Verapamil hydrochloride (VP), a highly hydrophilic drug with a short biological half-life, was incorporated into a series of Ch-based and/or Ch/Kc-PEC-based beads to control its release profile in vivo. The formulation of VP-loaded beads was optimized using stepwise statistical designs based on a prespecified criterion. Several characteristics of the prepared beads, such as entrapment efficiency (EE%), in vitro drug release, swelling ratio, size and surface microstructure as well as molecular interactions between the drug and formulation ingredients, were investigated. In vivo pharmacokinetic (PK) studies were carried out using the rabbit model to study the ability of the optimized VP-loaded beads to control the absorption rate of VP. Results revealed that the prepared superhydrophobic substrates were able to fabricate VP-loaded beads with extremely high EE exceeding 90% w/w compared to only 27.80% when using conventional ionotropic gelation technique. PK results showed that the rate of VP absorption was well controlled following oral administration of the optimized beads to six rabbits compared to a marketed VP immediate release (IR) tablet, as evidenced by a 2.2-fold increase in mean residence time (MRT) and 5.24-fold extension in half value duration (HVD) over the marketed product without any observed reduction in the relative oral bioavailability.In this work, ultrahigh drug-loaded chitosan (Ch)/K-carrageenan (Kc) polyelectrolyte complex (PEC) beads were formed in situ by cross-linking in a glutaraldehyde-saturated atmosphere and were prepared on superhydrophobic substrates fabricated by spraying glass surfaces with ready-made spray for domestic use (NeverWet Controlled-release multiparticulate systems designed to deliver highly hydrophilic drugs with relatively short biological half-lives offer many therapeutic and clinical benefits due to reduction in dosing frequency. In addition, they are well known to be uniformly distributed in the gastrointestinal tract (GIT), resulting in more uniform absorption with no risk of dose dumping compared to single unit systems ,2.Hydrophilic drugs are generally difficult to encapsulate using conventional encapsulation techniques due to their rapid diffusion from the prepared systems to the external aqueous phase, which makes their formulation more challenging ,4. AttemMany of the encapsulation techniques proposed to produce polymeric particulate systems have limited applications due to the mandatory use of organic/toxic solvents or high temperature, difficulty in complete solvent removal in addition to low drug EE .Inspired by the behavior of water droplets on the lotus leaf, many scientists have tried to mimic its ultra-hydrophobic surface. The combination of its hierarchical roughness with a low surface free energy is believed to be the main cause of the water repellency of this surface and its self-cleaning effect. Thus, scientists were able to fabricate superhydrophobic surfaces with high water contact angle (>150°) and low sliding angle (<10°) to be used in many life applications . AlthougN-isopropylacrylamide) beads for controlled release of encapsulated bioactive substances. Puga et al. [Several studies reported the use of superhydrophobic surfaces to formulate beads from hydrophilic polymers with high EE. Upon application of the aqueous polymer solution to the superhydrophobic surface, it forms a spherical shape, which can be easily hardened under mild conditions without any need for an external liquid phase ,9,10. Soa et al.  achievedHydrogels of natural polymers, such as chitosan (Ch), have been widely used in the preparation of controlled-release systems due to their biodegradability and biocompatibility . Ch espeVerapamil hydrochloride VP, A, the moIn an attempt to alter VP pharmacokinetic profile in vivo, superhydrophobic substrates were fabricated in this study and were utilized to prepare controlled-release ultrahigh VP-loaded Ch-beads and Ch/Kc-PEC-based beads. The formulated beads were prepared and hardened in a glutaraldehyde-saturated atmosphere to exclude the use of an external liquid phase in order to achieve maximum EE after tracking any drug diffusion to the superhydrophobic substrate. Ch B was com® spray was purchased from Rust-Oleum . Chitosan low molecular weight (MW) (50–190 kDa), medium MW (190–310 kDa) and high MW (310–375 kDa) were purchased from Sigma-Aldrich Inc. Al. . Kappa-Carrageenan was purchased from Acros Organics . Verapamil HCl was supplied as a gift from El-Nasr Chemical Co. . Glutaraldehyde solution (50% v/v) and glacial acetic acid were purchased from El Nasr pharmaceutical chemicals company .Glass slides (50 mm × 50 mm) were used in this study. NeverWet®” spray on clean glass slides. Briefly, glass slides were thoroughly cleaned and dried, and then three thin layers of “base coat” were applied with 30 min intervals between each application. After complete drying of the base coat, the “top coat” was sprayed as another three layers with 5 min intervals, and then the glass slides were left to dry in the air before use. The roughness of the surface was observed by scanning electron microscopy. The static contact angles (CA) of water, olive oil, VP-Ch and VP-Ch/Kc dispersions were measured by dropping a 5 µL droplet of each of the above-mentioned liquids on the treated glass surface using contact angle meter DM-701 interfaced with KYOWA analysis system FAMAS software version 3.4  The same measurements were repeated on untreated glass surfaces to compare results. Sliding angles (SA) were measured for each of the above-mentioned liquids by slowly tilting the treated glass slide until the examined liquid droplet started to move. Results were recorded as the mean values of three sample measurements [* and θ are the CA of the liquid droplet on the treated glass and untreated glass, respectively; f is the fraction of solid treated surface in contact with the liquid droplet and accordingly, (1-f) is the fraction of the trapped air beneath the liquid droplet.Superhydrophobic substrates were prepared by applying “NeverWeturements . The fraurements  as follo2 full factorial design was applied to evaluate the influence of the used Ch on the characteristics of the prepared beads. In this design, Ch molecular weight (MW) and concentration, each at three levels, were selected as the independent variables, whereas EE% and the rate of in vitro drug release from the prepared beads expressed as t90% were evaluated as the dependent variables. Dispersions of different grades of Ch in 1% acetic acid solution were prepared at various concentrations according to the design shown in v/v in water) at its bottom as cross-linking agent without any contact with the glass slides. Thus, hardening of Ch beads was performed in a glutaraldehyde-saturated atmosphere to exclude the use of an external liquid phase and reduce drug leakage. Traces of un-reacted glutaraldehyde were removed under vacuum [90% to yield the system with the highest desirability factor.A 3r vacuum . All ninw/v) was dispersed in 1% v/v acetic acid solution containing 80 mg of VP (w/v and high MW respectively). The resultant dispersions were stirred thoroughly until homogenous dispersions were obtained [90% of VP using Design-Expert® software, where the most desirable system was selected, as previously discussed, for further investigation.Polyelectrolyte complex (PEC) beads were prepared by complexing Ch with Kc to further prolong VP release from the beads . Simply mg of VP . The disobtained ,14. For max 278 nm [max. The EE% was calculated from the following equation,e is the amount of VP encapsulated within the beads and Df is the amount of free un-encapsulated VP.After complete solidification of the formulated beads, the superhydrophobic layer was peeled from the glass surface, immersed into 100 mL of water and stirred for 24 h to extract any residual amount of free un-encapsulated VP from the surface. On the other hand, the amount of the encapsulated drug was extracted with water for 24 h after complete crushing of the solidified beads. Extracted drug in water was measured using a UV Spectrophotometer  at λ®  bisects tablet was used as a reference tablet for in vitro release studies. VP dissolution from Isoptin® was evaluated from the tablet as a whole (80 mg) and upon dividing it into four quarters to assure the similarity in the dissolution behavior when the tablet is divided and suspended before oral administration to rabbits, as described under in vivo studies. Results showed that the dissolution profiles of Isoptin whole tablet compared to the divided tablet were highly similar, and the calculated similarity factor (F2) was found to be 69.58. All in vitro drug release studies were performed in duplicate.The release of VP from the beads was determined in a dissolution tester  following the USP paddle method. All studies were done in 300 mL 0.1 N HCl (pH 1.2) for the first 2 h to simulate gastric conditions, then 100 mL of 0.2 M tribasic sodium phosphate was added to raise the pH to 6.8 to simulate intestinal conditions for the rest of the release period while maintaining sink conditions . A weigh90%) was calculated and statistically analyzed.The release kinetics were studied by fitting the release profile to zero-order, first-order and Higuchi diffusion model . The tim0 and wt, represent the initial weight of the beads and the weight of the swollen beads at each time point, respectively. All experiments were performed in duplicate [The swelling behavior of two optimized systems (F8 and F8/3.58) obtained from both statistical designs were examined in three different dissolution media: 0.1 N HCl (pH 1.2), phosphate buffer (pH 6.8) and pH-change medium ,14. For uplicate .The size, surface structure and topography of optimized F8 and F8/3.58 beads were observed using SEM . The beads were mounted on metal grids and sprayed with gold , and photomicrographs were taken.−1.FTIR scanning was performed for VP, Ch, Kc, drug free-Ch beads, drug-free Ch/Kc-PEC beads in addition to F8 and F8/3.58 beads. 