GYNAUTO-STI study was carried out to assess the performance of a novel genital veil  as a genital self-sampling device to collect genital secretions to diagnose STIs by molecular biology as compared to reference clinician-collected genital specimens, in adult African women. Sexually transmitted infections (STIs) are highly prevalent in sub-Saharan Africa. Genital self-sampling may facilitate the screening of STIs in hard-to-reach remote populations far from large health care centers and may increase screening rates. The cross-sectionalLa Renaissance Plus”. A clinician obtained an endocervical specimen using flocked swab. Genital secretions were also obtained by self-collection using veil. Both clinician- and self-collected specimens were tested for common curable STIs  and genital Mycoplasma spp. by multiplex real-time PCR . Test positivities for both collection methods were compared by assessing methods agreement, sensitivity, and specificity. Adult women living in N'Djamena, the capital city of Chad, were recruited from the community and referred to the clinic for women's sexual health “C. trachomatis: 3 (1.2%), N. gonorrhoeae: 1 (0.4%), M. genitalium: 4 (1.6%) and T. vaginalis: 1 (0.4%)], while the prevalence of genital mycoplasmas was much higher (54.2%) with a predominance of Ureaplasma parvum (42.6%). Self-collection by veil was non-inferior to clinician-based collection for genital microorganisms DNA molecular testing, with “almost perfect” agreement between both methods, high sensitivity , and specificity . Remarkably, the mean total number of genital microorganisms detected per woman was 1.14-fold higher in self-collected specimens compared to that in clinician-collected specimens. A total of 251 women  were prospectively enrolled. Only seven (2.8%) women were found to be infected with at least one common STIs [ Veil-based self-collection of female genital secretions constitutes a convenient tool to collect in gentle way cervicovaginal secretions for accurate molecular detection of genital bacteria. Such sampling procedure could be easily implemented in STIs clinics in sub-Saharan Africa. Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis could be easily treated if properly detected. The World Health Organization (WHO) reports global numbers worldwide of new cases for C. trachomatis, N. gonorrhoeae, and T. vaginalis infections of 78, 131, and 142 million every year, respectively . In each inclusion site, peer educators contacted adult women in community churches and mosques or women association networks during a one-month period and proposed them to be included in the GYNAUTO-STI study after an oral explanation and collective awareness sessions on the objectives of the survey, mainly focused on sensitization on prevention strategies against STIs and cervical cancer. After oral consent, the selected women were invited, with paid transportation, to come to the clinic “La Renaissance Plus”, N'Djamena, which is one of the main settings for women's sexual health in Chad, and to participate in the study. Childbearing-aged and older women living in N'Djamena regularly attend the clinic “La Renaissance Plus” for gynecological examinations and for obstetrical services.The capital city N'Djamena comprises 10 districts, which include a variable number of neighborhoods. Twenty-three sites in neighborhoods of 5 districts randomly selected out of 10 were further chosen for study inclusion  or self-collection of genital secretions using the V-Veil-Up Gyn Collection Device [Method 2] according to the randomization.The gold standard Method 1 was carried out by a doctor using a flocked swab . Briefly, after placing the speculum (without lubricant prior to insertion), the physician used the swab to perform cervical sampling by introducing it into the cervical canal and performing 5 rotations before being removed and immediately placed in its plastic container. The swab was then placed in the cold (ice pack).For the self-collection Method 2, the study participant firstly received from a nurse a 15-minutes training on how to use the V-Veil-Up Gyn Collection Device for vaginal self-sampling . After i Hôpital Général de Référence Nationale, N'Djamena, Chad. Swabs and veils were further transported in frozen ice packs to the microbiology laboratory of the Hôpital Européen Georges Pompidou, Paris, France, for molecular analyses.Each genital sample was transported in ice packs within an hour after collection to be stored at -80°C at the virology laboratory of theμL of the elution buffer provided in the extraction kit and stored at -80°C before bacterial DNA detection, as described in part previously  and commensal mycoplasmas  were detected by molecular biology using the CE IVD-marked multiplex real-time PCR Allplex™ STI Essential Assay    and commensal mycoplasmas (group II) showed a trend to be slightly higher in self-collected specimens than clinician-collected specimens, but the trends were not statistically significant for all comparisons  . InteresP<0.001] .The N. gonorrhoeae, C. trachomatis, M. genitalium, and T. vaginalis) as well as genital mycoplasmas  colonization among community-recruited asymptomatic adult women living in N'Djamena, Chad. The results showed low incidence of common STIs, as only seven women (2.8%) were infected. The prevalences of genital mycoplasmas  were much higher in more than half women with a predominance of U. parvum. Furthermore, the genital microorganisms DNA diagnostic accuracy of a novel genital veil (V-Veil-Up Gyn Collection Device) was assessed as female genital self-sampling device to collect cervicovaginal secretions. All specimens collected by the veil were found positive for the β-globin gene, demonstrating the lack of PCR inhibitors or the presence of cellular DNA, which made STIs detection possible by molecular testing. The results showed high accuracy of the veil-based genital self-collection, which was non-inferior to clinician-based collection as reference for genital bacteria DNA molecular testing, with “almost perfect” agreement between the two collection methods, high sensitivity and specificity. Outstandingly, the mean number of detected genital bacteria was significantly higher when using veil-based collected genital secretions than clinician-collected cervical secretions by swab. Thus, the self-collection by veil allowed detecting 1.14-fold more genital bacteria than the swab-based collection, likely originating from cervical as well as non-cervical areas of the vaginal cavity, including vaginal cul-de-sacs, vaginal walls, and vulva. Taken together, veil-based self-collection of genital secretions appears a convenient tool to collect in a gentle way genital secretions for accurate molecular detection of female genital bacteria. Such sampling procedure could be easily implemented in STIs clinics in sub-Saharan Africa.The present study reports on the prevalences of common curable STIs , N. gonorrhoeae (0–3.1%), and T. vaginalis (0.4 – 17.6%) have been reported [Epidemiological data on the prevalences of common bacterial STIs in Chad have been until now poorly reported. In a cohort of 311 HIV-1-seropositive women, Mortier and colleagues found a prevalence of 1% foraginalis . Remarkareported –31. Thisreported . M. genitalium  are generally considered as commensals of the female uro-genital tract, as they may be found at rates up to 80% in sexually active women [ M. genitalium in this population would need further investigation to assess its clinical consequences and potential adverse pregnancy outcomes.The high rate of carriage of genital mycoplasmas in our study population is reminiscent of previous reports in women of reproductive age from neighboring countries, with prevalence rates of 65% in Cameroon  and 36% ve women , 38. How GYNAUTO-STI study, we have had the opportunity to evaluate the genital microorganisms DNA diagnostic accuracy of a novel genital veil (V-Veil-Up Gyn Collection Device) as female genital self-sampling device to collect cervicovaginal secretions.Taking into account that most adult Chadian women are living in remote rural areas, or far away of adequate healthcare facilities, self-collection of genital specimen carried out at home by women themselves could represent a relevant alternative allowing increasing the coverage of screening when coupled with adapted STI pathogens testing by molecular biology. In theκ coefficients were between 0.81 to 0.99 demonstrating “almost perfect” agreement between collection methods [ C. trachomatis and N. gonorrhoeae testing may be useful to contextualize our observations [ C. trachomatis or N. gonorrhoeae test results with home-based collection by vaginal swab  than with clinic-based  specimen collection  [ C. trachomatis or N. gonorrhoeae per 100 woman-years of follow-up for home-collected and clinic-collected specimens  in a prospective randomized controlled trial including high-risk young women followed up during 18 months [The percent of agreements between clinician-based collection and veil-based self-collection to detect any vaginal microorganisms was above 90%, and all Cohen's  methods . Althougrvations . A recens: 3041) , 39–44. 8 months . Finally a priori located at non-cervical vaginal areas .Because clinical management and research usually depend on single-point detection, it is important to use the collection method the most capable to detect genital microorganisms by molecular biology. For example, the cumulative presence of HPV in female genital tract is always greater than its point prevalence, suggesting that single-point sampling is less than 100% sensitive . OtherwiOur observations also point to the potential interest of using a supervised self-collection strategy among African women, in which oral counseling processes are aided at all times by a healthcare or non-healthcare professional as a counselor to understand the instructions for use, the genital anatomy, and provide counseling with a very high rate of acceptability and satisfaction of self-collection. The evidence of high acceptability for supervised strategies was previously reported for another self-collection of capillary blood or saliva during HIV self-testing, especially in resource-constrained settings . FinallyLa Renaissance Plus”, N'Djamena, were not patients from the clinic, avoiding the obvious bias of recruitment by health care facilities. Furthermore, 9 (3.5%), 19 (7.5%), and 8 (3.2%) study participants were HIV-, HBsAg-, and HCV-specific antibody positive, respectively, in accordance with the high endemicity of these three major chronic viral infections in Chad [Our study had several strengths. First, we tried to limit the selection bias of the study population by community-based recruitment in order to make this survey as much representative as possible of the female population in Chad. Thus, study participants referred to clinic “ in Chad , 49. How in Chad . Thirdly.5%), 19 .5%, and  in Chad , even in in Chad , 51, 52.In conclusion, the diagnosis and prevention of bacterial STIs is likely one of the most important public health challenges that Chad has to face in a near future. Self-collection of genital secretions using the V-Veil-Up Gyn Collection Device constitutes a simple and powerful tool to collect genital secretions for further molecular testing and screening of genital microorganisms that could be easily implemented in the national prevention program in Chad. In regions of the world where access to care is limited due to socioeconomic reasons or where clinician-collected samples may be limited due to personal and/or sociocultural concerns, self-collection method by inexpensive veil may provide a way to extend screening to an underserved population.


