Met (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5′- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNA The detection limit was calculated based on the linear range (0–500 nM eMet transcript), with concentrations of 1000 nM eMet excluded due to non-linearity. Data are presented as the means ± SEM of three independent experiments.2. HeLa cells were plated on collagen-coated glass-based dish. After 2 d, the medium was exchanged for fresh medium without phenol red, and the cells were then microinjected with the reaction mixture specified below containing 20 μM Cy5 C5 maleimide  as an injection marker. For the experiment, 10 μM eMet or iMet transcripts was renatured by incubating for 1 min at 85°C in buffer D containing 20% Dulbecco’s phosphate buffer saline (-) and 1 mM MgCl2, followed by slow cooling to 4°C. Each renatured tRNA transcript (10 μM) was incubated with 1 μM ISMBe1 in buffer D at 37°C for 2 h. Microinjections were performed using Eppendorf Femtojet microinjection equipment  and Femtotip microinjection capillary tips at 70 hPa. All imaging experiments were performed using an Olympus FV1200 confocal microscope . The fluorescence intensities of ISMBe1 and Cy5 were quantified by using FV10-ASW 4.2 software (Olympus). Data are presented as the means ± SEM of four independent experiments. p-values were calculated using Student’s t-test.HeLa cells were obtained from RIKEN BRC which is participating the National Bio-Resource Project of the MEXT, Japan. HeLa cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (Sigma) and 1% antibiotic-antimycotic solution  at 37°C under an atmosphere of 5% COTo detect endogenous eMet in living cells, the cells were microinjected with scrambled ISMB or ISMBe1. Imaging experiments were performed using an Olympus IX51 inverted microscope. The fluorescence intensities of scrambled ISMB and ISMBe1 were quantified using FV10-ASW 4.2 software. Data are the means ± SEM of 7 cells for scrambled ISMB and 10 cells for ISMBe1.2. Then, tRNA molecules were eluted with a linear gradient of KCl (0.3–2 M) in buffer E, and the fractions were analyzed by 7 M urea 8% PAGE. The eMet-specific radioisotope-labeled probe was prepared by using [γ-32P]-ATP  and T4 polynucleotide kinase (Toyobo). The sequence of the eMet-specific probe was as follows: 5ʹ-ATT TTT GGT GCC CCG TGT GAG GAT CGA AC-3ʹ. The tRNA mixture (2 μg) containing each fraction eluted from Q-Sepharose was hybridized with 8.5 pmol eMet-specific radioisotope-labeled probe in a buffer containing 10 mM Tris-HCl (pH 6.8) and 0.5 mM MgCl2 for 10 min at 95°C, followed by slow cooling to 25°C. The reaction mixture was resolved on 8% native PAGE, and then eMet/probe complex was detected by using BIOMAX MS Film (Sigma). The fraction containing mature eMet was collected and the concentration of mature eMet was calculated using ImageJ software , by comparison with the band intensity of 8.5 pmol eMet transcript/probe complex.HeLa cells were grown until 70–80% confluence. The cells were suspended in solution D  for total RNA extraction. Proteins in the total RNA fraction were removed by phenol-saturated sodium acetate (pH 5.2). Total RNA was collected by precipitation with 2-propanol. It was then applied to a Q-Sepharose column  equilibrated with buffer E containing 25 mM Tris-HCl (pH 7.4), 150 mM KCl, and 2.5 mM MgClRenatured mature eMet (500 nM) was incubated with 50 nM ISMBe in buffer C at 37°C for 2 h. The fluorescence spectra were acquired as described above. Data are presented as the means ± SEM of three independent experiments.Secondary structure of the eMet transcript was shown in Lys3 transcript (15-min reaction) was lower than that of ISMBe and ISMBi in the presence of tRNA transcript (2-h reaction)  Figs  and S2B on) Figs . In the on) Figs . By conton) Figs . These oTo determine the detection limit of ISMBe, the linearity of fluorescent response of each ISMBe molecule to a target transcript was examined. The fluorescence of each ISMBe molecule responded linearly to the increasing concentrations of the eMet transcript up to 500 nM . The detTo confirm that ISMBe specifically hybridized with the eMet transcript, eMet transcripts with one- and two-base mismatches were prepared . In addiAs shown in To detect endogenous eMet in living cells, ISMBe1 and scrambled ISMB were introduced into HeLa cells by microinjection. The fluorescence intensities of ISMBe1 and scrambled ISMB in living cells were similar at 120 min . Next, tWe were concerned that multiple nucleotide modifications of mature eMet might interfere with the hybridization of ISMBe1 and mature eMet , since tTo investigate whether ISMBe could detect mature eMet, the fluorescence intensities of ISMBe molecules were determined in the presence of mature eMet from fraction 13. The fluorescence intensity of scrambled ISMB in the presence of mature eMet remained at the background level . The fluIn the current study, to determine the effective target region of ISMB for detecting tRNA molecules, eight ISMB molecules targeted to the representative tRNA regions were designed. We showed that ISMBe molecules, particularly ISMBe1, targeted to the 5ʹ-region inclusive of the D arm, could be used to detect eMet transcript and mature eMet Figs . In addiLys3 transcript than those targeted to the anticodon arm and the T arm .,27.26,27he T arm . Furtherhe T arm ,29. ThisPreviously, we showed that the fluorescence increase of conventional MB targeted to the T arm, excluding the CCA sequence, is the lowest among the analyzed probes targeting various tRNA regions . On the Asp (Tm = 73°C) from yeast is more stable than the tRNAAsp transcript (Tm = 65°C) [As shown in Figs  = 65°C) . TherefoThe current study highlights some limitations of the devised method. First, ISMBe1 and ISMBe2 detected eMet transcripts with one-base mismatches . In addiIn summary, we demonstrated that ISMB targeted to the D arm region is the most suitable probe for detecting tRNA transcripts and mature tRNA. While the ISMB/short RNA complex was rapidly formed, the formation of the tRNA/ISMB complex required a relatively long time. Until now, tRNA localization has been mainly analyzed by the FISH method and fluorescence labeling of tRNA in mammalian cells ,17,38–40S1 Fig(TIF)Click here for additional data file.S2 Fig(A) The cloverleaf structure of iMet transcript is shown. Black lines are target regions of each designed ISMBi molecule. (B) The fluorescence intensities of ISMBi in the absence of target RNA (white bar) or in the presence of eMet transcript (black bar). The fluorescence intensities were normalized to digested ISMBe1. Data are the means ± SEM of three independent experiments.