2–3 mg of each sample was ground then mixed with 100 mg of potassium bromide, compressed into thin discs using a hydrostatic press and finally scanned with FTIR spectrophotometer  over wavelength range 4000–400 cm® tablets following oral administration of single doses equivalent to 10 mg/kg VP each [In vivo absorption studies were carried out in rabbits to compare the PK profiles of VP from F8/3.58 beads and Isoptin VP each . Male alF8/3.58 beads equivalent to the administered doses were filled into size “2” hard gelatin capsules, which were administered to the rabbits orally followed by water to ensure swallowing. The capsule acted only as a reservoir to contain the formulated beads. The capsule shell readily dissolved in dissolution media within a few minutes as observed in in vitro release studies. Similarly, Isoptin bisects tablets were first divided, and then an amount equivalent to 10 mg/kg was dispersed in water by sonication immediately before administration. A volume of the suspensions equivalent to the calculated dose was given to the animals through the oral route.Blood samples (3 mL) were withdrawn from the ear vein of each rabbit at predetermined time intervals: 0 (pre-dose), 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12 and 24 h post-administration of a treatment into heparinized glass tubes. The plasma was immediately separated from the blood cells by centrifugation at 4000 rpm for 15 min and stored frozen until analysis by liquid chromatography-mass spectrometry (LC-MS/MS).v/v)) and finally transferred to the auto-sampler vials, where 10 µL was injected into the LC-MS/MS.Plasma concentrations of VP were determined using a selective, sensitive and accurate LC-MS/MS method that was developed and validated before use . Briefly® tablets were estimated. The observed maximum plasma concentration (maxC) and time to reach maxC (maxT) were estimated directly from the plasma concentration-time profile. Elimination rate constant (k) was estimated from the terminal elimination line using the log-linear regression analysis, and the half-life (t1/2) was calculated as t1/2 = 0.693/k. The area under the curve (AUCt0-) was calculated using the trapezoidal rule from zero time to the last time of blood sample and the area under the curve from zero to infinity (AUC0-∞) was calculated as (AUC0-∞) = (AUCt0-) + Ct/k, where tC is the last measured concentration at time t. The mean residence time (MRT) was calculated from AUMC0-∞/AUC0-∞, where AUMC0-∞ is the area under the first moment curve. In addition, half value duration (HVD) was calculated and used to measure retard quotient (RD). RD is a parameter that gives the factor by which the half value duration (HVD) is extended in the sustained release (SR) formulation compared to the immediate release (IR) one, where it is calculated as the ratio between the HVD of SR system relative to IR system. A RD value of 1, 1.5, 2 and 3 indicates no, low, medium and strong retardation, respectively, of the SR system in vivo [The plasma concentration-time data were analyzed via non-compartmental PK model using Kinetica 2000 software  and PK parameters of VP post-administration of either F8/3.58 beads or Isoptin in vivo .t-test . For in vivo studies, statistical inferences were based on untransformed values for maxC and AUC variables and observed values for t1/2. The nonparametric Signed Rank Test (Mann–Whitney’s test) was used to compare maxT between the two treatment groups. The one-way analysis of variance (ANOVA) F-test was used for testing the equality of several means. For multiple comparison, the procedure used was the least significant difference (LSD). Statistical analyses were carried out using SPSS  or Design-Expert® software .All in vitro data points are the average of at least two independent experiments performed, and the values are expressed as means ± SD. Statistical significance of the experiments’ results was assessed by Student’s ®” spray on the glass slides, a rough structure was created on the surface, as shown in SEM images of Following application of “NeverWetWhen a water droplet was dropped on the surface of the treated glass, it showed a significantly higher CA (>150°) with lower SA (<10°) compared to untreated smooth glass surface A. The suThe results presented in 90% were statistically analyzed in a 32 full factorial design. Afterwards, the selected system from the first design was complexed with Kc to formulate PEC beads, which were then cross-linked with glutaraldehyde, as described before, to further control the release of VP from the formed beads. The effect of Kc concentration was studied through a response surface design to further select the optimized system.Ch is a cationic polysaccharide, which is soluble in acidic solution. It tends to harden into hydrogel upon covalent cross-linking with glutaraldehyde, glycol or glyoxal solution ,12. In aw/w (p < 0.05) the encapsulation of the drug into the beads, whereas Ch concentration did not show any significant effect (p > 0.05). High MW Ch showed significantly higher (p < 0.05) EE% than lower MW Ch (w/w) [In general, encapsulation of hydrophilic drugs into spherical systems is challenging and problematic due to the loss of drug to the external phase ,5,31. A w/w . Upon ster MW Ch . This miCh (w/w) .Evaluation of the in vitro rate at which the drug is released from a delivery system is important to determine the release mechanism as well as to predict in vivo release upon oral administration . In genew/w followed by a slower rate of release. This burst effect is common with hydrophilic drugs, where a rapid initial release of the drug for a short time occurs upon contact with the dissolution medium, followed by the desired release rate [In order to evaluate VP release from the prepared Ch systems, the beads were suspended in 0.1 N HCl followed by phosphate buffer, as previously described. All the prepared systems showed a high initial burst release ranging from 35% to 55% ase rate . Hydrophase rate . In addiase rate ,39. UnenKinetic analysis of the release data showed that all formulated systems followed Higuchi diffusion model , where p90% was calculated from the best-fitted equation (90% was significantly (p < 0.05) affected by Ch concentration. On the other hand, changing Ch MW did not have a significant (p > 0.05) effect on the release rate. Similar findings were observed by Yassin et al. [w/v resulted in a slower drug release with higher t90% value . According to this theory, increasing polymer concentration above a certain percolation threshold will no longer slow down the drug release because the gel layer formed above this concentration is coherent and homogenous, thus controlling the hydration of the system [However, further increase of Ch concentration from 1.5% to 2.0% e system . Similare system .90% with desirability factor of 0.703. F8 showed 98.54% EE (w/w) and t90% of 4.04 h. The optimized system (F8), which is composed of 1.5% w/v high MW Ch, was integrated into another design to form PEC with Kc to modulate the sustainability of VP release.Optimization results revealed that system “F8” is the optimized system when the optimization conditions were set to the highest EE% and longest tw/w of VP. Upon statistical analysis of the data, Kc concentration did not show any significant (p > 0.05) effect on the encapsulation of VP into PEC beads  longer t90% reaching 5.32 ± 0.65 h compared to 2.87 ± 0.24 h upon using 1.5% w/v , whereas Kc is a linear polysaccharide with one sulfate group (−OSO3−) per each disaccharide moiety. Thus, each monosaccharide of Ch (C6H11O4N with monomer wt. 161g/monosaccharide) will react with a disaccharide moiety of Kc (C12H18012S with dimer wt. 386g/disaccharide) to produce the PEC. By calculation, it was found that 1.5 g of Ch will complex with almost 3.59 g of Kc. Therefore, at Kc concentration higher than 3.59% w/v, there is no more complex formation and subsequently, no further retardation in drug release.Aiming to further control VP release from the formulated beads, Kc was added to Ch to produce VP-loaded Ch/Kc-PEC-based beads. Upon kinetic analysis of the release data, all the formulated beads still followed Higuchi diffusion model. The t1.5% w/v B. This rw/v of Kc when combined with system F8 previously selected from the first statistical design. This choice confirms the above suggestion that one monosaccharide of Ch interacts with a disaccharide of Kc, as previously discussed. The desirability factor of the suggested optimized system (F8/3.58) was 0.892 after setting the optimization conditions at the highest EE% and longest t90%. F8/3.58 beads showed an EE% of 91.99 ± 0.24% w/w and Higuchi diffusion model of release with t90% of 5.36 ± 0.40 h  are graphically illustrated in w/w) compared to F8 beads.The in vitro release profiles of the two optimized systems from both designs (F8 and F8/3.58) and the marketed VP tablets  stretching vibrations of Kc [3−) vibrations at 1261 cm−1 of Kc, which could be considered as evidence to the inclusion of Kc in PEC formation [−1 was slightly shifted, indicating that the amine group was protonated to a NH3+ group in the complex [3−) group of Kc and the NH3+ group of Ch.FTIR spectroscopy was employed for the detection of any chemical interaction among the formulation components and VP in the prepared beads by pointing out the characteristic molecular groups . Figure −1 ,47. The ns of Kc . Plain Cormation . In addi complex . Therefo−1), a sharp characteristic peak of C≡N at 2237 cm−1, C–H stretching of the benzene ring at 1593, 1516 and 1473 cm−1 and a strong C–O stretching vibrations of the aromatic ethers at 1261 cm−1 [VP spectrum e showed 261 cm−1 ,49.® tablets by monitoring VP plasma levels for 24 h post-oral administration to rabbits in a crossover design. The rabbit model was reported to be more suitable than dogs, rats and mice to explain VP clinical PK and drug interactions, which take place in rabbits in patterns very similar to those found in humans [The in vivo behavior of capsules filled with F8/3.58 beads were assessed and compared to Isoptinn humans .p < 0.05) higher plasma concentration up to 6 h post-administration, followed by fast elimination and rapid decline in drug concentration in the subsequent time intervals. On the other hand, F8/3.58 beads exhibited a slowly declining curve that maintained VP plasma concentration for a prolonged time.Plasma concentration-time curves of both F8/3.58 beads and Isoptin tablets are shown in n = 6) following the two treatments are summarized in maxT, k, t1/2, AUC0-t and AUC0-∞ estimates from both treatments were not statistically significantly different (p > 0.05). The insignificant difference between the values of both AUC0-t and AUC0-∞ is an indication of the absence of any decline in VP bioavailability from the developed sustained release formulation, although suffering from an extensive first-pass effect [maxC estimate observed from the marketed product (41.78 ± 21.49 ng/mL) compared to the mean estimate from F8/3.58 beads (10.41 ± 6.97 ng/mL) was expected due to the rapid and immediate release of VP from Isoptin, thus achieving a high plasma concentration. p < 0.05) MRT obtained from the F8/3.58 beads indicates a longer residence of the drug molecules in the body, confirming that the formulated beads offered a sustained release behavior in vivo, as previously observed in vitro. This result was further confirmed by the significantly higher HVD values calculated (p < 0.05) from SR F8/3.58 beads compared to IR Isoptin. The calculated RD ratio showed 5.24 times extension in the HVD, suggesting a strong retardation (RD > 3) of the formulated SR beads in vivo [The mean PK parameters (s effect . The sig in vivo . It was maxT estimates obtained from both treatments could be due to the initial burst release of VP from the beads, as previously described under In Vitro Release studies. Similar results were reported by other researchers during preparation of controlled-release systems where lopinavir-loaded nanoparticles showed 1.3-fold increase in MRT compared to free lopinavir, although exhibiting the same maxT [The similar ame Tmax . Similarame Tmax .It was also noted that the variability in plasma concentrations in the six rabbits following administration of F8/3.58 beads was less compared to Isoptin tablets during the absorption phase, which further confirms that controlled-release multiparticulate systems result in reduced inter- and intra-individual variation. Thus, VP-loaded Ch/Kc-PEC-based beads could be a promising cost-effective, scalable SR multiparticulate formulation that can be added to the industrial arsenal to alter the PK profile of VP and similar drugs to ensure more effective therapeutic and clinical applications.In this study, we demonstrated that ultrahigh VP-loaded Ch/Kc-PEC-based beads prepared on superhydrophobic substrates are promising formulations that could control the release of VP and extend its duration of action in vivo. The results obtained in this study suggest that the developed multiparticulate F8/3.58 system may be an alternative to conventional immediate-release VP tablets, which necessitate frequent administration. The proposed formulation is an easily modifiable delivery platform for ultrahigh encapsulation of water-soluble therapeutic molecules, the release profiles of which are usually difficult to control via conventional encapsulation techniques.