Pore-size distribution (PSD) is the most critical parameter for size-exclusion virus removal filters. Yet, different dry- and wet-state porometry methods yield different pore-size values. The goal of this work is to conduct comparative analysis of nitrogen gas sorption (NGSP), liquid-liquid and cryoporometry with differential scanning calorimetry (CP-DSC) methods with respect to characterization of regular and cross-linked virus removal filter paper based on cellulose nanofibers, i.e. the mille-feuille filter. The filters were further characterized with atomic force and scanning electron microscopy. Finally, the removal of the worst-case model virus, i.e. minute virus of mice  was evaluated. The results revealed that there is no difference of the obtained PSDs between the wet methods, i.e. DSC and liquid-liquid porometry (LLP), as well as no difference between the regular and cross-linked filters regardless of method. MVM filtration at different trans membrane pressure (TMP) revealed strong dependence of the virus removal capability on applied pressure. It was further observed that cross-linking filters showed enhanced virus removal, especially at lower TMP. In all, the results of this study highlight the complex nature of virus capture in size-exclusion filters. Virus removal filtration is a robust method to clear viruses and other microbial pathogens from liquids during manufacturing of protein-based pharmaceuticals and in water purification . The firThe most critical parameter for a size-exclusion filter is its pore-size distribution. Yet, different methods relying on various physical principles yield different pore-size values. This is because each method reflects the accessibility of pores to specific probing medium based on different assumptions. Normally, viruses are particles having a particle size between 20 and 200 nm, and therefore methods that can probe pores in this size range are particularly interesting for filtration applications. For the majority of mesoporous materials, i.e. materials having pores between 2 and 50 nm, nitrogen gas sorption porometry (NGSP) is the method of choice. The Barrett-Jonyer-Halenda (BJH) model, based on Kelvin equation, is applicable for NGSP and relates the pore-size distribution to emptying of pores from condensed liquid nitrogen as a function of surrounding nitrogen relative pressure. NGSP is an isothermal method performed on dry samples. However, from the point of filtration, application methods probing the pore-size distribution in the wet state are preferable. In this respect, two wet-porometry methods that are useful to probe mesoporous materials include liquid-liquid porometry (LLP) and cryoporometry with differential scanning calorimetry (CP-DSC). LLP is an isothermal method that is based on replacement of one pore-filling liquid with another immiscible liquid, wherein the pore size is related to applied pressure needed to replace two liquids with each other based on the Hagen-Poiseuille equation ,9. CP-DSAnother parameter that can affect the efficiency of virus removal is the applied transmembrane (TM) pressure during filtration ,12,13,14In this work, we focus on the characterization of the pore-size distribution and separation properties of a new type of nonwoven virus removal filter paper developed at Uppsala University, Sweden. The filter paper consists of 100% nanocellulose and is produced by hot-pressing technology, similar to papermaking, rather than by phase inversion that is utilized for membrane manufacturing ,17,18,19The goal of this article is to perform comparative analysis of various dry- and wet-porometry methods in the context of worst-case model virus, i.e. minute virus of mice (MVM), filtration.Based on the previous results, 33 μm filters were used for this study as they provide a relatively good balance between virus removal, mechanical stability, and flux performance .®, Merck Millipore, Burlington, MA, USA) using vacuum suction in a funnel. The cellulose cake on the filter was then removed and dried at 40 °C for 12 h using a hot-press .A 3 wt% Cladophora cellulose dispersion was prepared in deionized water using a high-energy ultra-sonicator  using 30 s pulses at 70% amplitude for 20 min. To cast the filter, 50 mL of the Cladophora cellulose dispersion was added to 300 mL of water. The dispersions were then drained over a 0.65 μm filter membrane  was used. The images was obtained using 0.8 kV acceleration voltage, and the sample was sputtered with Pd/Au prior to the analysis to limit electrostatic charging effects.A Bruker Dimension Icon atomic force microscopy  system with a Bruker silicon nitride ScanAsyst-Air probe  was used to obtain the images. The probe has a symmetric pyramid geometry with a nominal tip radius of 2 nm. The filter was mounted using a double adhesive tape on magnetic disc holders. Bruker’s ScanAsyst software  was used with the instrument running in peak-force taping mode to obtain the images.TM Silica-Alumina SSA 210 m2·g−1 (lot number: A-501-49) standard prior to analysis. The deviation between the pore-size mode of the calibration data from the nominal standard values was 0 nm. The sample was degassed for 6 h at 95 °C at 5 °C·min−1 ramp temperature prior to analysis. The pore-size distribution was calculated according to the Barrett-Jonyer-Halenda (BJH) method [Nitrogen gas sorption isotherms were obtained using ASAP 2020 instrument . The performance of the instrument was validated using Micrometrics) method  from thek is a shape factor, set to 1 with the assumption of cylindrical pores, γ is the surface tension, θ is the contact angle between the interface and the pore wall, ΔP is the TM pressure and r the effective radius.The liquid-liquid displacement method was used as proposed by Erbe  and BechQphase2(ΔP) is the volumetric flow of intrusion liquid when the wetting fluid is present in the membrane at a set pressure, Intrusion fluidQ(ΔP) is the volumetric flow of intrusion fluid in absence of wetting fluid present in the membrane, ΔP is the pressure, QF(r) is the flow weighted pore-size distribution, k is the shape factor which is set to 1 approximating a cylindrical pore shape, and γ is the interfacial surface tension between the two fluids, which has been reported as approximately 6.3 × 10−4 N at 22 °C [The flow weighted pore-size distribution was calculated using Equations (2) and (3) as described by S. Giglia et al. :(2)R(∆P)at 22 °C . The mea−1 rate to −20 °C and then heated to 5 °C at a heating rate of 0.7 K·min−1. The CP-DSC method was used as proposed by Landry . The samon-pkT is the difference of the depressed melting point to the true melting point of bulk water, r is the radius of the pore. mA, mB and mδ are probing liquid dependent constants. Herein, we use the following values for mA = 19.082, mB = −0.1207, and mδ = 1.12 calculated by Landry [V/dr) was calculated using Equation (5):Q/dt is the heat flow, dt/d(∆t) is the scanning rate of 0.7 K/min, d(∆on-pkT)/dr is the melting point depression from Equation (4), m is the mass of the sample, ∆fH(T) is the temperature-dependent melting enthalpy, and ρ(t) is the temperature-dependent density.For a porous material with a narrow pore-size distribution, the melting point depression can be related to a pore radius using Equation (4):y Landry . The difT is the current temperature and mT0 is the equilibrium melting temperature of water.The temperature-dependent melting enthalpy was calculated using Equation (6):T is the temperature in Kelvin.The temperature-dependent density of water was estimated using Equation (7):r2 = 0.99). The estimated function is then used as dQ/dt in Equation (5) to calculate the pore-size distribution.To achieve peak separation between bulk water and pore-confined water peaks, a second-degree Gaussian function is fitted to the peak of the confined water using Matlab (n = 5 for each volume 0.7 K·min−1 heat rate) and the mean peak temperature was calculated to 0.61 ± 0.1 °C.The