(TIF)Click here for additional data file.S3 Fig(A) Image of living cells injected ISMB at 120 min. (B) Increase in the fluorescence of scrambled ISMB (square) and ISMBe1 (triangle) in living cells are shown. Fluorescence intensity of each cell at 0 min was defined as 1.0. Data are the means ± SEM of 7 cells for scrambled ISMB and 10 cells for ISMBe1. Scale bar, 20 μm.(TIF)Click here for additional data file.S4 Fig(A) After anion exchange chromatography, each fraction was analyzed by 7 M urea 8% PAGE. (B) The complex of eMet with eMet-specific radioisotope-labeled probe was electrophoresed on 8% native PAGE, and was then detected by ethidium bromide staining (left) and by autoradiography (right).(TIF)Click here for additional data file.S1 TableF, fluorophore; Q, quencher. The underlined bases were designed to hybridize to the target region.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


CH) and Limousine (LIM), would be affected by their housing on concrete (CS) or rubber-covered (RCS) fully slatted floor. A total of 228 CH  and 115 LIM  were housed in four and two commercial farms, respectively, in groups of 9.0 ± 2.1 animals/pen with an average space allowance of 3.1 ± 0.2 m2. Draining gaps of CS and RCS pens were 16.9 ± 1.7% and 11.6 ± 1.2% of the total surface, respectively. Bulls of both breeds had similar initial body weight , and they were slaughtered when they reached suitable finishing. Charolais had a higher final body weight (BW) than LIM , and bulls of both breeds on RCS had higher average daily gain than on CS . The percentage of bulls early culled or treated for locomotor disorders were reduced by RCS only for LIM, while RCS tended to prevent the occurrence of bursitis for both breeds. During two 8-h behavioural observations, bulls on RCS performed more head butt/displacements and chases than on CS, and they reduced the frequency of abnormal lying down events. The use of RCS increased mounts’ frequency only in LIM, while its reduced drainage capacity impaired only the cleanliness of CH. Postmortem hoof inspection showed longer claw dorsal wall and diagonal lengths, and sharper toe angles for CH on RCS than LIM on both floors. Results of this study point out that fully slatted floors, regardless of being rubberized or not, are not suitable for bulls finished at a final BW above 700 kg due to their detrimental effects on health and welfare. The use of RCS could be recommended as an alternative to CS only if bulls are slaughtered at a lower final BW (around 600 kg), like in the case of LIM breed.Intensive fattening of late-maturing breeds on concrete or rubberized slatted floors is the prevalent beef production system in mainland Europe. The rationale behind this study is that specific beef breeds with different slaughter weights might have a diverse response to different flooring systems. The study aimed at assessing whether growth performance, health, behaviour and claw condition of two beef breeds, Charolais ( Intensive fattening of late-maturing breeds is the predominant beef production system in mainland Europe. These animals are often kept on concrete slatted floor during finishing, but recently, positive effects on their welfare were reported for rubberized slatted floors. However, research on these two flooring solutions has not investigated the response of specific beef cattle breeds with different slaughter weights like Charolais and Limousine. This study suggested that fully slatted concrete floors, bare or rubberized, impaired the health and welfare of heavy bulls like Charolais. Rubberized slats could be a recommended alternative only for lower body weight bulls like Limousine.et al., et al., et al. (CH) and Limousine (LIM) are the most imported French purebreds fattened in Italy. Charolais is generally slaughtered at a heavy BW (over 700 kg), due to its well-known growth potential, feed efficiency and carcass quality  with draining gaps of 16.9 ± 1.7 (SD) % of the total surface. In the second half of the experimental pens, concrete slats were covered with 30-mm thick of synthetic rubber (RCS) , designed to match the gap profile of the slats underneath and to allow the drainage of the manure with draining gaps of 11.6 ± 1.2% of the total surface.In each farm, half of the experimental pens had a fully slatted concrete floor . Dietary samples were collected throughout the study and analysed for DM and CP according to the methods of the Association of Official Analytical Chemists  were included in the study, and detailed numbers of pens and bulls considered per each farm according to type of floor and breed are reported in Table ts AOAC, . Analysi, et al. , while s, et al. . Diets oADG). A beef cattle market expert set the end of the finishing period according to the achievement of a suitable finishing of all bulls in each pen. Bull carcass weights were gathered from the slaughterhouse personnel. The same veterinarian belonging to the producers’ association was in charge of bulls’ health in all farms. Individual health status of the animals was daily checked throughout the finishing period. Visually sick/lame animals were temporarily removed from the fattening pens to a sick bay to receive pharmaceutical treatment until healing. The number of bulls that were treated for respiratory and locomotor disorders was recorded as well as the number of bulls that were early culled due to fatal or traumatic events or lameness. A trained fixed veterinarian performed an individual bull’s