZIKV, anti-ZIKV-PSBs were mixed with different amounts of ZIKV ) or related (West Nile virus (WNV) and dengue ) mosquito-transmitted viruses with our ECL-based immunoassay. As shown in ZIKV is present in human blood, urine and other body fluids1617in utero521920Mosquito transmitted viruses, such as DENV, Japanese encephalitis virus, and WNV are worldwide public health challenges5032+, and DPA) that have easily distinguishable excitation and emission propertiesAn ECL-based detection assay may be ideal for virus detection as it offers several advantages including ultrasensitivity, reproducibility, cost-effectiveness, and rapidness over conventional immunoassays and PCR-based assay. For instances, compared with fluorescence-based methods, ECL assay does not need a light source, which effectively frees the ECL from scattered-light and luminescent impurity interferences. Moreover, the specificity of the ECL reaction associated with an ECL label and a coreactant decreases problems with side reactions . This is supported by many recent studies5455In summary, we developed an ultrasensitive, simple-to-use ECL-based immunoassay for specific detection of ZIKV in human body fluids. This proof-of-concept study can be extended to the detection of other viruses or pathogens.All experiments were performed in accordance with guidelines reviewed and approved by the University of Southern Mississippi (USM) Institutional Review Board. Written informed consent was obtained from all human volunteers. All the experiments involving live viruses were performed by the certified personnel in the biosafety level 3 (BSL3) laboratories following the standard biosafety protocols approved by USM Institutional Biosafety Committee.4 beads μl−1) and streptavidin-coated super-paramagnetic polystyrene beads  were purchased from PolySciences Inc. and Dynal Biotech Inc., respectively. Other reagents were purchased from either Sigma or other commercial vendors.Monoclonal antibody (mAb) against ZIKV  specifically recognizes the E protein on ZIKV and was generated after immunization of mice with infectious ZIKVAedes albopictus cells. All virus stocks were titered in Vero cells (ATCC CCL-81) by plaque assay as previously describedA recent strain of ZIKV (PRVABC59) was obtained from B. Johnson . CHIKV (LR-OPY1-2006 strain) was provided by R. Tesh . WNV isolate (CT2741) and the four serotypes of dengue viruses  were provided by J. Anderson . WNV, ZIKV, and CHIKV were propagated in African green monkey kidney cells  and DENV  were propagated in C6/36 4 μl−1 of avidin conjugated PSB(RUB) beads was stored at 4 °C until use.Loading of ECL-labels to PSBs was performed as previously described29Biotinylation of antibody was performed as described4 to 100) were mixed with 40 μL of mAb-PSB(RUB) conjugated with virus-specific mAbs and allowed to form conjugates for 45 min at room temperature with gentle shaking in a Dynal mixer at 20 rpm. After washing one time with PBS, 10 μl of mAb-MB solution  was added and samples were incubated for 45 min at room temperature with gentle shaking in a Dynal sample mixer at 20 rpm to produce the sandwich-type PSB(RUB)- < Virus > MB aggregates (designated as PSB < ZIKV > MB). The PSB < ZIKV > MB aggregates were fixed in 4% paraformaldehyde (to inactivate virus) and resuspended in 500 μL of PBS, and placed in a Dynal magnetic separator for 5 min. With the tube on the Dynal magnet, the solution was carefully aspirated to remove the unbound fraction. The tube was removed from the magnet and aggregates were resuspended in 500 μl of PBS and again placed in a magnetic separator. This process of magnetic separation and washing was repeated for three times to minimize the possible adsorption of free (virus unbound) PSB(RUB) beads on the wall of the tube, which could produce significant non-specific background signal during ECL measurement.Viruses were diluted in PBS containing 2% BSA or biological fluids, such as human plasma and urine that were collected form healthy volunteers. Inclusion of 2% BSA minimized the non-specific binding of virus to the surface of beads. Diluted virus solutions (100 μl) containing different PFU of viruses (10+(10 mM AgNO3 and 0.10 M TBAP in MeCN) as the reference electrode were used. All three electrodes were incorporated in a 5 ml disposable glass vial cell containing assay samples. Before each measurement, the Pt electrode was polished with a 0.3 μm alumina slurry, and all electrodes were cleaned carefully by washing with excess water followed by rinsing with MeCN. The ECL intensities along with the cyclic voltammetry signals (CV) were recorded simultaneously in a home-built ECL instrument as described previouslyTo measure ECL-intensity, aggregates were dissolved in a 2.0 mL solution of 10 mM BPO-0.10 M TBAP in MeCN for optimized ECL measurements (see results for details). A three-electrode system with a 2.0-mm diameter Pt disk as the working electrode, a Pt wire as the counter electrode, and an Ag/AgECL label loaded PSBs and PSB-virus-MB aggregates were imaged in a Zeiss LSM510 META confocal microscope .ZIKV particles attached on the surface of beads  were quantified by RT-qPCR, as described previouslyAntibody conjugation to beads was analyzed by immunoblotting as previously describedHow to cite this article: Acharya, D. et al. An ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of Zika virus. Sci. Rep.6, 32227; doi: 10.1038/srep32227 (2016).