We assessed Zika virus seroprevalence among healthy 1–4-year-old children using a serum sample collection assembled in 2014 representing 30 urban sites across Indonesia. Of 662 samples, 9.1% were Zika virus seropositive, suggesting widespread recent Zika virus transmission and immunity. Larger studies are needed to better determine endemicity in Indonesia. Aedes mosquitoes. Because of the epidemic that swept through the Americas in 2016, Zika virus infection is known to cause microcephaly, as well as other congenital defects and Guillain-Barré syndrome . In the first tier, we tested serum samples diluted 1:10. Samples that suppressed >90% of Zika virus PFUs were considered potentially positive for Zika virus antibodies because DENV-specific antibodies, if present, could have cross-reacted and neutralized Zika virus. We then subjected the specimens considered potentially positive to a second PRNT90, in which we tested serum samples against Zika virus and all 4 DENV serotypes  that could differentiate Zika virus neutralizing antibodies from those produced in response to DENV infection were adapted from protocols developed by the CDC . The chaerotypes . Specimedy titer  Table. W90 screening, we detected possible Zika virus antibody in 73 (11.0%) of the 662 serum samples  is the international standard. In just 2% (12/662) of specimens, we could not determine whether the antibodies were Zika virus or DENV specific. When using the more conservative criterion of only classifying a sample as positive for Zika virus antibodies if no DENV-specific neutralizing antibodies are detected, the number of Zika virus antibody–positive samples decreases by only 6, leaving 54 samples still classified as Zika virus seropositive. Further evidence for the validity of the PRNT90 was that DENV neutralizing antibody–positive samples were negative for the presence of Zika virus neutralizing antibodies across a range of titers .Our assessment, involving use of the PRNT90 with samples from the remaining cohort (the 5–18-year-olds), but we expect higher percentages of nonspecific flavivirus seropositivity in the samples from this older age group.Although our data provide some evidence regarding geographic distribution, no information is presented regarding a specific threshold titer associated with clinical illness or a correlation between geography and titer. Further studies involving larger sample sets would be necessary to address these topics. The single age group, relatively small number of specimens, and limited number of sites, particularly from eastern parts of the country, do not give a comprehensive picture of endemicity throughout Indonesia. The small numbers of specimens available from most localities did not enable accurate estimation of the proportional differences between localities. We could perform PRNTMuch has been published on epidemic Zika virus, but little is known about the effect of Zika virus in endemic areas. Determining the prevalence of Zika virus in Indonesia can provide clues to its potential long-term public health significance in endemic settings. Mild or asymptomatic infection is common, and confusion with dengue during diagnosis probably accounts for how long Zika virus was unrecognized in Indonesia and other areas of Southeast Asia. Besides the need to better evaluate Zika virus incidence and distribution, a high priority for future investigations will be determining the extent of Zika virus–related birth defects. If, like other flaviviruses, a primary Zika virus infection results in lifelong immunity, infections during childhood could reduce a person’s risk for infection later in life and thus the incidence of Zika virus–related birth defects. This knowledge provides clues for understanding future patterns of Zika virus transmission in the Americas.Description of methods and plaque reduction neutralization test 90% titers for Zika virus and dengue viruses 1−4 of samples from 1−4-year-old children (n = 72), Indonesia, October–November, 2014.


Erinaceus roumanicus). The complete genome of the virus, called H14-hedgehog/2015/HUN (GenBank accession number MN044446), is 4,118 nucleotides in length with a readthrough stop codon of type/group 1 in ORF1 and lacks a poly(A) tract at the 3′ end. The predicted ORF1-RT (RdRp) and the capsid proteins had low (31-33%) amino acid sequence identity to unclassified tombus-/noda-like viruses (Hubei tombus-like virus 12 and Beihai noda-like virus 10), respectively, discovered recently in invertebrate animals. An in vivo experimental plant inoculation study showed that an in vitro-transcribed H14-hedgehog/2015/HUN viral RNA did not replicate in Nicotiana benthamiana, Chenopodium quinoa, or Chenopodium murale, the most susceptible hosts for plant-origin tombusviruses.Tombusviruses are generally considered plant viruses. A novel tombus-/carmotetravirus-like RNA virus was identified in a faecal sample and blood and muscle tissues from a wild northern white-breasted hedgehog ( Recently, the discovery of novel viruses has been dramatically enhanced by the use of culture-free viral metagenomics and next-generation sequencing techniques . HoweverTombusviridae) are positive-sense, single-stranded RNA viruses  that are currently thought to be plant viruses with relatively narrow host range . ORF2  had 33% aa sequence identity  to the capsid protein of the unclassified Beihai noda-like virus 10 (KX883117) from tiger crab [A specimen pool containing three faecal samples from northern white-breasted hedgehogs was subjected to viral metagenomics analysis. After Tombusviridae and Providence virus (family Carmotetraviridae)  sequence of ORF1-RT showed the distant phylogenetic position of H14-hedgehog/2015/HUN from the representative members of the family Based on these results we compared the complete genome sequence of H14-hedgehog/2015/HUN with the viral metagenomic reads (N = 79) matching Providence virus and found that they were completely identical to the corresponding canonical domain IV of the RdRp region of H14-hedgehog/2015/HUN  of the cloned H14-hedgehog/2015/HUN was used to inoculate N. benthamiana plants. Inoculated leaves were tested for the presence of the virus at 1 and 6 hours post-inoculation (hpi) and 10 and 24 days post-inoculation (dpi) by Northern blot using the virus-specific radioactively labeled probe. The strongest signal was detected at 1 hpi, and its intensity decreased steadily, reaching zero at 24 dpi. Although a signal could be detected after 10 dpi, it disappeared later, showing that it was only the signal of the remaining RNA from the initial transcript rather than a replicated virus  of the potential plant origin of strain H14-hedgehog/2015/HUN, an rus Fig. A.Fig. 3 were used as a template for Using the SCR-F/R screening primer pairs, only the faecal specimen collected from animal H14 was RT-PCR positive for the study virus . Available tissue samples collected from hedgehogs H14 were also tested for H14-hedgehog/2015/HUN virus by RT-PCR using the SCR-F/R primers. Two (blood and muscle tissue) of the five tissue samples were RT-PCR positive, and both were confirmed to contain H14-hedgehog/2015/HUN virus sequences by direct nucleotide sequencing. Sequence comparisons showed 100% nucleotide sequence identity within the amplified 368-bp-long RdRp region between the H14 sequences originating from faeces, blood and muscle.in vivo by experimental inoculation of plants with viral RNA.A large number and genetically diverse viruses were detected by viral metagenomics and next-generation sequencing methods from faecal samples from a wild northern white-breasted hedgehog. Collectively, these viruses have a wide known  host range, including bacterial, plant, fungal, insect and vertebrate hosts. In addition, numerous genetically diverse tombus- and tombus-like viruses were also identified in these faecal samples. After the characterization of a novel dicipivirus from these specimens , this stTombusviridae. On the other hand, the genome organization of H14-hedgehog/2015/HUN has features similar to those of Providence virus in the family Carmotetraviridae [H14-hedgehog/2015/HUN shows high genetic divergence in ORF-RT relative to its closest known relative in the family aviridae . Based oaviridae , the ORFaviridae . Whetherin vivo experimental plant RNA inoculation. We could not demonstrate replication of H14-hedgehog/2015/HUN virus in N. benthamiana, C. quinoa, or C. murale, the most susceptible plant systems for tombusvirus replication [Initially, because of the potential dietary origin of this genome in hedgehog faeces, we looked for a potential plant host for H14-hedgehog/2015/HUN. To investigate the replication potential of H14-hedgehog/2015/HUN in plants, we used lication . We therlication , 8.In summary, we have described a novel RNA virus from faecal, blood and muscle samples of a vertebrate animal that initially exhibited a distant relationship to plant viruses and viruses detected in invertebrates. Based on the results of this study, plant tropism could not be detected for H14-hedgehog/2015/HUN. Further investigations are needed to determine whether this genome is of dietary origin and does not replicate in hedgehog cells or is capable of replication in either vertebrate or invertebrate  cells.