melting temperature onset of bulk water, representing the water outside of the pores, was measured and calculated as follows. The DSC endotherm was measured for 5 different volumes of water viz. 1, 2, 4, 6, and 10 µl (The mille-feuille filter (45 mm in diameter) was placed in a filter holder (Advantech KST-47) with underlying general-purpose filter paper support. The filter holder was filled with 200 mL deionized water. The rate of flow was measured for a set volume of 50 mL for each pressure viz. 1, 2, 3, 4, and 5 bars. The rate of flow was monitored gravimetrically by collecting the outflowing liquid on an analytic balance, connected to LabX software , at 20 s intervals. All measured values were normalized per bar in order to visualize the compaction effect of the filters.50) at 8-fold dilution. EPT was used for samples with high viral loads. LVP was performed for samples with expected low virus loads, provided that such indications had been demonstrated during EPT.Virus spiking tests were performed similar to the flux measurement as described. The filter holder was filled with 200 mL of 0.1% bovine serum albumin in phosphate buffer solution, spiked with MVM. Five fractions of 40 mL each were collected during the filtration experiment. The virus titres before and after filtration were evaluated using end-point titration (EPT) and large-volume plating (LVP) methods. The MVM titres were evaluated from observed cytopathogenic effect (CPE) and tissue culture infectivity dose , and sufficient cell density. Upon adding of virus-containing solution, both EPT and LVP plates were incubated at 37 ± 1 °C and a COThe CPE was verified microscopically as shown in Each individual well at varying dilutions was tested, and positive wells were documented. The results for EPT were then analyzed using the Spearman-Kärber formula (Equation (8)) as described previously ,29.(8)lY0: decadic logarithm of highest dilution factor of the sample, which causes the infection of all parallel cultures (= 8)d: decadic logarithm of dilution step (= log103);iP: observed reaction rate starting and including the rate at Y0;v: decadic logarithm of volume conversion factor (= log105).In the LVP assay, the lowest dilution is analyzed on multiple wells. With this, a higher total volume of the original samples is screened for residual infectivity.If virus-induced changes in 15 to 50% of all wells of the LVP assay are observed, the virus titer is calculated according to the Spearman-Kärber formula (see above). It is assumed that a 1:3 higher concentrated dose compared to the dose analyzed leads to an infection of all parallel cultures. Only two reaction rates are reported: the reaction rate determined by the LVP and the reaction rate of the virtual 1:3 higher concentrated dose (= 1).If virus-induced changes are observed in only a few wells of the LVP  for a sample, the virus titer is calculated according to Equation (9):D: predilution factor of the sample;pn: number of virus-positive wells;n: number of all wells tested;wV: sample volume per well (0.2 mL).If no virus-induced changes are observed for a sample, the virus titer is determined by the Poisson distribution (Equation (10)) at the 95% confidence limits:p: 0.05;v: tested sample volume in mL;V: process fraction volume in mL.The reduction of viral load, expressed as a logarithmic reduction value (LRV), represents the capability of a virus clearance, calculated using Equation (11):C0: log10 total virus load spiked start materialnC: log10 total virus load related filtrateThe titer was measured for each fraction individually, and the confidence interval for each measurement point in the duplicate run was 95%.The mille-feuille filter is a nonwoven wet-laid filter paper produced by hot-pressing cellulose nanofibers. The pores in the filter paper are made up by the voids between cellulose nanofibers. These pores can be visualised through AFM imaging for both regular and cross-linked filters as shown in A distinctive feature for mille-feuille filter is its layered architecture, which can be visualized by SEM. As it was mentioned earlier, the choice of the appropriate method for pore-size characterization is not straightforward. 3 for the cross-linked compared to 0.43 g/cm3 for the regular filter. Based on the SEM images of cross-sections, we hypothesize that cross-linking reduces the space between individual nanocellulose sheets. The calculated pore-size distributions for the three methods can be seen in In general, it is concluded that there is not a significant difference between the methods regarding detected pore size. It should be noted that the NGSP and LLP managed to detect the upper pore size cut-off around 35–40 nm, which is of importance for virus removal filtration. From the point of practical utility, the LLP method was less practical to implement compared to the CP-DSC method. Firstly, a larger sample was required per test due to the size of the suitable filter holder. Secondly, the procedure was performed manually and, therefore, it was laborious and time-consuming. Because of the large sample size, the method further required relatively large quantities of liquids with specific composition and viscosity. In general, higher variability of data was observed compared to the other automated methods. Contrary to LLP, CP-DSC method was preferable because it was automated and did not require large sample size or large volumes of liquid. As a result, the produced data showed less variability than that seen with LLP. Among the drawbacks of the CP-DSC, the uncertainty related to determination of the freezing point for bulk water needs to be highlighted. Another drawback is the poor peak separation due to the highly asymmetrical structure of the filter. In all, the CP-DSC method is currently more preferable for a reliable determination of the pore-size mode in the filter paper. Developments in automated commercial LLP instruments will likely improve the quality of obtained data with this method.The measured normalized flux rates of water for the regular and cross-linked filter paper are presented in To further characterize the performance of the filter, removal of worst-case model parvovirus, i.e. MVM  suspended in a 1 wt% bovine serum albumin (BSA) solution, was investigated using the regular and cross-linked samples. It should be noted that the different methods did not show large differences in the pore size between filters but virus removal capability was greatly affected by the cross-linking and pressure gradient. The enhanced wet strength and compactness of the cross-linked filter in the wet state accounts for the observed increase in virus removal capability as discussed above in In this work, comparative analysis of dry- and wet-porometry methods was performed to characterize regular and cross-linked virus removal filter paper using 3 independent methods. The results of the study suggest that wet-porometry methods yield a slightly larger pore size than that detected by NGSP. CP-DSC method was found particularly useful to characterize the PSD in the wet state. NGSP, CP-DSC, and LLP did not detect substantial pore-mode shift between regular and cross-linked filters. The effect of TM pressure on the efficiency of MVM removal was also investigated for the first time for regular and cross-linked filters. TM pressure of >3 bar was needed to achieve ≥4 LRV removal for noncross-linked samples. Cross-linking the cellulose nanofibers substantially improved the MVM removal efficiency at lower pressures enabling ≥4 LRV removal at 3–4 bar. This is particularly interesting since neither of the characterization methods could detect any change in the pore size for the cross-linked sample. In all, the results of this study highlight the complex nature of virus capture in size-exclusion filters, involving not only steric hindrance but also other parameters, such as hydrodynamic constraint at high pressures and also possibly weak-type surface interactions.