health check the last month of finishing in all farms. Each bull was visually inspected from the feeding alley, and the occurrence of front and hind leg problems such as bursitis (swelling), alopecia and lesion/wound was recorded as binary variables (presence/absence) according to the Welfare Quality® Assessment protocol for cattle  .All the animals were slaughtered in the same abattoir owned and managed by the producers’ association. Front and hind feet of a minimum of nine bulls/floor type/farm were randomly chosen and inspected z et al. , dorsal ICC) were calculated for behavioural data gathered during the two observation sessions to assess agreement between observers a posteriori both for breed and type of floor effects using MedCalc Statistical Software . Statistical analyses of variables expressed as proportions regarding treated and early culled bulls were performed using χ2 tests (with the Marascuilo procedure) to verify their association with the type of floor within breed. Variables gathered as binary regarding bulls’ cleanliness and health were expressed as percentages of bulls. When the prevalence resulted ≥ 1%, they were tested for association with the type of floor within breed using the one-way logistic regression analysis , and the odds ratio (OR) and 95% confidence intervals (CI) were calculated using RCS as term of comparison. Claw measurements were analysed using a mixed model  that considered the effect of breed, type of floor and their interaction as fixed and of farm as random effects, with the Bonferroni adjustment option. Regarding the normality and homoscedasticity of the errors, the hypotheses of the linear model were graphically assessed by visual inspection of the studentized residuals, and all variables met the model assumptions.Pen was the experimental unit for bulls’ growth performance, continuous behavioural data and events. The single animal was the statistical unit for data regarding carcass weights, time to lying down, clinical traits, cleanliness and claw dimensions. Initial and final BW, ADG, days of fattening and carcass weight were analysed using a mixed model that considered the fixed effect of breed, type of floor and their interaction, with farm nested within breed as random effect and the Bonferroni adjustment option. Continuous behavioural data gathered using the scan-sampling technique were expressed as percentage of bulls performing each behavioural activity per scan per pen, while events were expressed as number of events performed per bull during the observation session at the pen level. These data were processed using a mixed model that considered breed, observation session, type of floor and breed × type of floor interaction as fixed effects, considering farm as a random effect, the observation session as repeated option  and the Bonferroni adjustment option. Intraclass correlation coefficients , and bulls kept in RCS pens had higher BW than those kept in CS pens . There were no breed and type of floor effects on the duration of the fattening. Average daily gain tended to be higher for CH than LIM bulls , and it was significantly higher for bulls housed on RCS than for those on CS floor . Carcass weights of CH bulls were heavier than those of LIM bulls , and it increased for bulls housed on RCS than for those on CS floor . No breed × type of floor interactions were observed for bulls’ growth and slaughter performance . The proportion of bulls lying with one front limb extended was higher for CH than LIM bulls , and for bulls housed on CS than for those housed on RCS floor . Breed and type of floor had no effect on the proportion of bulls lying with two limbs extended or with lateral recumbency, or resting. Among them, only the proportion of bulls with lateral recumbency varied for the observation session effect, being more frequently at the end than at the beginning of the fattening . Both breeds performed eating and ruminating activities with a similar frequency, and the type of floor did not affect them . Breed × type of floor interactions for the occurrence of events are shown in Figure P < 0.05) was observed for head butt/displacement and chasing events that were recorded with a higher frequency in bulls of both breeds on RCS compared to CS floor . The use of RCS floor increased the frequency of mounting events only in LIM bulls while slipping events did not differ according to both breed and type of floor. Regardless of breed, bulls kept in CS pens had a higher frequency of unsuccessful lie-down attempts  and of abnormal lie-down events  than bulls in RCS pens. Slipping events and unsuccessful lie-down attempts occurred more frequently for bulls at the beginning than at the end of the fattening . A significant breed × type of floor interaction . In particular, lying-down duration was longer for LIM bulls housed on CS floor, lower for CH and LIM bulls on RCS floor and intermediate for CH bulls on CS floor.During the two observation sessions the agreement between observers was good both for breed and for the type of floor, since the ICC calculated on the percentages of all behavioural variables were ≥0.70. Observation of continuous behaviour showed a similar proportion of CH and LIM bulls standing, and there were no type of floor and observation session effects on standing behaviour Table . SternalCharolais bulls housed on RCS floor had longer dorsal wall lengths of both front and hind claws compared to CH bulls on CS floor and LIM bulls on both floors Table . Diagonaet al.  than CH bulls. The problem of hoof overgrowth of these animals recorded on RCS floor might be prevented by introducing a certain percentage of a more abrasive surface in the floor pen. Results of this study show that, despite the positive growth performance, health and welfare of CH bulls finished at a final BW above 700 kg were impaired by their housing on both concrete or rubberized slatted floors. Therefore, alternative flooring systems to the fully slatted floor should be tested by future studies in order to improve health and welfare of these heavy animals.