Chronic cough is a distressing symptom and a common reason for people to seek health care services. It is a symptom that can indicate underlying tuberculosis (TB) and/or chronic airways diseases (CAD) including asthma, chronic obstructive pulmonary disease (COPD) and bronchiectasis. In developing countries including Malawi, provision of diagnostic services and clinical management of CAD is rudimentary, so it is thought that patients make costly and unyielding repeated care-seeking visits. There is, however, a lack of information on cost of illness, both direct and indirect, to patients with chronic cough symptom. Such data are needed to inform policy-makers in making decisions on allocating resources for designing and developing the relevant health care services to address universal coverage programmes for CAD. This paper therefore explores health seeking costs associated with chronic cough and explores information on usage of the coping mechanisms which indicate financial hardship, such as borrowing and selling household assets.This economic study was nested within a community-based, population-proportional cross-sectional survey of 15,795 individuals aged 15 years and above, in Dowa and Ntchisi districts. The study sought to identify individuals with symptoms of chronic airways disease whose health records documented at least one of the following diagnoses within the previous year: TB, Asthma, COPD, Bronchitis and Lower Respiratory Tract Infection (LRTI). We interviewed these chronic coughers to collect information on socioeconomic and socio-demographic characteristics, health care utilization, and associated costs of care in 2015. We also collected information on how they funded their health seeking costs.We identified 608 chronic coughers who reported costs in relation to their latest confirmed diagnosis in their hand-held health record. The mean care-seeking cost per patient was US$ 3.9 (95% CI: 3.00–5.03); 2.3 times the average per capita expenditure on health of US$ 1.69. The largest costs were due to transport (US$ 1.4), followed by drugs (US$ 1.3). The costs of non-medical inputs (US$ 2.09) was considerable (52.3%). Nearly a quarter (24.4%) of all the patients reportedly borrowed or/and sold assets/property to finance their healthcare. CCs with COPD and LRTI had 85.6% and 62.0% lower chance of incurring any costs compared with the TB patients and any patients with comorbidity had 2.9 times higher chance to incur any costs than the patients with single disease. COPD, Bronchitis and LRTI patients had 123.9%, 211.4% and 87.9% lower costs than the patients with TB. The patients with comorbidity incurred 53.9% higher costs than those with single disease.The costs of healthcare per chronic cougher was mainly influenced by the transport and drugs costs. Types of diseases and comorbidity led to significantly different chances of incurring costs as well as difference in magnitude of costs. The costs appeared to be unaffordable for many patients. Tuberculosis and chronic airway diseases (CAD) including asthma, chronic obstructive pulmonary disease (COPD) and bronchiectasis represent a major disease burden to most developing countries, particularly in Sub-Saharan Africa –4. COPD It has been estimated that, asthma morbidity and mortality accounts for around 1% of all disability-adjusted life years (DALYs), equivalent to 16 million DALYs lost per year worldwide . A cruciFurthermore, COPD is characterized by airflow limitation that is not fully reversible but nonetheless treatable. The World Health Organization (WHO) estimates that 65 million people have moderate to severe COPD with 5% of all (3 million) global deaths being attributed to COPD . BronchiHealth seeking costs associated with chronic cough (CC) are estimated to rank as one of the highest among chronic diseases due to the significant health care utilisation associated with these conditions ,10. SeveNumerous studies have been published evaluating health seeking costs of CC in high income countries –16, 17 hThis is important given that in low income settings, relatively small costs on health can be disastrous and lead to catastrophic healthcare expenditure –12. ThisThe main objective of this paper is to explore health seeking costs incurred by chronic coughers starting from the onset of symptoms, to diagnosis and the management of the illness. This is the cost of the whole care-seeking process which includes pre-diagnostics and diagnostic costs, treatment costs, hospitalisation costs as well as guardians (accompanied persons) costs. To reach the main objective, we set out to estimate the direct costs  associated with chronic cough, estimate variations in these costs across socio-economic and socio-demographic groups and to identify coping mechanisms with detrimental effects on economic status of the chronic coughers.This study was reviewed and approved by The College of Medicine Research and Ethics Committee (COMREC), which is situated within The University of Malawi, College of Medicine. This is a local Institutional Review Board which approves research projects in Malawi. This was a trial with the following approval number: P.07/13/1424 . ParticiThis cross-sectional community-based survey was conducted in 2015 (January to June) in rural areas of two districts, namely, Dowa and Ntchisi, in the central region of Malawi. A total of 15,795 individuals were included in the survey where household was the unit of approach. Among other goals, this survey intended to identify individuals at age 15 years and above with evidence or symptoms of chronic cough/wheeze, and a diagnosis in their health passport. It was also intended to collect data on health seeking costs that chronic coughers incur before getting a diagnosis, after diagnosis, treatment, hospitalisation as well as guardian costs and how health seeking costs incurred by chronic coughers were financed. i.e. financial coping mechanisms.We administered a validated patient cost questionnaire recommended by the Stop TB Partnership  using prhttps://www.oanda.com/currency/converter/. We collected information on how much was spent for the preceding 12 months at the time of the survey for the utilized health services e.g., pre-diagnostics and diagnostic costs, treatment costs, hospitalisation costs as well as guardian costs. The specific items costed include administrative, laboratory, X-ray, drugs, travel, food as well as accommodation.Data analysis was done in STATA v13.1. Direct medical and non-medical costs associated with chronic cough were summarised using means accompanied by their 95% confidence intervals. An estimate of variations in costs based on socio-demographic and socio-economic status was done by looking at how much each category  of chronic coughers paid to access healthcare. For coping mechanisms, we calculated the absolute number of chronic coughers who sold assets and/or borrowed money to finance health seeking costs. Costs were reported in United States Dollar (US$) using April 2015 exchange rate (US$1 = MK450) from OANDA—Two-part regression analysis was applied to estimate the effect of individuals’ sociodemographic characteristics on the direct healthcare costs for seeking healthcare due to chronic cough. The direct costs was a limited dependent variable and was continuous over most of its distribution but had a mass of observations at zero values. The decision of spending for healthcare and its magnitude might not be statistically independent , 28 ApplThere were a total of 608 people, with confirmed diagnosis of CAD in their health card/passport who reported costs in relation to their health care seeking, with a total mean cost of US $3.9 as illustrated in The magnitude of costs was also dependent on the kind of diagnoses such that some diseases were costlier than others. For instance, Further analysis shows that there were also variations in costs incurred by chronic coughers with respect to symptoms such that those with breathlessness paid slightly higher costs than those with other symptoms as seen from It was also observed that there were variations in costs with respect to sociodemographic and socioeconomic conditions of the chronic coughers . The resMost chronic coughers who reported health seeking costs had primary level education and their cost was slightly higher than those without any formal education and those with secondary education and above . Most ofOur two-part multiple regression model found that the presence of diseases and comorbidity led to significantly higher chance of incurring any healthcare costs  than the farmers. No other socioeconomic or demographic factors showed any significant influence on costs for healthcare of the CCs.In addition to income and savings, two major coping mechanisms for health care payments were observed, namely borrowing and selling assets/properties. Our findings in Most chronic coughers sold household assets to cover health care costs and the majority of costs came from selling farm products (58.8%) and livestock (30.4%). See A total of 608 chronic coughers reported health care costs in relation to their latest illnesses and the mean total cost was estimated to be US$ 3.9 in a 12-month period. The largest share of these costs was due to transport (US$ 1.4), followed by medicines (US$1.3). Considering that the majority of the population where the study was done is poor, the 0.42% of the total household consumption expenditure is a huge burden. The variations in costs could be described by demographic and socioeconomic characteristics of the investigated chronic coughers. We observed that chronic coughers at age of 65 years and above had the largest costs which reduced with younger ages. This was probably due to the fact that most elderly people required a guardian to go to the health facility where they sought health care and the costs drove upwards mainly due to transport, food and accommodation. We also noted that the costs were higher for widows (US $5.5) than those who were married, divorced/separated and those who had never been married. However, no clear reasons of such disparities could be identified. Our multivariate analysis showed that patients with different diseases had significantly different chance of incurring healthcare costs and that costs varied depending on the type of diseases and well as the presence of comorbidity.Our findings, however, were consistent with previous studies, which investigated chronic coughers’ pathways to seeking health care and the associated costs they incurred in other low-income settings ,21,23,25We do not have scope of estimating the incidence of catastrophic costs of households due to limitation of data particularly on household income or consumption expenditure. However, using a proxy measurement of financial protection, this study confirmed that many chronic coughers sold assets/property and borrowed money to pay for health care. Such findings gave an indication that the costs of care, even if not very high in absolute terms, were not affordable for many poor households and might have brought economic impoverishment to some on a temporary basis if it was not persistent.We were aware of a limitation of this study regarding recall bias. We used a cross-sectional survey covering a 12-month period, which might be subject to recall bias while our main variables of interest dealt with costs of healthcare. Although the bias might be low with the reported costs of the current visits, we suspect that the costs of previous visits might suffer more from this recall bias.Despite this limitation, the study does present important insights into health seeking costs of chronic coughers and illuminates some coping mechanisms that can cause or perpetuate poverty. First, while selling of assets can help raise money to cover chronic cough related expenses, it is important to note that asset sale involving disposal of factors of production like land may increase the vulnerability of households. Second, borrowing money from family, friends or money- lenders may represent a reasonable trade-off overtime. However, they can increase the economic vulnerability of households in the long term, particularly for large health seeking costs .We propose that further studies on costs of chronic coughers should be done so that costing data can inform policy makers in the design and provision of universal coverage programs for core chronic cough health services. Future studies should also look at management of CAD and TB in relation to cost implication. Addressing major chronic cough challenges that threaten health and economies alike in low-income settings requires information on the type and extent of costs incurred by different groups  of individuals affected by chronic cough, which can contribute with more knowledge than that found by this current study. The results from the current study, however, can add to the knowledge of the policy makers in designing and provision of universal coverage programs for core chronic cough health services  and 12The costs of healthcare per chronic cougher was mainly influenced by the costs of transport and drugs and these costs varied with demographic and socioeconomic characteristics of the patients. Types of diseases and comorbidity led to significantly different chances of incurring costs and the magnitude of costs varied significantly. Though the costs were not reportedly very high as a share of total household consumption expenditure, it appeared to be unaffordable for many patients.S1 Data(XLSX)Click here for additional data file.


Integrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors  that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza virus replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates. The positions of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of virus-regulated pathways , or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining human PPI networks with virus-host PPI data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow, even after the removal of known abundance-degree bias within PPI data. We have isolated a subnetwork of the human PPI network that connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes distinct from those associated with virus-interacting proteins. Selecting proteins based on subnetwork topology, we performed an siRNA screen to determine whether the subnetwork was enriched for virus replication host factors and whether network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins—more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization. Viruses such as influenza virus hijack and reprogram host cellular machinery to perform virus replication tasks. Influenza outbreaks have a major impact on public health and the global economy each year , 2. WhilUnderstanding virus-host interactions in the context of the human protein-protein interaction (PPI) network will provide a global perspective into how influenza virus manipulates host proteins and aid in identifying host factors involved in influenza virus replication for drug targeting –15. ThHere, we demonstrate a method of integrating virus-host protein interaction data into a human PPI network to prioritize host proteins as antiviral drug target candidates. First, we analyzed a set of 1,292 human proteins identified previously as having interactions with influenza virus proteins , 299 of Studies have shown that proteins in network positions that are essential for information flow within a PPI network  are more likely to be associated with diseases , 35 or dt test P value for comparing log-scaled non-virus-interacting and virus-interacting data < 10−16).Virus proteins were significantly more likely to interact with host proteins that were in positions of high regulatory importance in the human PPI network. For every protein, the degree (number of neighbor proteins) and betweenness  , subnetwork , and the total network . After fitting degree and abundance to a linear model, the bias due to abundance was removed . Therefore, despite known biases ingrained in PPI data, virus-interacting proteins bind more proteins than non-virus-interacting proteins.Knowing documented bias between protein abundance and protein degree , an anal10.1128/mBio.02002-18.1FIG S1FIG S1, TIF file, 1.2 MB.Protein abundance versus protein degree for the total network’s original degree (A) and adjusted degree (B). Each plot is fit with a linear model in red, demonstrating that the correlation found in the original degree is not present after value adjustment. Download Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02002-18.5TABLE S1Table S1, XLSX file, 0.8 MB.Degree, betweenness, and abundance data for all proteins in the PPI network. Each protein  is listed with degree, betweenness, and virus protein binding partner information . Sheet A shows the total network and also contains abundance data from Geiger et al. , and adjCopyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P value for log-scaled data < 10−16). Comparing median betweenness after the removal of proteins with a betweenness of zero, virus-interacting proteins still had a significantly higher betweenness though the population medians were closer in value . The tendency for virus proteins to bind host proteins that had a higher degree and betweenness was consistent when analyzing the interaction partners of each virus protein separately . This indicates that influenza virus proteins selectively interact with human proteins that can strongly regulate cellular behavior. These results are consistent with published findings for HCV and dengue virus . Generally, ∼25% of the randomly sampled proteins are connected by two or fewer interactions, while 88.7% of PB1-interacting proteins, 90.0% of HA-interacting proteins, 98.2% of NS1-interacting proteins, and 79.6% of all host proteins that interact with any influenza virus protein are connected by two or fewer interactions. Collectively, these results support that viral proteins are selectively targeting closely clustered host proteins.We found that virus-interacting host proteins are very significantly clustered within the PPI network. The set of proteins that interact with a viral protein are significantly more closely clustered in the network than expected by chance  that appear within a protein complex and compared that fraction to the fraction of all protein pairs  in the PPI that appear in a protein complex. Mammalian protein complex information was downloaded from CORUM  . We founNetwork analysis of virus-interacting host proteins demonstrates that viral proteins preferentially interact with closely connected host proteins that are in positions central to information flow across the human PPI network, suggesting that it may be possible to exploit network positions to prioritize potential antiviral drug targets. We hypothesized that there exists a subnetwork consisting of pathways that connect virus-interacting proteins to key cellular machinery that is likely to be significantly enriched for host factors. We further hypothesized that the topology of host factors within this subnetwork may provide an additional advantage in identifying host factors.P value of <0.01. The viability of siRNA-transfected cells was assessed using Cell-Titer Glo assay, and downregulation of mRNA levels for the hit proteins was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Of the 264 previously identified host factors tested, 71 significantly downregulated virus replication. Of the 71, 21 were identified to directly interact with influenza virus proteins. In all, 50 of the host factors downregulated virus growth and do not directly interact with the virus. We labeled these proteins as “internal-essential” host factors.To evaluate these hypotheses, we first performed an siRNA screen of host factors identified in a previous genome-wide screen for influenza virus host factors to identify key host factors that do not interact directly with the virus . Poor re10.1128/mBio.02002-18.6TABLE S2Table S2, XLSX file, 0.1 MB.Effects of siRNAs targeting host factors identified to be important for influenza virus replication by Karlas et al.  on virus production. Note that two siRNAs were used per Entrez Gene ID. Sheet 2, labeled “Untested host factors,” lists host factors that were identified in the Karlas screen but were not evaluated in this study. Sheet 3, labeled “Effect on virus replication,” includes virus titers observed in HEK293 cells. Download Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the post hoc analysis P < 0.01). While the degree of connecting proteins does not shift significantly between the total PPI network and the virus subnetwork  and the set of proteins that interacted with any of the virus proteins (“VB”) are included for comparison. Download FIG S2, TIF file, 0.3 MB.The distributions of the degree (a) and betweenness (b) of the interaction partners of each of the 11 virus proteins. The Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A functional enrichment analysis was performed using DAVID 6.8’s Functional Annotation tool . Previou10.1128/mBio.02002-18.7TABLE S3Table S3, XLSX file, 2.3 MB.DAVID functional annotation tool results for virus-interacting proteins and connecting proteins of the influenza virus subnetwork. Full results include the clustering, chart, and table outputs from DAVID 6.8. Download Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the n = 39) and lowest betweenness (n = 39) and conducted another in vitro virus replication assay. HEK293 cells were again transfected with siRNAs targeted to each of the 78 candidate protein’s genes, and the procedure described previously was performed to determine the proportion of the connecting proteins tested that are host factors of influenza virus replication. The hit rate is defined as the proportion of proteins tested that significantly downregulated virus replication.To evaluate the hypothesis that the “connecting” proteins are likely to be host factors and to simultaneously evaluate whether network topology can improve host factor identification, we selected 78 proteins of the subnetwork with the highest  , in the P = 0.024) and in the whole-genome screen (P < 2.2 × 10−16) but not significantly distinct from the rate observed in rescreening the Karlas host factors (P = 0.21). When considering the connecting proteins based on their betweenness, the high-betweenness connecting proteins had a hit rate of 0.39 (15/38) which was significantly higher than the hit rates observed in the virus-interacting and whole-genome screens . The high-betweenness protein hit rate was higher than the rate observed in the screen of host factors by Karlas et al. (P = 0.16). The low-betweenness connecting protein hit rate was lower than that of the high-betweenness connecting proteins (12/39 = 0.31). The difference in hit rates between high- and low-betweenness proteins was not significant (P = 0.57). In all, the screening results suggest that proteins connecting virus-interacting proteins to host factors of influenza virus replication are highly enriched for host factors themselves—significantly more so than proteins that directly interact with virus proteins. However, the topological information from betweenness does not significantly improve host factor identification.The siRNA screen of the connecting proteins found that connecting proteins were significantly enriched for host factors, but there was no statistically significant advantage in selecting proteins by betweenness . Of the s et al. , but not10.1128/mBio.02002-18.8TABLE S4Table S4, XLSX file, 0.02 MB.Effects of siRNAs targeting host factors