As the leading cause of death worldwide, viruses significantly affect global health. Despite the rapid progress in human healthcare, there are few viricidal and antiviral therapies that are efficient enough. The rapid emergence of resistance, and high costs, as well as the related side effects of synthetic antiviral drugs, raise the need to identify novel, effective, and safe alternatives against viral diseases. Nature has been of the most exceptional help and source of inspiration for developing novel multi-target antiviral compounds, affecting several steps of the viral life cycle and host proteins. For that matter and due to safety and efficacy limitations, as well as high resistance rate of conventional therapies, hundreds of natural molecules are preferred over the synthetic drugs. Besides, natural antiviral agents have shown acceptable antiviral value in both preclinical and clinical trials.This is the first review regarding molecular and cellular pathways of the virus life cycle, treatment strategies, and therapeutic targets of several viral diseases with a particular focus on anthocyanins as promising natural compounds for significant antiviral enhancements. Clinical applications and the need to develop nano-formulation of anthocyanins in drug delivery systems are also considered. As unique obligate pathogens mostly dependent on live organisms, viruses are becoming a leading cause of death in life-threatening diseases . Due to In spite of tremendous advancements in antiviral drugs, there are several problems with the existing antiviral treatments, including safety and efficacy limitations, as well as their high costs . The catThis is the first review regarding the current antiviral approaches and alternative, natural antiviral compounds while tackling particular attention to anthocyanins. We also focused our attention on the need to develop nanoformulation to improve the anthocyanin delivery system.Antiviral drugs can be categorized into the inhibitors of fusion, uncoating, nucleic acid synthesis, integration, protease, and release .The interaction between the virus and the host cell membrane or receptor(s) is the first phase of the viral life cycle called fusion/entry. Fusion/entry inhibitors have been more provided for human immunodeficiency virus (HIV) treatment  and respThe low pH of the endosome activates M2 proton channels following virus entry to acidify the viral interior and weaken the electrostatic interaction to allow viral uncoating . UncoatiFollowing uncoating, nucleic acid synthesis is the third step of the viral life cycle, which is mediated by viral enzymes, including RNA polymerase, DNA polymerase, and reverse transcriptase. So, these enzymes have been considered as alternative targets in many viral infections such as HBV , herpes Inhibitors that specifically target integration have also been approved for the treatment of HIV infection. Since integrase is a necessary enzyme for the replication step involving the integration of host cell DNA to the viral genome, it has become a validated target for developing anti-HIV agents . New intViral proteases also signify an appropriate target for the development of novel antiviral agents against HIV and hepatitis C virus (HCV) infections . FurtherNowadays, using a combination therapy of protease inhibitors with reverse transcriptase inhibitors, called highly active antiretroviral therapy (HAART), significantly enhanced the clinical outcome, reduced mortality, and morbidity, and greatly improved the life expectancy in HIV-1-infected individuals. HAART cannot eradicate the virus from HIV-infected cases. Drug-resistant, long-term use toxicity, and related high-cost limit the use of HAART in infected individuals . These aThe release of new viruses from the host cell is the final step of the virus infection cycle. Some drugs inhibit this phase through acting on the viral protein involved in such a process, which have been approved for the treatment of InfV infection . The haeFrom the antiviral drugs restrictions point of view, there is no suitable vaccine or therapeutic option for many viral infections. Conventional antiviral drugs are limited by the inadequate response, the rapid development of resistance, and adverse effects. It is well established that using even standard and the same doses of antiviral drugs may result in different inter-individual serum concentrations and clinical outcomes, the reasons for which may be multifactorial including differences in concomitant medications, underlying diseases, treatment compliance, genetic factors, and gender-related metabolism. This noticeably influences the antiviral efficacy, consequent resistant variants, and the toxicity incidence. Drug hypersensitivity reactions, which vary in clinical manifestations, severity, and frequency, are among other adverse effects of a few antiviral drugs  , showed the potential antiviral effects . InhibitTherapeutic antibodies have also shown promising effects against viral infections, including RSV, HIV, avian InfV, and rabies viruses .Nowadays, ribozymes, aptamers, and RNA interference (RNAi), specifically small interfering RNAs (siRNAs) are of other nucleic acid-based antiviral therapies to target HIV, HCV, HBV, Marburg virus, Ebola virus and influenza viral genes . BesidesAs novel cellular targets of licensed antiviral drugs, DNA terminase, helicase, and primase have a promising future. Letermovir has shown prophylactic effects against HCV in phase 2 and 3 of a clinical trial, by influencing the synthesis of the viral terminase complex . It did in vitro and in vivo antiviral activities. Among them, only a few have been approved for clinical use by western health  connected to a heterocyclic ring with oxygen function (C), which is also connected to a third benzyl ring (B). Part C with three carbon bridges is connected to parts A and B Figure 2Glycosidic bonds are formed at 3 or 5 positions or both, and produce the monosaccharides, including glucose, galactose, rhamnose, and arabinose, as well as di-saccharides, and tri-saccharides, which could possess acylated side chains with aliphatic or cinnamic acids Figure 3This is the cause of the structural diversity of anthocyanins with the difference in stability and color. The fundamental differences would be the total number and position of OH groups, nature, number, and position of connected sugars, the carboxylates in aliphatic chain or aromatic forms or acylated groups linked to the sugar in the structures, and the position and place of these connections    . Knox eted cells . R. nigr surface , interfe surface  and compin vitro . As Ikutin vitro . AltogetSolanum stenotomum and S. tuberosum were surveyed potential sources for natural red colorants, especially anthocyanins and contained pelargonidin-3-rutinoside-5-glucoside acylated with p-coumaric acid as major anthocyanins , human herpes virus type 1 (HHV-1), and vaccinia virus strains Western Reserve (VACV-WR). According to the results, the extract of S. paniculatum inhibited the replication of HHV-1, but exhibited no effect on VACV-WR and EMCv (S. nigrum seeds (SNS). As reported by the authors, the chloroform extract of SNS reduced the expression or function of HCV NS3 protease in a dose-dependent manner. Furthermore, NS3 may have extra effects involved in interference with host cell functions, like inhibition of protein kinase A-mediated signal transduction or cell transformation. Besides, the antiviral activity of SNS is a target for therapeutic development of new antiviral compounds for acute and chronic HCV, where NS3- mediated processing of the polyprotein is essential for HCV RNA replication and maturation  have shown cyanin as a major anthocyanin of this species of edible legumes . According to the findings, the anthocyanins fractions were involved in the antiviral activity of V. angularis, which were affected by maceration conditions, and the anthocyanin-deficient white bean did not show antiviral effects into the exudate fluids. Moreover, the antiviral activity was still observed after heating for 10 min. Further studies on the antiviral activity of the extract affect preliminary adsorption phase and the preliminary phase of viral replication. The antiviral activity of V. angularis was not only restricted to RV, but the HSV and InfV were affected as well  . In spitRubus coreanus  exhibited the presence of some anthocyanin structures, which were tentatively identified as cyanidin 3-O-xylosylrutinoside, 3-O-sambubioside, cyanidin 3-O-rutinoside, delphinidin 3-O-rutinoside, delphinidin 3-O-glucuronide and pelargonidin 3-O-rutinoside ) fruits with different distributions and contents   . Cyanidiocarpon) . In anotocarpon) . As an aVitis vinifera (grape)  . In a re (grape) . Previou (grape) . Similar (grape) . In the  (grape) . TherefoCyanidin-3-arabinoside and cyanidin-3-galactoside were found as two major anthocyanins in cocoa seeds (CS), which contained 0.02–0.4% of defatted CS powder . Kamei eLycium barbarum (wolfberry) were reported by Zheng et al., including petunidin-3-O-galactoside-5-O-glucoside, petunidin-3-O-glucoside-5-O-glucoside, delphinidin-3-O-rutinoside(cis-p-coumaroyl)-5-O-glucoside, delphinidin-3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside, petunidin-3-O-rutinoside (caffeoyl)-5-O-glucoside, petunidin-3-O-rutinoside (cis-p-coumaroyl)-5-O-glucoside, petunidin-3-O-rutinoside (trans-p-coumaroyl