The genus capripoxvirus consists of sheeppox virus, goatpox virus, and lumpy skin disease virus, which affect sheep, goats, and cattle, respectively. Together capripoxviruses cause significant economic losses to the sheep, goat, and cattle industry where these diseases are present. These diseases have spread into previously free bordering regions most recently demonstrated with the spread of lumpy skin disease virus into the Middle East, some Eastern European countries, and Russia. This recent spread has highlighted the transboundary nature of these diseases. To control lumpy skin disease virus, live attenuated viral vaccines are used in endemic countries as well as in response to an outbreak. For sheeppox and goatpox, live attenuated viral vaccines are used in endemic countries; these diseases can also be contained through slaughter of infected animals to stamp out the disease. The thermostability, narrow host range, and ability of capripoxviruses to express a wide variety of antigens make capripoxviruses ideal vectors. The ability to immunize animals against multiple diseases simultaneously increases vaccination efficiency by decreasing the number of vaccinations required. Additionally, the use of capripoxvirus vectored vaccines allows the possibility of differentiating infected from vaccinated animals. Arboviruses such as bluetongue virus and Rift Valley fever viruses are also responsible for significant economic losses in endemic countries. In the case of Rift Valley fever virus, vaccination is not routinely practiced unless there is an outbreak making vaccination not as effective, therefore, incorporating Rift Valley fever vaccination into routine capripoxvirus vaccination would be highly beneficial. This review will discuss the potential of using capripoxvirus as a vector expressing protective arboviral antigens. Capripoxviruses represent a genus of the poxviridae family under the subfamily chordopoxviriniae; the genus includes three animal virus species that have a devastating impact on sheep, goats, and cattle in Africa, Asia, and most recently Eastern Europe –4. The vIt is believed that SPPV was first reported in the second century in central Asia before spreading to surrounding countries and Europe , 6. SPPVAlthough LSDV is not an arbovirus, insect and arthropod vectors spread the disease through mechanical transmission. The most likely vectors involved in transmission of LSDV are stable flies, mosquitos and hard ticks , 14. SinThe most effective and widely used vaccines against capripoxviruses are live attenuated vaccines . These lDue to regulatory issues related to trade, preventative vaccinations against capripoxviruses are not in use in disease free countries , 19. In Arboviruses are a diverse group of arthropod-borne viruses that are able to replicate in arthropods and vertebrate hosts –26. ArboCulicoides midges exclusively transmit BTV to ruminants (Bluetongue virus (BTV) is a virus in the family reoviridae under the genus orbivirus that causes bluetongue disease, an OIE listed hemorrhagic disease, in wild and domestic ruminants –30. BTV uminants , 34. In uminants , 35, 36.uminants . Mortaliuminants , 38. In uminants , 39, 40.Culloides midges  vaccines or inactivated vaccines, neither of which is available for all serotypes of BTV , 46. MLVInactivated or killed vaccines have also been commercially available to immunize against BTV. They are inactivated chemically, using heat, or through exposure to UV or gamma radiation , 49, 50.in vitro  and disabled infectious single cycle vaccines. Recombinant subunit vaccines use a specific protein expressed in vitro . Notablyin vitro , 51. VP5in vitro . Virus lin vitro , 54. VLPin vitro .Disabled infectious single cycle/animal (DISC/A) vaccines have also been developed against BTV. These vaccines generally lack an essential gene, which results in an inability to replicate in the host cells for more than one cycle. A DISC vaccine lacking VP6, a structural protein has been successfully produced and has been experimentally shown to provide immunity against challenge in sheep –58. AlthRecombinant vectored vaccines are live attenuated virus vaccines modified to express genes encoding antigens to elicit protective immunity. Many viral vectors have a limited capacity to express foreign antigens. Therefore, it is important to select the best antigen(s) to elicit protective immunity following vaccination. For BTV, it has been shown from the use of subunit vaccines that structural proteins VP2, VP5, and VP7 confer protective immunity, VP2 being the most effective . MultiplRift Valley fever virus (RVFV) is an enveloped segmented negative stranded RNA virus of the family Bunyaviridae, genus Phlebovirus , 70. It RVFV has been responsible for devastating outbreaks throughout the African continent and has most recently been reported in the Arabian Peninsula , 73, 74.Aedes spp. being the primary vectors  and LSDV in cattle. The recombinant vaccine was generated in lamb testicular cells using LSDV. The cells were then transfected with plasmid DNA containing the fusion (F) protein of RPV and a selectable marker (gpt) to replace the TK gene of LSDV; recombinant virus was then isolated through rounds of plaque purification . This vaWallace and Viljoen  generateHomologous recombination is a commonly used method of editing genomes and has been used to successfully delete or add antigen-encoding genes into capripoxviruses. Recombinant capripoxvirus vaccine generation using homologous recombination is achieved by infection of permissive cells with a capripoxvirus vector followed by a transfection with a transfer plasmid. The transfer plasmid contains selectable markers and the gene of interest with flanking regions for a non-essential capripoxvirus gene, often thymidine kinase (TK) –116, 120Esherichia coli (E. coli) β-galactosidase gene, use of green fluorescent protein (GFP) genes and/or the use of E. coli xanthine phosphoribosyltransferase (gpt) gene  gene , 120. Efpt) gene . The propt) gene . The secpt) gene   gene .CRISPR refers to clustered regularly interspaced short palindromic repeats. CRISPR is found in prokaryotes where it functions as a defense system to attack invading foreign DNA where the foreign DNA is inserted following the CRISPR sequence and CRISPR associated (Cas) genes to produce guide RNAs that then target the sequence of foreign DNA for destruction should it ever be re-introduced into the prokaryote , 134. CRA novel method of recombinant poxvirus generation has recently been demonstrated involving large scale gene synthesis . The proDifferentiating infected from vaccinated individual (DIVA) vaccines are possibly the most promising means to control and monitor the spread of rapidly spreading infectious diseases in small ruminants and cattle. Previously known as marker vaccines, DIVA vaccines refer to genetically altered conventional vaccines, which have at least one antigenic region missing . This reCurrently there is no DIVA vaccine and companion diagnostic test for capripoxviruses, although there are molecular based methods available to discriminate between vaccine and wild type viruses –144. TheThe continuing spread of lumpy skin disease into previously free regions is leading to more countries where all capripoxvirus members are present. Since these viruses cannot be differentiated using serology, the only method to identify the specific virus is PCR and/or sequencing. The historical method used to identify the virus used the ruminant host that the virus was isolated from to characterize the virus. This worked generally well; however, there is an exception where this method did not identify the virus properly . The seqCapripoxvirus vectors can be tailored to include antigens for specific disease agents in the region. This is especially important in the case of BTV where there are 29 serotypes present, designing vectored vaccines based on geographically prevalent and cross-reactive serotypes is crucial to maximize the protective capability of a multivalent vaccine. The limit of the number of foreign antigens expressed simultaneously in a capripoxvirus vector is currently unknown; however, it is likely more than two antigens. In addition, it is possible that VLPs can be expressed using a capripoxvirus vector, however, this has not be demonstrated to date. It is possible to develop a capripoxvirus vector encoding the protective antigen GnGc from Rift Valley fever virus along with protective VLPs from bluetongue to generate a multivalent vaccine to protect sheep, goats and cattle from these diseases.Capripoxvirus vectors have tremendous potential for use as multivalent vaccines to protect sheep, goats and cattle from arboviruses, capripoxviruses and other devastating diseases such as peste des petits ruminants. The difficulty in vaccinating animals against arboviruses such as Rift Valley fever virus is the cyclical nature of the disease where producers do not have the resources to vaccinate for a disease that may or may not occur. Using a multivalent capripoxvirus vaccine can alleviate these issues by having a vaccine that can protect against Rift Valley fever together with endemic capripoxvirus diseases that occur much more frequently . The useMT wrote the first draft of the manuscript. SB wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.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.