with high or low betweenness in the virus-host subnetwork on virus production. Download Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 7.2 × 10−05 and P = 1.1 × 10−05, respectively; odds ratio = 1.4 and 1.5, respectively). There is no significant difference in the enrichment of host factors between connecting proteins and virus-interacting proteins . To ensure the host factors identified in the study of Karlas et al. , and no significant difference in the enrichment of host factors between connecting proteins and virus-interacting proteins was found (P = 0.49).To determine whether host factors identified in previous screens are enriched within the virus subnetwork, we compiled a list of host factors of influenza virus replication identified in at least one of six previous screens , 47–51s et al.  were not10.1128/mBio.02002-18.9TABLE S5Table S5, XLS file, 0.1 MB.Hit lists of genes identified in six independent genome-wide screens. Studies include König et al. (2010), Brass et al. (2009), Shapira et al. (2009), Hao et al. (2008), Karlas et al. (2010), and Sui et al. (2009). Download Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Network approaches have demonstrated their potential impact on health-related research, including gene/protein characterization and drug design and side effects , 36, 52,Virus-interacting proteins are closely clustered in the network, which may be a result of attempts by the virus to manipulate specific biological functions . This mFunctional enrichment analysis of the subnetwork reinforces that while virus-interacting proteins are associated with virus replication processes, proteins within the constructed subnetwork are associated with immune response to viral infection. Results for virus-interacting proteins largely build on the per protein discussion of virus-host interactions found in previous work , identifThe conclusion that host-virus interaction data can be leveraged to improve virus replication host factor discovery is unlikely to be affected by off-target concerns associated with siRNA screens. Though off-target concerns often challenge siRNA studies, changes to experimental protocols (such as requiring multiple siRNA hits per targeted gene or changing siRNA concentrations) can only moderately improve false-positive rates –58. ThThe variability and incompleteness of PPI data as well as the limited agreement between influenza virus replication screens are well-known concerns for network-based drug target discovery. False discovery of virus-host interactions and the possibility that virus-host interaction data are skewed toward well-studied networks could also have an effect on the clustering result in virus-interacting proteins. However, the enrichment of host proteins important for influenza virus replication within the constructed virus subnetwork demonstrates that even with these possible shortcomings, PPI network analyses have the power to identify important host factors for influenza virus replication. The antiviral protein candidate screen performed in this study further supports the use of PPI data during candidate prioritization with significant hit rates against virus-interacting proteins and randomly targeted proteins.The observation that betweenness does not significantly improve host factor prediction suggests that alternative topology measures should be considered. There were several reasons why betweenness was selected. Biological pathways are known to have several alternative routes to maintaining cellular operations, a key feature of biological robustness –61. BiAnother interesting continuation of this study would identify the cause of the effect of connecting proteins on virus replication. The mechanism by which each host factor is regulating virus replication may offer additional clues for drug candidate prioritization efforts. Overall, this PPI-based study provides insight into the network characteristics of virus-host interactions and supports the idea that the influenza virus evolved to interact with host proteins in dominant network positions in order to maximally manipulate host cells.Protein-protein interaction (PPI) data were downloaded from the Human Integrated Protein-Protein Interaction rEference (HIPPIE) database   as appropriate. Prop.test and fisher.test both compare outcome proportions between binomial groups with the latter being more precise for small group sizes. Graphics were produced with either the default graphing features of R or with the ggplot2 library .2. Virus plaque titers were determined by plaque assay in Madin-Darby canine kidney (MDCK) cells. MDCK cells were cultured in Eagle’s MEM (Gibco) with 5% NCS at 37°C in 5% CO2.Influenza A/WSN/33 virus (WSN) (H1N1) was generated using reverse genetics . HEK293 siRNA treatment procedure, cell viability, and virus titer determination are described in detail in Watanabe et al. . BrieflyTC method using beta-actin as an internal control. Primer sequences are available upon request.To confirm the downregulation of host genes by their respective target siRNAs, quantitative reverse transcription-PCR (qRT-PCR) experiments were performed. 10.1128/mBio.02002-18.10TABLE S6Table S6, XLSX file, 0.04 MB.Primers used for qPCR. Download Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the t test was used to compare the mean virus titers in cells treated with gene-specific siRNAs with those in cells treated with AllStars Negative Control siRNA. Holm’s method for multiple comparisons was then applied to the P values.For each set of siRNAs, the virus titers from cells treated with siRNAs were normalized by the titers obtained from cell treated with AllStars Negative Control siRNA . siRNAs 10.1128/mBio.02002-18.3FIG S3FIG S3, TIF file, 0.6 MB.Boxplot of the degree and betweenness distributions for connecting (candidate) proteins, virus-interacting proteins, and internal essential proteins. Black lines indicate the median values for the populations. Download Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02002-18.4FIG S4FIG S4, TIF file, 0.2 MB.The mean log fold change (LFC) versus the mean fold change (FC) in cell viability for all 156 gene-specific siRNAs tested. Blue and green points highlight data corresponding to the AllStars Negative Control siRNA and siRNA against influenza virus NP gene (positive control), respectively. The LOESS regression curve (red dotted line) shows that virus growth was not dependent on cell viability. Download Copyright © 2018 Ackerman et al.2018Ackerman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Phascolarctos cinereus) are native Australian marsupials whose populations are in decline from a range of threats. Infectious diseases caused by the bacterium Chlamydia pecorum and other pathogens are of particular concern. We analysed 26 poly-A selected RNA-sequencing libraries from a data set designed to study the immune response of koalas to ocular chlamydial infection. Using virus discovery techniques, we identified the coding-complete genome sequence of a novel picorna-like virus, denoted Burpengary virus, that was most common in south-east Queensland. Notably, abundance measurements of the virus across all 26 libraries revealed an inverse relationship between abundance and ocular disease in koalas, suggesting that the co-infection of Burpengary virus and Chlamydia pecorum is inhibited.Koalas ( Phascolarctos cinereus) is an iconic Australian marsupial species under threat from a number of anthropogenic factors such as loss of habitat due to deforestation and urbanisation, attack from domestic animals such as cats and dogs, and vehicle collisions [Chlamydia pecorum, a bacterium that infects the eyes and urinary tract causing blindness, infertility and death [The koala  and two gammaherpes viruses [Chlamydia, such that KoRV might cause immunomodulation and increase susceptibility to chlamydial disease [Chlamydia might be infected with other viruses, we employed virus discovery techniques based on bulk RNA sequencing [C. pecorum [In addition to  viruses ,6. KoRV  disease . To detequencing ,9,10. Th pecorum .n = 13), and those without (n = 13), were assembled and analysed [de novo using Trinity (v.2.5.1) [https://web.expasy.org/translate/). The coding-complete genome sequence of a picorna-like virus was identified in the assembled contigs of four libraries, while partial sequences were identified in nine other libraries. Bowtie2 (v.2.2.5) [A total of 26 previously determined poly-A selected RNA-seq libraries of ocular tissue sampled from koalas with evidence of gross pathology consistent with koala chlamydiosis  . Hits tov.2.5.1)  to deterv.2.2.5)  was thenv.2.2.5)  against Picornaviridae. Accordingly, all Picornaviridae polyprotein amino acid sequences available within the RefSeq database as of January 2019 were compiled and aligned to the novel protein sequence using MAFFT (v.7.300) [To determine the phylogenetic relationship of the novel virus identified here to previously characterised viruses the amino acid sequence of the assembled polyprotein was aligned to other viruses within the family v.7.300)  employinv.7.300) , with 10The abundance of the virus in each library was determined using Bowtie2 (v.2.2.5)  to alignC. pecorum infectious load was assessed in conjunctival swabs from each animal following DNA extraction as previously described [C. pecorum species-specific LAMP assay was modified for qPCR analysis [C. pecorum. All reactions were run in duplicate and were carried out on a Rotor Gene Q real-time PCR machine in a 20 µL reaction volume. The reaction mix consisted of 10 µL QuantiTect mastermix containing SYBR Green I chemiluminescent dye, 1 µL of 10 µM each forward and reverse primer, 3 µL dH2O and 5 µL sample DNA template. Cycling conditions included an initial denaturation of 94 °C for 10 min, with 35 cycles of denaturation (94 °C for 15 s), annealing (57 °C for 30 s) and extension (72 °C for 25 s). Negative (dH2O) and positive (cultured C. pecorum DNA) controls were included in each run. Infection loads were expressed as a number of gene target copies/µL.escribed . A