acid)-5-O-glucoside, petunidin-3-O-glucoside -5-O-glucoside, petunidin-3-O-rutinoside (feruloyl)-5-O-glucoside, and malvidin-3-O-rutinoside (cis-p-coumaroyl)-5-O-glucoside  supplementation reduced the pathology and symptoms of InfV infection through attenuation of inflammatory cytokines, such as TNFα and IL-6, in the mice that were nourished with LWB for four weeks and thereupon treated with InfV A while maintaining the same diet. However, it raised IL-2 through increasing the systemic lymphocyte T  and were revealed by A. melanocarpa fruit juice (AMFJ) were cyanidins, which were mainly in the form of aglycone  is a rich source of anthocyanins -5-glucoside and peonidin (aglycon) (Thymus vulgaris), rosemary , prunella (Prunella vulgaris), peppermint (Mentha x piperita), lemon balm , and sage  against HSV. The inhibitory activities of phenolic compounds against HSV type 1 (HSV-1), type 2 (HSV-2), and HSV-1 acyclovir-resistant strain (ACVres) were assayed in vitro, which indicated a high antiviral effect against three strains of HSV time-dependently. According to the results, the extracts had efficacy in anti-HSV before adsorption stage and, in contrast, had no efficacy in the replication stage  . In theion stage . TherefoSambucus nigra) extract. From the pharmacological mechanistic point of view and according to the molecular docking of this compound, it bound with the 430-cavity adjacent to the residues of InfV neuraminidase. Since this antiviral compound bound remote from Asp 151 and Glu 119, two residues known to regulate the neuraminidase resistance, this compound was shown to have potential as a new class of antiviral drugs against the InfV without this susceptibility , dengue virus (DENV), and zika virus (ZIKV). The results showed that delphinidin reduced WNV. Further assays discovered that delphinidin generally interfered with the attachment, adsorption, and entrance stages of the viral cycle and has a direct effect on the virus particles executing a virucidal activity. The same inhibitory activity of WNV variants was reported with acidic pH variation in the endosomal system for viral fusion. It was also shown that anthocyanins exhibiting antiviral activity against WNV is initiated by a virucidal activity instead of an inhibitory activity of viral fusion that was dependent on pH. Delphinidin also decreased the viral activity of DENV and ZIKV. Finally, delphinidin was reported to disrupt the viral activity of three viruses of the flaviviruses family in the cell culture .Swaminathan et al. assessed the activity of cyanidin, delphinidin, pelargonidin as three anthocyanins against 430-cavity of InfV neuraminidase and their binding site using molecular docking and mass spectrometry. Regardless of structural similarity, these compounds had differences not only in the position but also in the number of OH groups in the benzyl substituent connected to the chromenylium ring. The precise distinction in the binding site features was exhibited by molecular docking and mass spectrometry. Delphinidin and cyanidin, with the most number of OH substituents, bound firmly and had more inhibitory properties than pelargonidin with only one OH substituent at 4´ position. Finally, the position and number of OH groups of anthocyanins were shown to affect their antiviral activity .Finally, according to the importance of purified antiviral anthocyanin in combating viral diseases, clarifying the exact pharmacological mechanisms of action could pave the road for the treatment of viral infections.Sambucus nigra L.), has been used in treating the viral disease like colds and flu from past to present (Hippophaë rhamnoides L. (sea buckthorn) berries indicated an immunomodulatory activity and positive effects on health  and 2.5 h (urine) . The metvia exclusive targeting of specific sites , carboxymethyl chitosan (CMC) and β-Lactoglobulin (β-Lg) were provided. The results showed that nanocomplexes encapsulation significantly improved the stability and bioavailability of anthocyanins in simulated GIT .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.+/taurocholate co-transporting polypeptide; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; InfV, Influenza virus; HSV-1/2, herpes simplex virus-1/2; NtRTIs, nucleotide reverse transcriptase inhibitors; NRTIs, nucleoside reverse transcriptase inhibitors; NNRTIs, non-nucleoside reverse transcriptase inhibitors; ORF, open reading frame; HAART, highly active antiretroviral therapy; NA, neuraminidase; NAIs, neuraminidase inhibitors; gp, glycoprotein; TLR-3 or 7, toll-like receptor-3 or 7; IFNγ, interferon-gamma; PD-1, programmed death 1, CTLA-4, cytotoxic T-lymphocyte antigen 4; TIM-3, T-cell immunoglobulin and mucin domain-containing molecule 3; RNAi, RNA interference; siRNAs, small interfering RNAs; DDX3, Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3; NS3h, non-structural protein 3 helicase; NS5B, non-structural protein 5B; VZV, varicella-zoster virus; NF-кB, nuclear factor-kappa B; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, EMCv, encephalomyocarditis virus; HHV-1, human herpes virus type 1; VACV-WR, vaccinia virus strains Western Reserve; SNS, Solanum nigrum seeds; TA, total anthocyanins content; RV, rabies virus, HPLC-DAD, High-performance liquid chromatography-diode array detector, CV-B1, coxsackievirus B1; PV-1 poliovirus type 1; HRSV-A2, human respiratory syncytial virus A2; FCV-F9, feline calicivirus-F9; MNV-1, murine norovirus-1; FBVS, foodborne virus surrogates; RCS, black raspberry seed; C3G, cyanidin-3-glucoside; MA, Morus alba; MAJ, Morus alba juice; MAS, Morus alba seed; Ad36, adenovirus 36; MCP-1, monocyte chemoattractant protein‐1; CJ, cranberry juice; BT2, bacteriophage T2; BT4, bacteriophage T4; GSE, grape seed extract; AJ, apple juice; GJ, gastric juice; CS, cocoa seeds; NK, natural killer cell; LWB, lacto-wolfberry; AMFJ, Aronia melanocarpa fruit juice; PJ, pomegranate juice; ACVres, HSV-1 acyclovir-resistant strain; WNV, west nile virus; DENV, dengue virus; ZIK, Zika virus; EBE, elderberry extract; CRP, c-reactive protein; Bid, two times a day; Qid, four times a day; GIT, gastrointestinal tract; CHC, chitosan hydrochloride; CMC, carboxymethyl chitosan; β-Lg, β-Lactoglobulin; CH, chitosan; CS, chondroitin sulfate; PLGA, poly lactic-co-glycolic acid; PEG, polyethylene glycol.HIV, human immunodeficiency virus; RSV, respiratory syncytial virus; NTCP, Na


Honey bee colonies suffer from various pathogens, including honey bee viruses. About 24 viruses have been reported so far. However, six of them are considered to cause severe infection which inflicts heavy losses on beekeeping. The aim of this study was to investigate incidence of six honey bee viruses: deformed wing virus (DWV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), sacbrood virus (SBV), kashmir bee virus (KBV), and black queen cell virus (BQCV) by a reverse transcription polymerase chain reaction (RT-PCR). A total of 250 adult honey bee samples were obtained from 50 colonies from eight apiaries situated in three different parts of the country . The results showed the highest prevalence of DWV followed by SBV and ABPV, and one case of BQCV. A comparison with homology sequences available in GenBank was performed by phylogenetic analysis, and phylogenetic relationships were discussed in the context of newly described genotypes in the uninvestigated South Eastern region of Europe. In conclusion, the present study has been the first to provide sequencing data and phylogenetics analyses of some honey bee viruses in Bulgaria. Apis mellifera L., Hymenoptera: Apidae) is a species of crucial economic, agricultural and environmental importance. Many factors lead to dramatic reduction of bee populations, reduction of bee production, and thus to disruption of the process of pollination of farming crops and wild plants (including certain endemic ones). Among such factors are known diseases with economic significance affecting the bees and their brood, such as bacterial, parasitic and viral , and Indian origin (BQCV and SBV) of most described honey bee viruses as well as their introduction in the West direction toward Europe and worldwide .Despite the importance of viral diseases on honey bee colonies health, the sequencing and phylogenetic data related to the origin and spreading of viruses on the Balkan Peninsula are scarce .Bulgaria as a part of the Balkans and a bordering region between the Middle East and Europe is an interesting point for understanding the dissemination and introduction of honey bee viruses. Therefore, in this study, we have reported the first survey of six honey bee viruses  in honey bee colonies in Bulgaria. A phylogenetic analysis was performed to reveal the geographic distribution of different virus strains.n = 20), Sofia district , and Smolyan district , , while in the last region a native Bulgarian honey bee named A. m. rodopica exists. There was no bias concerning honey bee samples collection.Samples of adult worker bees were collected from 50 colonies originating from eight apiaries in three locations in Bulgaria: Rousse district , , during While in the mountainous part of the country (Smolyan district) the honey and pollen diet is very diverse (consisting mainly of meadow flora), in the flat regions (Rousse and Sofia district) there is monofloral honey and pollen . The apiary density in the mountainous part of