Lichen planus (LP) is a chronic inflammatory, immunological, mucocutaneous disease can affect skin, genital and oral mucosa. Oral lichen planus (OLP) is the most common noninfectious, chronic inflammatory oral disease affecting 1-2% of the general adult population. World Health Organization (WHO) classifies OLP as a potentially malignant disorder. Epstein Barr virus or human herpesvirus-4, is a member of the herpes virus family and one of the most ubiquitous viruses known to human, infecting approximately 90% of the world’s adult population. The virus often infects B lymphocytes resulting in a wide spectrum of mucocutaneous and systemic diseases, ranging from mild lesions to aggressive malignancies. The aim of this study was to investigate expression of the EBV encoded RNAs EBER1 and EBER2 in oral and genital lichen planus and compare results with normal tissues in situ hybridization which is considered the golden standard for detection of EBER.A total of 68 biopsies, 25 oral LP, 26 genital LP, 10 oral controls and finally 7 genital controls were analysed using situ hybridization.All samples had RNA as shown by the control slide, whereas no case contained neither EBER1 nor EBER2.Based on results from our study EBV is not involved in aetiology of lichen planus. Key words:Mucosal lichen planus, Epstein - Barr virus. Lichen planus (LP) is a chronic inflammatory, immunological, mucocutaneous disease with different clinical appearances ,2. LP caWorld Health Organization (WHO) classifies OLP as a potentially malignant disorder . The malEBV or human herpesvirus-4, is a member of herpes virus family and one of the most ubiquitous viruses known to human, infecting approximately 90% of the world’s adult population. Like all herpes viruses EBV may remain in the host organism for life, but in most cases does not cause any harm in healthy carriers . The virPrimary EBV infection occurring in children is often asymptomatic . In contEpstein Barr virus encoded RNA can be detected by different methods but Epstein Barr virus-encoded RNA in situ hybridization is considered the gold standard for detecting and localizing latent Epstein Barr virus in tissue samples ,17. The A total of 68 biopsies were collected in this study. These biopsies consisted of 25 oral LP, 26 genital LP, 10 oral controls (OC) and finally 7 genital controls (GC) . All 51 Samples, were retrieved from the archives at the Department of Clinical Pathology, University Hospital, Umeå. The study was approved by the Ethical Review Board at Umeå University (dnr 2013-252-32M).For EBER-in situ hybridisation (EBER-ISH) a commercially available kit was used. From each samples two sections were cut and in one of the sections EBER1 and EBER 2 was detected using the Inform EBER Probe . At the same time section number 2 was incubated with an RNA Positive Control Probe  in order to make sure mRNA was present in the samples. The NBT-BCIP Detection Chemistries  was used for detection. Stainings were performed using a Bench Mark Ultra  according to the supplier’s protocols.All biopsies were assessed by to investigators (ME and KD) independently. In cases of disagreement slides were discussed in a joint session until consensuses was reached.All samples had RNA as shown by the control slide, whereas no case contained neither EBER1 nor EBER2  this virus has been the subject of study in many kinds of cancer. The virus often infects B lymphocytes resulting in a wide spectrum of mucocutaneous and systemic diseases, ranging from mild lesions to aggressive malignancies (Since the discovery of the EBV, the first identified human tumour virus by Epstein (Epstein gnancies .EBV is one of the most ubiquitous viruses known to human infecting approximately 90% of the world’s adult population. Like all herpes viruses EBV may remain in the host organism for life, but in most cases it does not cause any harm in healthy carriers . The oraEBER represent two RNA species, EBER1 and EBER2, encoded from two separate but homologous viral genes. EBER transcripts are expressed in latently infected cells at levels approaching a million copies per cell . BecauseReviewing literatures reveals rather few papers with divergent results depending on different method and choice of tissue. In this study in contrary to all other studies we could not detect occurrence of EBER expression in epithelium in oral and genital lichen planus as well as healthy controls. Yildirim and co-workers used immunohistochemistry, staining OLP slides with EBV primary antibody EBV/LMP-Ab-1 and found that 35% of OLP patients were positive and suggested that EBV may play a role in the ethiopathogenesis of OLP By using PCR or ELISA some investigators detect antibodies against EBV in approximately 25% of LP patients -23 whileUsing different PCR techniques, requires initial homogenizing of tissue and extraction of DNA. This eliminates the possibility of determining which cell type harboured the virus, and allows for false positive results due to contamination with laboratory EBV DNA or EBV-infected lymphocytes in the specimen. The positive result for EBV by solution PCR could have been due to EBV-infected lymphocytes rather than LP epithelium.Despite intense research in the field of lichen planus there are some crucial questions that remain unanswered among those premalignant potential of lichen planus and some authors investigate expression of EBV in lichen planus and somehow connect it to LP´s pre- malignant potential.Results of our study is based on Epstein Barr virus-encoded RNA in situ hybridization which is considered the golden standard for detecting and localizing latent Epstein Barr virus in tissue samples ,17.EBER transcripts are systematically expressed in every EBV-infected tumour, and in lymphoid tissues taken from patients with infectious mononucleosis, and in the rare infected cell representing normal flora in healthy virus carriers. In fact, oral hairy leukoplakia is the only EBV-related lesion that lacks EBER .Based on results from our study EBV is not involved in aetiology of lichen planus.