recenanalysis . The F3 C. pecorum status and geographic location). These RNA-sequencing libraries included samples from 26 individual koalas in varying stages of ocular health grouped into five categories based on disease state and presence or absence of C. pecorum following qPCR screening. Specifically, (i) H1 indicates individuals with no signs of ocular disease and the absence of C. pecorum; (ii) H2 indicates individuals with no signs of disease but with C. pecorum detected by PCR; (iii) G1 are individuals with acute active disease but an absence of symptoms of chronic ocular disease; (iv) G2 indicates individuals with chronic, active disease; and (v) G3 are individuals with chronic, inactive disease with little or no discharge but evidence of corneal scarring.A total of 26 sequencing libraries were chosen from a larger data set originally generated to analyse the immune response of koalas to ocular chlamydial infection  , was identified in 15 libraries and a coding complete genome was assembled. A single ORF was predicted, suggesting a polyprotein organisation, with is consistent with the genome structure of other viruses within the family e family . Analysi support .C. pecorum infection (C. pecorum with the virus and host genes. The abundance of C. pecorum was, however, measured using PCR and gene copies per µL of DNA extract from each conjunctival were recorded for each sample (The abundance of Burpengary virus was measured as reads per kilobase million, and compared to that of koala GAPDH and KoRV to determine if virus abundance was associated with the general abundance of transcripts within each data set. This revealed no association between the abundance of GAPDH or KoRV and Burpengary virus . Importanfection . Due to h sample .Koala samples were collected across south-east Queensland and north-east New South Wales, Australia. To tentatively determine if Burpengary virus was associated with a particular geographical region, the collection locations were mapped and collection points were coloured by virus abundance . This reInfectious diseases are a significant driver of morbidity and mortality in wild koalas . The datC. pecorum relative to the abundance of Burpengary virus, KoRV or the housekeeping gene GAPDH [As the sequencing libraries were poly-A selected during library preparation, bacterial sequences are necessarily eliminated, such that we were unable to measure the abundance of ne GAPDH . This alne GAPDH , the mosne GAPDH .C. pecorum when animals develop acute or chronic ocular disease, although this clearly needs verifying with a larger sample size. It is possible that infection with the bacterium may block viral replication in a similar manner to that of Wolbachia—a genus of arthropod-associated intracellular bacteria known to block the replication of a variety of RNA viruses in Drosophila and some mosquito species [Aedes aegypti mosquitoes [Interestingly, Burpengary virus was identified almost exclusively in samples collected from healthy koalas showing no signs of ocular chlamydiosis. This tentatively suggests that there is a mechanism, currently unknown, that may prevent the co-infection of ocular tissues with the novel virus and  species ,29, inclsquitoes ,31. A pesquitoes . This masquitoes ,34. Innasquitoes .C. pecorum.These results highlight our limited knowledge of the diversity of novel microorganisms present in unique Australian fauna such as the koala. The clinical impact of Burpengary virus on koala health at the individual and population levels is unclear, and like many other studies of this marsupial is limited by the logistical challenges associated with sampling these animals in the field. The apparently inverse association with chlamydial ocular disease appears to add another dimension to the complex and multi-factorial nature of chlamydial disease in this host . Future


Lettuce chlorosis virus-SP (LCV-SP) , is a new strain of LCV which is able to infect green bean plants but not lettuce. In the present study, high-throughput and Sanger sequencing of RNA was used to obtain the LCV-SP full-length sequence. The LCV-SP genome comprises 8825 nt and 8672 nt long RNA1 and RNA2 respectively. RNA1 of LCV-SP contains four ORFs, the proteins encoded by the ORF1a and ORF1b are closely related to LCV RNA1 from California (FJ380118) whereas the 3´ end encodes proteins which share high amino acid sequence identity with RNA1 of Bean yellow disorder virus . The genomic sequence of RNA2 consists of 8 ORFs, instead of 10 ORFs contained in LCV-California isolate. The distribution of vsiRNA  along the LCV-SP genome suggested the presence of subgenomic RNAs corresponding with HSP70, P6.4 and P60. Results of the analysis using RDP4 and Simplot programs are the proof of the evidence that LCV-SP is the first recombinant of the family Closteroviridae by crossover recombination of intact ORFs, being the LCV RNA1 (FJ380118) and BnYDV RNA1 (EU191904) the origin of the new LCV strain. Genetic diversity values of virus isolates in the recombinant region obtained after sampling LCV-SP infected green bean between 2011 and 2017 might suggest that the recombinant virus event occurred in the area before this period. The presence of LCV-SP shows the role of recombination as a driving force of evolution within the genus Crinivirus, a globally distributed, emergent genus. Lettuce chlorosis virus (LCV) belongs to the genus Crinivirus, family Closteroviridae. Viruses included in this family are the largest among the known plant viruses and present ssRNA, positive-sense genome [Closteroviridae has been classified into three genera based on vector transmission and phylogenetic relationships: Closterovirus, Ampelovirus, and Crinivirus. Recently, a new genus named Velarivirus has been proposed [Crinivirus include segmented genomes, are whitefly-transmitted and limited to the phloem [e genome . Family proposed . All meme phloem .Crinivirus are considered emerging epidemics worldwide [Bemisia tabaci and affecting lettuce and sugar beet crops [Closterovirus gene array coding for a heat shock protein homolog , a 50–60 kDa protein (ORF5), the major  and the minor coat protein  [Many members of genus orldwide . LCV waset crops . The comet crops  and compet crops . LCV RNA, ORF 8) ,8. FurthCucurbit chlorotic yellow virus (CCYV) and with Bean yellow disorder virus (BnYDV), [Crinivirus that infects Leguminosae family crops [LCV shows high sequence similarity with (BnYDV), ,9; the lly crops . LCV wasly crops . In 2014ly crops . Recentlly crops ,13.Bromo mosaic virus [Potyviridae where crossovers have been described at ORFs and 3´UTR sequences [Viral RNA-RNA recombination and/or reassortment of genomic segments (pseudo-recombination), between virus strains or virus species, increase the genetic variability and is a powerful process in virus evolution, responsible for the emergence of new viral strains or species . RNA recic virus  and it sequences –21,16.Closteroviridae, homologous recombination has been described at CP, P20 and in defective RNAs and sgRNAs in the Closterovirus member Citrus tristeza virus (CTV) [[Ampeloviruses Grapevine leafroll-associated virus 3 (GLRaV-3) and GLRaV-11 [Closterovirus members share a core of conserved genes, they present high variability in the set of ORFs located in the 3´end of RNA or in the 3´end of RNA 1 if the genome is bipartite, as is the case of the genus Crinivirus [Crinivirus have been described to encode RNA silencing suppressor (RSS) proteins. The presence of mechanisms capable of increasing genetic variability such as genome recombination may have important evolutionary implications because some of these proteins have been characterized as RSS proteins [Within the family s (CTV) [–24 and aGLRaV-11 . Althouginivirus . Some geproteins , 27–30.Plant viruses are strong inducers and targets of RNA silencing, and generally high levels of virus-derived small interfering RNAs (vsiRNAs) are accumulated during virus infections. Characterization of vsiRNAs by deep sequencing has primarily been carried out in experimental host plants and less so in horticultural species .Crinivirus, LCV-SP, originating from a crossover of intact ORFs between LCV-RNA1 and BnYDV-RNA1 as parentals, and analyse a population of isolates collected in the area. The implications in emergence of Crinivirus epidemiology are discussed.In this study we describe a recombinant The owners of the orchards granted permission to collect the samplesBetween 2011 and 2017, samples of green bean leaves showing symptoms that could be attributed to LCV-SP  according to the manufacturer’s instructions. Partial length viral sequence of RNA1 and RNA2 of LCV genome was determined by RT-PCR using a primer walking strategy with a set of degenerate primers based on LCV  and BnYDV  sequences. Partial sequences of LCV-SP RNA1 and RNA2 previously described [de novo assembly function of VELVET v. 1.2.08 implemented in the SCBI Picasso server (http://www.scbi.uma.es) with a k-mer = 18. Partial RNA1 and RNA2 LCV-SP sequence obtained for Sanger sequencing was used as a reference for contigs sequences. RNA1 and RNA2 consensus draft sequences obtained were ensured through RT-PCR amplification using specific primer pair based on the assembled genomes (not shown). The 5´/3´ SmartTM RACE cDNA Amplification Kit  was used to complete the 5´ and 3´ends. PCR fragments were cloned in pGEM-T vector and at least two cDNA clones were sequenced in both directions. To obtain the profile distribution of LCV-SP virus-derived small interfering RNAs (vsiRNAs), the 21 and 22 nt reads of the small RNA library were aligned to the assembled LCV-SP genome using MISIS-2 [Small RNA purification from the Almeria LCV-SP isolate infected tissue was obtained using the miRCURY RNA isolation kit , according to the manufacturer’s instructions. Small RNA library was prepared using the Illumina TruSeq Small RNA Kit . The library was sequenced on the Illumina deep sequencing platform using the services provided by the Centre for Genomic Regulation . Sequencing adapters and reads shorter than 18 nucleotides (nt) were removed using GENEIOUS and reads ranging from 18 to 24 nt were selected. These reads were assembled into contigs larger than 33 nt using the  MISIS-2 .Deep sequence from LCV-SP dsRNA was used to confirm the LCV-SP RNA1 and RNA2 genomic sequences. For that, double-stranded RNA was purified from another LCV-SP isolate collected in 2014  followedhttp://www.ncbi.nlm.nih.gov) using GENEIOUS. The e-value cut-off was set to 10–5 so high confidence matches could be reported. The numbers of reads aligning to LCV or BnYDV genomes were annotated to confirm the presence of sequences belonging to both virus species.The contigs obtained from both massive deep sequencing were conducted to BLAST analysis against the GenBank database in NCBI (5´TCACAGCCGAGATCAACAGAG 3´ (nt 7170–7190) and LC-Bn51R 5´ACAACAGATCAAAATCCACAATG 3´ (nt 7906–7928) which were designed to amply part of the 3’ end of the LCV-SP RNA1 genome corresponding to the partial region of ORF1b and P26 proteinsLCV-SP isolate "Almeria" virions were purified after adapting the methodology described for CTV by . For thaLettuce infectious yellows virus , Cucurbit yellow disorder virus , Cucurbit chlorotic yellow virus , Bean yellow disorder virus , Beet pseudo yellows virus , Tomato chlorosis virus  and Potato yellow vein virus , were retrieved from the GenBank database and compared with the complete sequence of LCV-SP; the genome sequence from the Closterovirus Citrus tristeza virus  was used as outgroup. The pairwise percentage of nucleotide sequence identity was performed using the Clustal W algorithm present in MEGA v 7.01 program [7 generations, sampling every 1000 trees and with a burn-in of 25% and chain heated to 0.2.For phylogenetic analysis, the complete genome sequences from the criniviruses, LCV ,  program . Approprp-value cut-off (α = 0.05). Only recombination events detected by four or more methods were considered as significant. The results obtained by RDP4 were confirmed using a boot scanning method in the SimPlot program v.3.5.1 [Recombination Detection Program 4 (RDP4) was used to detect potential recombination events in LCV-SP, likely parental sequences, and localization of recombination breakpoints . The pro v.3.5.1 .5´GCATTCAAGAAATTGTGGGATG 3' (nt 7517–7538) and LCVSP-50R 5´ATATTAATGTAATTCTACGGTC 3´ (nt 8738–8759) which amplified a region of LCV-SP encompassing 1242 nt corresponding to the entire region of the P26 and P6 proteins. Reverse transcription of the isolates was carried out with a reverse primer using Superscript II (Invitrogen) at 42°C for 60 min. Subsequently, DNA fragments were amplified by PCR with Expand High Fidelity PCR System  with the following conditions: 95°C for 3 min, 35 cycles of denaturation for 20s at 95°C, annealing for 30s at 55°C and extension for 40s at 72°C followed by one final extension cycle for 5 min at 72°C. PCR products were cloned into pGEM-T vector . The clones were bidirectionally sequenced. Nucleotide alignments were performed using Clustal W in MEGA v. 7.01 [The crossover recombination event was confirmed in several isolates by RT-PCR. Eleven isolates were obtained in the surveys from 2011 (3), 2012 (2), 2013 (1), 2014 (2), 2015 (1) and 2017 (2). They were analysed by RT-PCR after RNA extraction using the primers LCVSP-44F  v. 7.01 ,40  was performed. The set of primers LC-Bn56F 5´GATTTTGGATTTGAAGC 3´ and LC-Bn51R (described above) amplified 739 and 769 bp fragments of LCV-SP RNA1 and BnYDV RNA1 genome, corresponding to nt 7190–7928 and 7294–8062, respectively by RT-PCR. Next, the PCR products were digested with KpnI (Promega) following the manufacturer's instructions. Band patterns were analysed on a 2% agarose gel.In order to determine analytically the presence of the recombinant Crinivirus named Tetterwort vein cholorosis virus (ALE18214).Walking strategy and Sanger sequencing allowed us to obtain 7109 bp and 7821 bp corresponding to 80% and 90% of complete genome of RNA1 and RNA2 of LCV-SP. The LCV-SP genome was completed by deep sequencing from small RNAs and RACE strategy. The genome was confirmed through RT-PCR amplification with specific primers  and consClosteroviridae coding for a HSP70h , and three diverged copies of the capsid protein: the ortholog of the coat protein homolog , the CP  and the CPm  which exhibit more than 95% amino acid sequence identity with RNA2 of LCV FJ380119. Percentages higher than 93% are shown in the case of both Chinese LCV isolates (KX685959 and KY430286). RNA2 of LCV-SP encodes also three putative small proteins: P5.6 , P6.4  and P9 , which show 92%, 100% and 96% nucleic acid identity with those in LCV FJ380119 and 88%, 100% and 96% nucleic acid identity with LCV isolates KX685958 and KY430286. Two putative small proteins in the RNA2 of LCV-Californian isolate (FJ380119), P6 in the 5´ end, and P4.8 in the 3´end, which have unknown function are absent in LCV-SP and in the Chinese LCV isolates. The last ORF of RNA2 (ORF9) encodes a putative P27, which shows a high amino acid identity with its counterpart proteins in the LCV genome sequences in GenBank: 98% with the Californian isolate FJ380119 and 96% with the Chinese isolates KX685958 and KY430286.The genomic sequence of RNA2 consists of 8 ORFs, instead of the 10 ORFs contained in LCV-California isolate, 9 ORFs as described in LCV-Nj and 8 in LCV-CN (KX685959 and KY430286) [Crinivirus [The putative 5’UTR of LCV-SP RNA1 and RNA2 are 72 and 269 nt in length, and share 99% and 96% of nucleotide identity with LCV FJ380118 and FJ380119, respectively. The first 5 nucleotides, GAAAT, are identical in both RNAs. This feature has been described in the Californian LCV isolate and in other members of genus RT-PCR products, produced from purified LCV-SP virions using the three sets of primers LCVSP-1F/LCVSP-2R, LCVSP-3F/LCVSP-4R and LCVSP-54F/LC-Bn51R yielded fragments of the expected sizes of 434, 463 and 759 bp, corresponding to the partial region of RNA2-HSP70, RNA1-ORF1a and RNA1-ORF1b and P26, respectively After removal of adapters and reads shorter than 18 nt, deep sequencing from small RNA library produced a set of 44,578 million reads, which were subjected to the novo assembly, generating 19,739 contigs. A total of 113 and 125 sequences of these contigs assembled with LCV-SP RNA1 and RNA2, respectively. Crinivirus arising by crossover recombination of P26 and P6 belonging to the 3´end of RNA1 of BnYDV (EU191904) and discarding the presence of any Californian or Chinese-related LCV in the plants.The result of BLASTN is shown in The profiles of vsiRNAs aligned to LCV-SP RNA1 and 2 were analysed. The 21-nt vsiRNA class population was most prevalent, followed by the 22-nt . From this 21-nt population, 58 675 and 80 904 reads matched to LCV-SP RNA1 and 2 genome. In the case of 22-nt class population, 53 932 and 68 146 matched with LCV-SP RNA1 and 2. The analysis of 21 and 22-nt vsiRNA strand polarity showed similar pictures, 53% of 21 and 22-nt vsiRNA class populations aligned to LCV-SP RNA1 were negative, whereas more than 50% of 21-nt and 22-nt vsiRNA class populations aligning to LCV-SP RNA2 were positive . The profile distribution of both vsiRNAs populations aligning to the LCV-SP RNA1 and RNA2 genome components were also very similar. The distribution of 21-nt vsiRNAs along LCV-SP RNA1 and 2 genome is shown in the Crinivirus show two well differentiated lineages; one comprising LCV-SP, LCV, CCYV, BnYDV and CYSDV and the other is formed by BPYV, ToCV, PVYV and LIYV. Then, two clades were generated where the divergence is clear  of LCV, BnYDV, CCYV and LCV-SP resulted in four recombination events detected  is much higher than in event 1 . LCV RNA2 was identified for six methods as a putative recombinant in event 4 with LCV-SP RNA2 and CCYV RNA2 as major and minor possible parents respectively . These sequences confirmed the presence of the recombinant LCV-SP in the south of Spain since at least 2011. Values of genetic distance sequences at non-synonymous positions for both ORFs were of the same magnitude . The ratKpnI after RT-PCR with the primers LC-BnF/LC-BnL, which amplified partial sequence of RdRp and P26 in both viruses, yielded two fragments of 582 y 157 bp when the virus was LCV-SP and a single band of 769 bp when the virus present was BnYDV. This RFLP test is then capable of distinguishing yellowing disease originating from recombinant or non-recombinant virus isolates ; P6, and P4.8, located in the 5´and 3´end of the Californian isolate, respectively, and with unknown function, are absent in LCV-SP. Both putative proteins are also absent in the LCV Chinese isolates recently reported ,13, whic3´UTRs sequences in both genomic RNAs are highly conserved, sharing 72% of nucleotide sequence identity. This feature is common among criniviruses  and it aHigher number of 21-nt class vsiRNAs populations with respect to 22-nt support the prevalence of a Dicer like protein 4 (DCL4) in the production of vsiRNAs in beans, as has been suggested in others plants ,55. 21- Crinivirus, which shows the lowest variability within the family Closteroviridae [Sweet potato chlorotic stunt virus (SPCV) and Beet pseudo yellows virus (BPYV) have been reported as examples of recombination-mediated gene gain [Crinivirus, an emergent genus and, as we have demonstrated, with more recombination possibilities than previously shown.Recombination analysis completed with RDP4 and Simplot programs, demonstrates the existence of natural crossover recombination of intact ORFs between LCV-RNA1 and BnYDV-RNA1 (event 1). The recombinant virus, LCV-SP, shifted its host range by infecting green bean crops and not lettuce, its original host ,5. No otoviridae , only Swene gain ,26. The S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 TableLCV-RNA1: FJ380118; BnYDV-RNA1: EU191904; LCV-SP-RNA2: MG489895; CCYV RNA2 904629. Parentals in the putative recombinants 2 and 3 are unknown. NA: not available.(DOC)Click here for additional data file.S1 FigLane 1: LCVSP-1F/LCVSP-2R; Lane 2: LCVSP-3F/LCVSP-4; Lane 3 LCVSP-54F/LC-Bn51R.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.