the country is from 5 to 10 km away, while in the flat regions of the country this distance is rather shorter (up to 1 km). In the Rhodope mountains, apiaries consist of 10 to 25 hives, while in flat regions the number of hives per apiary is up to 100.The presence of the Varroa mite was not checked for each colony at the time of sampling, but all honey bee colonies are treated with acaricides during the autumn period.A. m. carnica, A. m. ligustica and A. m. macedonica have been imported from Central Europe and reared mainly in the flat regions in the country. In the Rhodope mountains, the local honey bees (A. m. rodopica) are undergoing selection control as part of the national biodiversity conservation. Beekeepers are encouraged to rear only the local honey bee, and crossbreeding with other races is not allowed.Different subspecies like From each colony, five adult bees  were collected individually, following the method described by The experimental design had been carried out under permissions and the guidelines of the Bulgarian Academy of Sciences and the Bulgarian Ministry of Environment and Waters (no. 627/30.03.2015).20 primers  according to the manufacturer’s instructions.The frozen samples were crushed in a mortar and were homogenized in an RL lysis buffer . After homogenization, the samples were centrifuged for 3 min at 15,000× g to remove unhomogenized particles. An aliquot of supernatant was used for the extraction of total RNA according to the manufacturer’s recommendations. The quality of the extracted total RNA was checked by electrophoresis and spectrophotometry. An average of 2 µg of the total RNA was used for copy DNA (cDNA) synthesis using Oligo(dT)The PCR mixture contained 25 µL of NZYTaq 2× Colourless Master Mix , 0.4 µM of each virus specific primer (FOR/REV), 1 µL of template cDNA in a total volume of 50 µL. All RT-PCR amplifications were carried out using a Little Genius thermocycler  under the following conditions: initial denaturation at 94 °C for 5 min; 35 cycles  and final extension at 72 °C for 10 min. The PCR products were visualized on 1% agarose gel with GreenSafe Premium . The fragment size was determined using Gene-Ruler™ 100 bp Ladder Plus . The primers used for detection of the viruses  had been described by AF150629, .All obtained DNA sequences were manually edited and aligned with reference complete viral genomes  by usin_003784, . The obtg MUSCLE , and theg MUSCLE  in each http://vassarstats.net/prop1.html), , .The data for RT-PCR detection of six bee viruses in the 50 hives are shown in Varroa destructor, which might explain why this virus was not detected in Varroa-free colonies in Australia (MG599458–MG599464). In the Balkans, this virus was found in Greece , and three in Spanish isolates   . The EurQ385502) . The phyQ385502) .It may be concluded that the Bulgarian DWV sequences shared a common origin with the Turkish virus strains as well as with those in other Mediterranean countries.MG649495–MG649499. To date, in the Balkans, this virus has been detected once in Greece  and Australia . The esY465679, . SBV froY465679,   .The phylogenetic analysis of the Balkan SBV revealed new European genotypes, introduced in a different way in Europe.MG649502) showed the highest homology with European BQCV strains from the Czech Republic (95%) and Hungary (93%)  , Fig. 3MG649502 ry (93%) . The lasry (93%) . The resry (93%) . Considery (93%) .In all clusters there exist different BQCV genotypes from China and Korea. This may be explained by the fact that there are at least three virus strains that infected honey bees in Europe imported from Central Asia .It is obvious that the genetic diversity of viruses in Europe is supported by the geographic spreading in Europe, possibly by independent transfer of infection without co-infections. Our data enlarge the geographic spreading of CSE BQCV strains in South-Eastern direction.V. destructor  with ABPV genotypes from Hungary (V. destructor (Varroa (ABPV is also frequently found in honey bee colonies. Apparently, this virus plays a role in cases of a sudden collapse of the honey bee and is transmitted by the parasitic mite structor . In the structor , Serbia structor  as well structor  as descrstructor  and sequ Hungary , (Bakony Hungary  and from Hungary . The est Hungary . Up to n Hungary . There a Hungary . The UK  Hungary . To datestructor . This vi (Varroa .Our findings have enlarged the existing data on the geographic spreading of ABPV up to South-Eastern Europe, where this virus was detected for the first time.We suggest that the different levels of virus infections may be partly explained by the climatic conditions in the mountainous and the plain regions. The Rhodope Mountains climate is rather colder than the other two investigated regions and, possibly, with a lower rate of infection with Varroa mites. This conclusion was supported by other studies which have observed the lowest infection with Varroa, Nosema and Virus in the regions with colder climate in Europe .The present study revealed the first sequenced data reported for the Balkan Peninsula and phylogenetic data about geographic distribution and genetic diversity including new data sets in the Balkans. The phylogenetic analysis of ABPV and BQCV has shown a close relation to Central-European virus strains that differ from West-European and Polish genotypes. The Bulgarian samples of SBV seem to form a new distinct clade within the European strains with a higher homology to the isolates from the Pacific region. The multiple genotypes of DWV are present in Bulgaria and are most closely related to the Mediterranean and Turkish DWV strains.


Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural elements involved in mediating the viral life cycle, including cyclisation, replication, and encapsidation. Here we report on a comparative genomics approach to characterize evolutionarily conserved RNAs in the 3 Flaviviruses are small, single-stranded positive-sense RNA viruses that are typically transmitted between arthropod vectors and vertebrate hosts. They are endemic in tropic and sub-tropic regions and represent a global health threat, although humans are considered dead end hosts in many cases.Flavivirus within the Flaviviridae family comprises more than 70 species, which are organized into four groups, each with a specific host association: Mosquito-borne flaviviruses (MBFVs) and tick-borne flaviviruses (TBFVs) spread between vertebrate  and invertebrate (mosquitoes and ticks) hosts, whereas insect-specific flaviviruses (ISFVs) replicate specifically in mosquitoes and no-known-vector flaviviruses (NKVs) have only been found in rodents and bats, respectively. This natural host-range-based classification is in good agreement with sequence-based phylogenetic clustering, mainly because all flaviviruses share a common genome organization [The genus nization . Conversnization  and trannization  are fundEmerging and re-emerging MBFVs such as Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Yellow fever virus (YFV) or Zika virus (ZIKV) are the causative agents of large-scale outbreaks that result in millions of human and veterinary infections every year . LikewisTBFVs form a monophyletic group consisting of a single serocomplex, although pathology and clinical manifestations vary among different viruses. They comprise more than a dozen of recognized species and separate into three groups: Mammalian tick-borne flaviviruses (M-TBFV), seabird tick-borne flaviviruses (S-TBFV) and the Kadam virus group. See  for a coDiptera and are typically divided into two groups [Aedes spp. mosquitoes and the other associated with Culex spp. mosquitoes, respectively [ISFVs naturally infect hematophagous o groups : Classicectively . They laectively . Insect-ectively . In thisectively .NKVs represent an ecologically and phylogenetically diverse set of viruses which have been isolated exclusively from vertebrates (mainly bats and rodents), without evidence for transmission by arthropod vectors. They form a non-monophyletic group among flaviviruses and are typically divided into bat- (B-NKV) and rodent-associated (R-NKV) groups, see Conserved RNA structures in the untranslated regions (UTRs) of RNA viruses are of particular interest because they mediate the viral life cycle by promoting or enhancing replication, as proposed for elements in both 5′UTRs  and 3′UTs [′UTRs ,16,17,18s [′UTRs . Tandem wo hosts . Likewiswo hosts .Viral RNA genomes are different from procaryotic and eucaryotic mRNA. In addition to coding for and regulating the viral machinery, viral genomic RNA (gRNA) exhibits functional regions that act upon different stages of the viral life cycle. The 10-12kB flaviviral gRNA is capped, but non-polyadenylated and encodes a single open reading frame (ORF). Upon translation, a polyprotein is produced, which is then cleaved by viral and cellular enzymes into structural and non-structural proteins . The ORFUpon flavivirus infection, accumulation of both gRNA as well as viral long non-coding RNAs (lncRNAs) is observed. These lncRNAs, which have been referred to as subgenomic flaviviral RNAs (sfRNAs)  are stabachinery ,31. Mechpossible . In partpossible .Xrn1 stalling results in dysregulation of cellular function with the aim of promoting viral infections. In this regard, functions of sfRNA in modulating cellular mRNA decay and RNAi pathways  as well