To date, epidemiological studies at the index site of the 2013–16 west African Ebola outbreak in Meliandou, Guinea, have been restricted in their scope. We aimed to determine the occurrence of previously undocumented Ebola virus disease (EVD) cases and infections, and to reconstruct transmission events.This cross-sectional seroprevalence survey of the adult population of Meliandou used a highly specific oral fluid test and detailed interviews of all households in the village and key informants. Each household was interviewed, with all members prompted to describe the events of the outbreak, any illness within the household, and possible contact with suspected cases. Information for deceased individuals was provided by relatives living in the same household. Symptoms were based on Ebola virus Makona variant EVD case definitions . For antibody testing, we used an Ebola virus glycoprotein IgG capture enzyme immunoassay developed from a previously validated assay. A maximum exposure level was assigned to every participant using a predetermined scale. We used a generalised linear model (logit function) to estimate odds ratios for the association of sociodemographic variables and exposure level with Ebola virus infection. We adjusted estimates for age and maximum exposure, as appropriate.Between June 22, and July 9, 2017, we enrolled 237 participants from 27 households in Meliandou. Two households refused to participate and one was absent. All adults in participating households who were present for the interview provided an oral fluid swab for testing, of which 224 were suitable for analysis. In addition to the 11 EVD deaths described previously, on the basis of clinical description and oral fluid testing, we found two probable EVD deaths and eight previously unrecognised anti-Ebola virus IgG-positive survivors, including one who had mild symptoms and one who was asymptomatic, resulting in a case fatality of 55·6% (95% CI 30·8–78·5) for adults. Health-care work  and level of exposure  were independent risk factors for infection.vs 11 cases). We show the first serological evidence of survivors in this population (eight anti-Ebola virus IgG seropositive) and report a case fatality lower than previously reported (55·6% vs 100% in adults). These data show the high community coverage achievable by using a non-invasive test and, by accurately documenting the beginnings of the west African Ebola virus outbreak, reveal important insight into transmission dynamics and risk factors that underpin Ebola virus spillover events.Ebola virus infection was more widespread in this spillover population than previously recognised (21 US Food and Drug Administration, Wellcome Trust, and German Research Council. We searched PubMed and Web of Science for articles published between Dec 1, 2013, and Dec 31, 2018, using the keywords “Ebola” AND “Meliandou” OR “Guinea”. We selected articles collecting data or describing events at the index site of the outbreak. No language restrictions were used. Despite extensive diagnostic, molecular, and phylogenetic research into the transmission of Ebola virus during the 2013–16 west African outbreak, no quantitative study or diagnostic methods have been employed at the suspected index location of Meliandou village in Guéckédou prefecture of south-eastern Guinea. Previous studies in this area, conducted during the outbreak, were necessarily limited and did not include serological investigation. Past studies on Ebola virus spillover events in other locations have relied on immunoassays with questionable specificity. During the west African outbreak, a new high specificity capture assay capable of detecting anti-Ebola virus immunoglobulins from oral fluid samples demonstrated the occurrence of mild and asymptomatic infections, and facilitated non-invasive serological studies with high population coverage.Added value of the studyThis study provides the first evidence of Ebola virus-infected survivors from the index site of the west African outbreak. A thorough retrospective epidemiological study done concomitantly among the resident adult population also greatly expands our understanding of the initiating events including transmission dynamics, probable transmission chains, lower case fatality rates, and the presence of both mild and asymptomatic cases.Implications of all the available evidenceOur conclusions highlight the importance and potential of deploying appropriate quantitative serological tools. The acceptability and specificity of this approach alongside careful epidemiological investigation provide comprehensive understanding of transmission dynamics in Meliandou. These data thoroughly characterise the initiating events of an Ebola virus outbreak and show that it spread further within this community than previously appreciated. They also show that it is possible to access a large proportion of a deeply affected community by building up trust and using an acceptable and non-invasive approach.Although severe clinical manifestations dominate during human EVD outbreaks, both minimally symptomatic and asymptomatic infections occur, particularly among contacts of cases.The true incidence of subclinical infections and their contribution to transmission dynamics are not fully understood.The Ebola virus Makona variant (Zaire species), which emerged from Guinea in 2013, caused the largest recorded outbreak of any Ebola virus species in humans (28 625 cases and 11 325 deaths notified)22We sought to enrol all family members residing in Meliandou who were aged 18 years or older at the time of the study. Meliandou is a rural village of 30 households living in 75 buildings, all belonging to the Kissi ethno-linguistic group. The village is encircled by a 100–200 m perimeter of forest and is about 12 km by uneven road from the nearest major urban centre (Guéckédou). All residents were checked by village leaders and local health-care workers before registration to confirm that they had been resident during the period of the outbreak in this area . Information on EVD exposure and symptoms was collected retrospectively. Interviews were done for all households on the basis of qualitative and quantitative approaches used in Sierra Leone, as described previously.Permission for the study was granted by the Guinea Comité National D'Ethique Pour La Recherche en Santé, the ethics committee of the London School of Hygiene & Tropical Medicine, and the UK National Health Service National Research Ethics Service. All participants gave written informed consent before interviewing and sample collection.Participants were initially denoted as being possible cases on the basis of symptoms reported during interviews . PossiblAfter each group interview, all adult participants provided an oral fluid swab using Oracol Plus collection devices . Swabbing was demonstrated by field staff and participants were directly observed firmly rubbing the sponge tip along the upper and lower gums for 90 s. Swabs were sealed and placed on ice in a cool box. The same day, swabs were centrifuged at 