The genomic architecture of flaviviruses has been extensively studied to understand the molecular principles required for sfRNA production. Chemical and enzymatic probing methods , togetheBFVs WNV , YFV [39BFVs WNV , Murray BFVs WNV , ZIKV [4BFVs WNV  and receBFVs WNV , highligur group ,45,46,47ur group . To furt(NCBI) refseq (https://www.ncbi.nlm.nih.gov/refseq/) and genbank (https://www.ncbi.nlm.nih.gov/genbank/) databases on 28 May 2018. We downloaded all complete viral genomes under taxonomy ID 11051 (genus Flavivirus) and filtered for TBFV, ISFV and NKV species listed in refseq annotation was not available for a species, we selected the longest complete genome from the genbank set as representative sequence. In total, the data set is comprised of 86 ISFV, 275 TBFV and 27 NKV isolates, respectively. The number of isolates with available 3Viral genome data for the present study were obtained from the public National Center for Biotechnology Information Flavivirus (MAFFT[iq-tree [The polyprotein/coding sequence (CDS) regions of most flaviviruses can be aligned consistently, however, UTRs typically show large variance both in length and sequence composition, rendering them ill-suited for phylogeny reconstruction. A phylogeny of all members of the genus avivirus  was therus , i.e., statistical models of RNA structure that extend classic Hidden-Markov-Models (HMMs) to simultaneously represent sequence and secondary structure. CMs, as implemented in the  package  allow foomycetes .locARNA [infernal default parameters are typically not optimal. Adjusting parameters, in particular disabling both heuristic filtering and local end detection, however, allowed our CMs to find homologs with strongly conserved secondary structures in presence of large sequence deviation from the original sequence the CM was built from. Likewise, cmsearch E-values turned out unsuitable for assessing hit quality in case of major sequence divergence. We therefore employed a cutoff approach, requiring a hit to form at least 75% of all base pairs listed in the CM in order to be considered significant.Here, structural multiple sequence alignments of the viral 3 locARNA  and CMs  locARNA . All 3′UlocARNA-generated structural alignments of full UTR sequences were cut manually into blocks corresponding to conserved secondary structures. Alternatively, we employed RNALalifold from the ViennaRNA package [Beside characterization of RNAs with homology to known structurally conserved elements, we aimed at identifying novel elements, considering both thermodynamic stability and sequence covariation as evolutionary traits. In this line,  package  to compuViennaRNA scripting language interface for thermodynamics calculations, the ViennaNGS [RNAaliSplit package [R2R [ETE3 framework [The above approach is implemented as a set of custom Perl and Python scripts for semi-automatic characterization and annotation of conserved RNAs in viral UTR sequences. Internally, these scripts build on the iennaNGS  suite fo package  for spliage [R2R  for visuramework  for treeSeveral flaviviruses have previously been studied in great detail, yielding a varied landscape of repeated RNA sequence and structure elements within the 3ications ,58,59. MThe comparative approach applied in the present study outperforms single sequence predictions by considering consensus structures formed by all sequences. This allows us not only to confirm previously described RNA structures but also to elucidate hitherto unrecognized tandem repeats in many species. In this line our results can help in understanding the complex evolution of flavivirus 3RNALalifold calculations were used as seeds for identification of conserved RNA structures. Based on recent experimental evidence for the existence of xrRNAs in TBFVs, ISFVs and NKVs, and previously characterized conserved RNA elements in flaviviral 3The four ecologic groups of flaviviruses show a varied 3iviruses . The eleMacFadden et al.  suggeste species a.The full structural alignment of the 3and OHFV ,59. We kFV [′UTR d.locARNA alignment and subsequent RNAalifold consensus structure prediction indicates strong secondary structure conservation with frequent structure-conserving sequence covariations. Taken together, this suggests good evidence that respective regions in TBFVs harbor a putatively structured and functional xrRNA-like RNA , Quang Binh virus (QBV), Mosquito flavivirus (MSFV), Palm Creek virus (PCV), Culex theileri flavivirus (CTFV) as well as a few other species with only partial genome sequence availability  and is ase pairs b,c. We t of CxFV  with theInterestingly, a screen of all available CMs in Parramatta River virus (PaRV) revealed five xrRNA-like elements a, with eConversely, the 3Our data suggests that the 3to TBFVs , do not to TBFVs  we const species . For CLBIsolated almost exclusively from mosquitoes, dISFVs do not seem to infect vertebrate cells, despite their phylogenetic proximity to MBFVs . This asAn unusual species within this group is Ecuador Paraiso Escondido virus (EPEV), which has been isolated from New World sandflies and has been classified as insect-specific virus. EPEV phylogenetically appears at the root of the Entebbe bat virus group (ENTVG), a clade comprised of the three NKVs Entebbe bat virus (ENTV), Sokoluk virus (SOKV) and Yokose virus (YOKV). While all of these viruses contain homologs of conserved stem-loop (SL) and dumbbell (DB) elements found in MBFVs, ENTVG species may have lost their vector dependence .Rather than forming a monophyletic group, the no-known-vector flaviviruses can be separated into two distinct lineages, which are closely related to either TBFVs or MBFVs, respectively . Two addWe found multiple hits of this CM at various loci within the 3itecture a. Surprielements .Conversely, there seems to be no generally conserved 3ted NKVs c. While Earlier work suggested that xrRNAs from TBFVs and tick-borne related NKVs fall into a more general structural class of xrRNAs . FollowiStructural alignment and consensus structure prediction revealed all high confidence hits to fold into a common secondary structure a,b. WhilWe further investigated whether any high confidence hits from I.xrRNA1/2 in cISFVs could be aligned to the generalized xrRNA model. Although both cISFV xrRNAs c bear soIn addition to learning xrRNA features, a more generalized CM enabled us to detect xrRNA-like structures  that could not be found previously.Flavivirus genus [Flaviviridae family, however, with major differences in xrRNA structure and sfRNA characteristics. While MBFV produce a 300-500nt sfRNA that corresponds to degradation products of the gRNA 3Mediated gRNA decay in the form of exoribonuclease resistance seems to be a pervasive strategy employed by viruses to circumvent host immune responses. Evidence of sfRNA production following incomplete Xrn1 degradation has not only been observed in different members of the us genus , but alsral gRNA .Bunyaviridae and Arenavividae [Tombusuviridae and Luteoviridae families [Moreover, recent studies have identified xrRNA functionality in several phylogenetically distant RNA viruses, such as animal-infecting, segmented viruses of the avividae  familiesfamilies ,66. The families  could shfamilies ,68 proviRepeated RNA elements appear to be a hallmark of flavivirus 3In this contribution we set out to identify homologs of known exoribonuclease stalling elements and novel conserved structures. To this end, we computationally characterized homologs of experimentally verified xrRNA in tick-borne and no-known vector viruses that seem to form a coherent class of RNA structures with capability to stall exoribonucleases . Likewise, we identified another class of xrRNAs in classic insect-specific flaviviruses (I.xrRNA1/2) which appears to be only distantly related to the former class. In the same line, we predicted a set of novel conserved elements in cISFVs that appear in quadruples and do not coincide with other insect-specific elements .Aedes spp. associated xrRNAs fold into a similar Y-shaped substructure, although the exact fold varies significantly among individual species.While we did not focus on studying the evolutionary history of these groups of elements in detail, our data suggests that many elements share ancestral roots. This is supported by the observation that at least the tick-borne, no-known-vector and https://github.com/mtw/ITNFV-Data.We compiled a set of covariance models that can be used for rapid screening assays in the identification and characterization of novel flaviviruses. All models are available from GitHub via A major problem is the limited availability of diverse 3