1500 g for 10 min to extract oral fluid and stored immediately at −20°C. Swabs were transported to Conakry, Guinea, at a maximum temperature of −15°C and shipped to the UK on dry ice for analysis at Porton Down, UK. Positive controls of oral fluid samples were provided by two PCR-confirmed survivors of Ebola virus infection (based in Guéckédou town) with persistent neutralising anti-Ebola virus IgG titres recorded as part of survivor studies. Serum samples were also acquired from the positive controls, one local negative control, and two suspected EVD survivors from Meliandou. Four UK-based volunteers also provided negative control samples.We detected human anti-Ebola virus (Zaire) IgG using an enzyme-linked immunosorbent anti-Ebola virus glycoprotein IgG capture assay  that was developed as a commercially available assay from the validated assay described previously.Epidemiological data were double-entered into EpiData  and exported into RStudio (version 1.0.13) for analysis. We used a generalised linear model (logit link function) to estimate odds ratios for the association of sociodemographic variables and exposure level with Ebola virus infection (defined as anti-Ebola virus IgG seropositive or died of suspected EVD during the outbreak period). We calculated p values via likelihood ratio tests . We adjusted estimates for age and maximum exposure, and assessed model assumptions for violations. For additional capture assay analysis to assess the association between sample volume or precipitate and normalised optical density we used unadjusted linear regression and Kruskall-Wallis non-parametric methods.The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between June 22, and July 9, 2017, we enrolled 237 participants from 27 households in Meliandou. One household was absent and two refused. 237 adults (≥18 years) were interviewed and gave swabs, of whom 117 (49·4%) were men and 120 were women (50·6%), with a mean age of 29·8 years. 38 adults from participating families were absent for survey activities. The most common occupations were subsistence farmers (40·4%), housewives (19·8%), or students or unemployed (23·5%).32 possible cases were identified via the interviews on the basis of reported symptoms. Of these, 13 people had died and ten were confirmed by key informants to have had EVD-like symptoms at the time, resulting in 23 suspected cases (S1–23). The remaining possible cases with reported symptoms were not confirmed by the key informants (pre-fixed U01–09). Of the 23 suspected cases, three deaths were in children and one survivor with confirmed symptoms was younger than 18 years so no oral fluid sample was collected.After we checked sample integrity and the volume extracted after centrifugation, 224 of 237 oral fluid samples were available for testing. During assay optimisation, the magnitude of normalised optical density responses in the positive control oral fluid samples  fell below a priori defined cutoffs for seroconversion .7 An altEight of 224 oral fluid samples were seropositive  including six of the nine suspected cases in adults , table 1Among unconfirmed cases (U01–09), the median normalised optical density of oral fluid was 0·68 (95% CI 0·67–0·70), compared with 0·71 (0·65–0·94) among all other oral fluid samples. Three suspected cases in which symptoms were confirmed  remained under the seropositive cutoff with normalised optical density values of 1·0, 0·73, and 0·64, respectively.By use of interviews and a population of cases defined by immunoassay results, including eight anti-Ebola virus IgG positive survivors, all seronegative patients or suspected cases that were not tested, and 13 suspected EVD deaths (including the three children who died during the outbreak), we generated a transmission chain that affected ten households .Figure 2The index case arose in December, 2013, in a boy aged 2 years (S1) whose first contact with Ebola virus reservoirs was possibly insectivorous bats.While in hospital, S4 was cared for by several family members (S7–S10) and her body was returned to Meliandou for burial. The next patients, S5 and S6, were the first cases in individuals residing outside the home of the index patient. Both were buried in Meliandou within 2 days of each other. S6's son  later died with EVD-like symptoms. The funerals of S4–S6 are believed to have spread the infection to other local villages.The next cases arose in the family of those caring for S4 during her stay in hospital and burial (S7–S10). S7, who also had high exposure to S3 and lived in the index household, was cared for in the home of a friend (S13) while symptomatic. S7 died and was buried in Meliandou. The asymptomatic seropositive patient (A01) attended the funeral of S7 but was not involved in the preparations of the body. The other family members who cared for S4 in hospital (S8–S10) all subsequently developed EVD symptoms. S8 and S10 returned to their home village (Dawa), where they were buried. S9 travelled to Conakry, where he developed symptoms and died.Although S11 and S12, who were health-care volunteers, were not previously reported as EVD cases, both developed EVD-like symptoms . S11's son (S19) cared for her and developed unreported EVD-like symptoms; he subsequently left Meliandou to seek traditional remedies and survived. During the same week, S13 developed symptoms after caring for S7, including contact with body fluids, but survived without admission to hospital.The local health-care worker (S18) developed EVD-like symptoms, including fever, vomiting, diarrhoea, red eyes, and blurry vision, but survived. He was involved in the care of all affected patients and in several burials. S18 was cared for exclusively by his wife (S23), who was incapacitated with EVD-like symptoms but survived.The last two suspected EVD deaths in Meliandou (S21 and S22) had safe burials by health authorities and external non-governmental organisations . S21 was probably exposed to Ebola virus outside Meliandou, while preparing for burial the body of a patient with suspected EVD in another village. Patient U01, who reported mild nausea, vomiting, and diarrhoea, cared for S21 and participated in her funeral. S22, who was the last individual to die, had several possible routes of infection: providing care in the same compound as S6 and S17, interacting with her symptomatic brother (S19), and assisting in the funeral of S7.None of the first ten patients (S1–10) survived, after which only three patients died and eight survived , 92 reported high-level exposure to suspected cases , with a further 45 sharing residence or providing care for a symptomatic patient without direct contact level 3; . All EVDIn the univariable analysis, Ebola virus infection was most common among women, with increasing age, and in those with a formal or informal health-care responsibility in the community . After avs 11) and the identification of eight seropositive survivors. Although initial zoonotic and human-to-human transmission within the index household have already been documented,This cross-sectional