Addition of ispaghula in alginate showed a significant increase (p < 0.05) in the drug loading (42% w/w) and entrapment efficiency (84% w/w). Fourier-transform infrared spectroscopy confirmed the presence of ispaghula and zein coating in the alginate beads as well as the ibuprofen loading. Scanning electron microscopy revealed better spherical geometry in the beads with ispaghula. The surface morphology of the uncoated beads was rough due to crystalline and surface drug. The zein coating has produced a smoother surface and particle adhesion. Differential scanning calorimetry has shown a reduction in drug crystallinity. Alginate beads extended the drug release for 4 h and the presence of zein extended the release for 6 h.The purpose behind the work was to fabricate alginate beads with better drug loading and extended drug release. Ispaghula was used to enhance the drug loading while zein was employed to extend the drug release. Ibuprofen was employed as a model drug in this study. Ibuprofen-loaded alginate beads with and without ispaghula were prepared using vibration technology and coated with zein. The beads prepared with alginate alone were shown to have loading and entrapment efficiencies of 35% and 70%  As crosslinking progresses, the drug tends to come out from the alginate beads due to shrinkage of gel network [Sodium alginate has been extensively used in drug delivery because of its biocompatibility and gel-forming ability with divalent ions . Beads c2) , ispaghu2) , locust 2)  and star2)  in order network .Plantago ovata, and is a hydrophilic material capable of retaining water. The ispaghula husk contains mucilage, and its hydrolysis yields d-xylose, l-arabinose, d-galactose and d-galacturonic acid [In the present investigation, we have studied the impact of the addition of ispaghula on the physicochemical properties of alginate beads. Ispaghula is a husk obtained from nic acid . Though nic acid ,10, thernic acid ,12. The nic acid . It is lPlantago ovata) was purchased from Sidhpur Sat-Isapgol factory, Sidhpur, Gujarat, India. Ibuprofen was purchased from Lianyungang Zhongyi International Trade Co. Ltd. . All other chemicals are analytical grade and obtained from RM Chemicals, Malaysia.Sodium alginate from brown algae and zein from maize were purchased from Sigma-Aldrich, Malaysia. Ispaghula  fitted with a nozzle (750 µm) and a vibration frequency of 600 Hz with an electrode voltage of 1000 mV. The air pressure was maintained between 500 to 700 mbar to generate beads. A curing time of 30 min was allowed for the crosslinking of the beads. After that, the beads were separated using a nylon mesh and washed with water several times to remove unreacted CaCl2 and finally dried at 40 °C until constant weight. Dried beads were kept in glass vials and stored in the desiccator at room temperature.5 g of sodium alginate was dissolved in 250 mL of water using magnetic stirring at room temperature (100 rpm). 5 g of ibuprofen was taken in a mortar; a small amount of alginate solution was added and triturated well to form a lump-free thick paste. More amounts of alginate solution were added (approximately 25 to 30 mL) to make a pourable mixture. While stirring (200 rpm), ibuprofen–alginate mixture was added to the rest of the alginate solution to form a lump-free suspension. The suspension was immediately dripped into 5% 5 g of sodium alginate was dissolved in 150 mL of water using magnetic stirring. 500 mg of ispaghula husk was stirred in 100 mL of water overnight using a magnetic stirrer at room temperature. The viscous solution produced was filtered through a muslin cloth to remove debris and undissolved material. The clear viscous ispaghula solution was added to 150 mL of alginate solution and mixed well. The beads were prepared as explained in the previous section.w/v solution of zein was prepared in 80% v/v of ethanol. 1 g of respective beads was placed on a nylon mesh strainer (14 cm diameter) with a holder to produce a uniform layer. 5 mL of zein solution was sprayed in small quantities at the surface of beads using a manual atomizer. The spraying was done to wet the entire surface of the beads and the coating was dried by blowing hot air (40–50 °C) using a conventional hair dryer . The beads were rotated by gently shaking the strainer and spraying/drying was continued to the completion of 5 mL coating solution in order to produce a uniform coating over the surface of the beads.A 10% Ibuprofen content in the microcapsules was determined via an extraction method. 300 mg of beads was immersed in 100 mL of phosphate buffer (pH 6.8) at room temperature overnight. The solution was subjected to sonication with a Hielscher UIP500hd ultrasonic homogeniser at 600 Hertz amplitude for 1 min to break the beads and dissolve the drug. The resulting solution was filtered using Whatman filter paper No. 40. The filtrates were made up to 100 mL with phosphate buffer (pH 6.8) and estimated with UV–vis spectrophotometer  at 264 nm. The theoretical percentage of drug loading (TDL) was calculated using the following equations .TDL = (The percentage of drug entrapment efficiency (DEE) was calculated according to the following equation.−1 in a Varian 640-IR FTIR  spectrophotometer using an attenuated total reflection accessory.The beads were powdered using a mortar and pestle. The infrared spectra of the powdered samples were measured between 600 and 3800 cmA field emission scanning electron microscope  was used to observe surface morphology and the shape of the beads. Cross-section of the beads was done by using a sharp stationary blade after soaking the beads in water for about an hour. The samples were fixed in stubs using double-faced copper adhesive tape and were coated with a thin layer of platinum using a Q150R S rotary-pumped sputter coating system  before being observed.Differential scanning calorimetry (DSC) analyses were performed in a temperature range from 50 to 200 °C on a DSC 1 . Approximately 5 mg of each respective sample was weighed and hermetically sealed into the aluminium crucible. The heating rate was 10 °C/min, and the nitrogen flow rate was 50 mL/min.The release studies were carried out to find the ability of the zein coating to extend the drug release from the beads. Ibuprofen is poorly soluble in acidic pH and hence pH 6.8 is used as the dissolution medium. The dissolution studies were carried out in a USP (United States Pharmacopoeia) dissolution tester  by a paddle method and using 900 mL of phosphate buffer pH 6.8 as the release medium. Beads equivalent to 200 mg of drug were individually filled in hard gelatin capsules. Capsules were dropped in each vessel, and the dissolution rates were measured at 37 ± 0.5 °C and 50 rpm. The samples were withdrawn at regular intervals, and the drug content was estimated at 264 nm using UV-visible spectrophotometer.p-value of 0.05 was identified as statistically significant. Uncorrected Fisher’s LSD (Least Significant Difference) multiple pairwise comparisons of the data were conducted to determine the statistical significance between the beads.Drug loading, entrapment efficiency and drug release data were analysed using repeated-measures one-way ANOVA assuming Gaussian distribution and using the Geisser–Greenhouse correction . The w/w, respectively. As shown in p < 0.05) in the drug loading (41.8 ± 1.2% w/w) and entrapment efficiency (83.7 ± 2.4% w/w). Further, multiple pairwise comparisons confirmed the significant increase (p < 0.05) in drug loading and entrapment efficiency of alginate beads in presence of ispaghula. It could be due to increased viscosity of the system which prevents drug loss during the crosslinking process. A similar effect was reported [The preparation conditions were optimised after making several trial batches. The beads prepared with alginate alone were shown to have an entrapment and loading efficiency of 70.3 ± 3.3% and 35.2 ± 1.6% reported  for the −1 indicated the stretching vibrations of asymmetric and symmetric C=O in the carboxylic acid groups in alginate molecules. A sharp peak at 1030 cm−1 and a small peak at 2922 cm−1 reveal C–O stretching and C–H aliphatic stretching vibrations of sodium alginate. Absorption bands of the FT–IR spectrum of ispaghula  spectrum of alginate shows characteristic peaks A reportespaghula B appearemolecule . The FT–molecule . The FT–−1, corresponding to the stretching vibration of the cyclic dimerized hydroxyl groups. The bending –OH absorption at 1231 cm−1 reveals free hydroxyl groups, and a peak at 1185 cm−1 indicates C–O stretching vibrations. Characteristic peaks at 1710 and 2945 cm−1 were observed for C=O and –OH stretching, respectively, for the carboxylic acid group in the ibuprofen. The peaks in 1508, 1456 and 780 cm−1 are associated with the vibration in the skeleton of benzene. These peaks were in accordance with our previous publication [The FT–IR spectrum of ibuprofen A has sholication . All theThe surfaces of ibuprofen-loaded alginate and alginate–ispaghula beads were rough due to the presence of crystalline drugs A–D. The DSC thermogram of ibuprofen  has showp < 0.05). Further pairwise comparisons using uncorrected Fisher’s LSD indicated the significant difference between the release pattern of uncoated and coated alginate beads. However, the release profile of alginate and alginate–ispaghula beads has shown insignificant difference (p > 0.05).As shown in Alginate beads are one of the simple ways to encapsulate pharmaceuticals for extended drug delivery. However, alginate beads possess problems such as low drug loading and faster drug release due to their hydrophilic nature. Alteration of surface by hydrophobic materials might be useful in obtaining a slower drug release. An attempt is made in the present investigation to improve drug loading and sustain drug release in the alginate beads. Addition of ispaghula in alginate beads significantly increases the drug loading and entrapment efficiency. This method could be useful in enhancing the drug loading of water-soluble drugs in alginate beads. Further, coating alginate beads using zein could sustain the drug for an extended period.