seroprevalence survey used detailed investigation and non-invasive immunological tools to document Ebola virus transmission at the index site of the largest recorded outbreak of human EVD. Although an outbreak investigation was done in 2014, no diagnostic or immunological methods have been previously used in Meliandou.Identifying survivors enhances our understanding of the transmission dynamics of Ebola virus during this spillover event. After the initial cases in the index family, the virus propagated across numerous households, stemming from a series of high-level exposures to cases in the absence of any preventive interventions. Both traditional funeral practices and contact with symptomatic patients or their body fluids were important in the dissemination of Ebola virus infection, as was also reported in rural areas during the course of the wider outbreak.We found evidence of asymptomatic and minimally symptomatic cases of Ebola virus infection in Meliandou, adding to the evidence on the prevalence of these disease states during the west Africa outbreak.The case fatality  in Meliandou was lower than previously reported (100%).The use of a non-invasive immunoassay based on oral fluid to detect anti-Ebola virus IgG was acceptable to the community and ensured high participation. Although the assay was previously validated in a comparable setting,This study has important limitations. Owing to the time that has elapsed since the outbreak, recall bias is likely, and is only partly offset by using group interviews and key informants. Serology was restricted to adults (aged ≥18 years), so children who had mild or no symptoms might have been missed, which underestimates these disease states.Our findings from Meliandou provide important documentation of the initiating events of the 2013–16 outbreak of Ebola virus in west Africa. Future research into Ebola virus and other emerging diseases will benefit from the use of acceptable non-invasive sampling to further our knowledge of mild and asymptomatic infection and transmission among populations at risk of Ebola virus spillover events. Such information can improve our understanding of the natural history of Ebola virus and contribute to establishing appropriate and sustainable surveillance systems to prevent communities like Meliandou from suffering the long-term effects of Ebola virus and related outbreaks.


Compared to animal and plant viruses, fungal viruses have been relatively less studied. However, the recent development of associated methodologies including sequencing technologies has led to the rapid advancement of research on viruses infecting fungi or fungi-like organisms. Fungi can cause human/animal diseases, and they are the major causative agents of crop plant diseases. Fungi harbor highly diverse viruses, many of which constitute distinct virus families/groups that are not present in other organisms. Of note is that there are also some virus taxonomical groups comprising both plant and fungal viruses, suggesting an active and recent horizontal transfer of viruses between the two kingdoms. Therefore, fungal virology is particularly significant for its applicative, fundamental, and evolutionary implications. The Research Topic “Frontiers in Fungal Virus Research” is aimed to present the most recent progresses on different aspects of fungal virology. This topic contains 13 research and two review articles, covering a variety of topics including virus identification, virus-mediated modulation of fungal pathogenicity, virus-virus interactions, and host defense.Khalifa and MacDiarmid identified a novel totivirus from Trichoderma koningiopsis. Another isolate belonging to the same species also was found from Clonostachys rosea distinct from T. koningiopsis, indicating a very recent natural horizontal transmission of this virus between unrelated fungi. It is interesting to further investigate how this virus is transmitted across different fungal species. Using RNA-seq analysis, Picarelli et al. revealed a high diversity of fungal viruses infecting Rhizoctonia solani isolated from Zoysia japonica (a cultivated grass) in Brazil. Most of the viruses show low sequence identity with other viruses, which include four large contigs of putative viral RNA that could not be assigned to any existing clade of viruses present in the databases. This suggests that they all belong to possible new viral species. This study provides a sound foundation for further understanding the diversity of fungal viruses. Kartali et al. identified novel totiviruses from Umbelopsis ramanniana. This work is particularly interesting and important because it is the first report on fungal viruses infecting this fungal genus, and this fungal species is an oleaginous fungus, a potential oil source for biodiesel production. Wang et al. characterized two novel viruses from Sclerotinia sclerotiorum each related to members of the order Tymovirales and the genus Botybirnavirus. Neither of them confers hypovirulence to the host. Moreover, they detected the accumulation of viral-derived siRNAs in S. sclerotiorum, indicating that RNA silencing operates against these viruses in the fungus. Li et al. characterized a novel hypovirulence-inducing hypovirus from Alternaria alternata isolated from an apple tree. Moreover, this virus also confers hypovirulence in Botryosphaeria dothidea, the causal fungal pathogen of apple white rot. Thus, this virus is a potentially valuable material as a bivalent biocontrol agent against fungal diseases in apple tree. Wang et al. identified a novel virus related to members of the genus Chrysovirus, family Chrysoviridae from Penicillium crustosum, a pathogen of citrus tree. Interestingly, this virus does not affect the growth or morphology of the fungal host but it decreases the fungal resistance against a fungicide. Certainly, this virus is a good molecular tool to study the mechanism of fungal resistance against fungicide. Jiang et al. identified a novel partitivirus from an opportunistic human pathogenic fungus, Aspergillus flavus. Infection by this virus causes significantly abnormal colonial and spore morphologies, but does not significantly affect its host virulence. It is important to further explore the presence of viruses in this important human-pathogenic fungus. Using RNA-seq analysis, Zhu et al. characterized viruses that are present in a hypovirulent strain of Sclerotium rolfsii. Interestingly, this fungal strain harbors various new viruses belonging to several established virus families and also some novel viruses that could not be assigned to any of the existing families or orders. Chun et al. identified a novel partitivirus from Trichoderma harzianum. Trichoderma species are known for their ability to inhibit other fungi, and therefore they are used as biocontrol agents for controlling common soil-borne plant pathogens. The important finding in this study is that although the infection by this partitivirus does not affect growth or morphology of its fungal host, it enhances antifungal activities of T. harzianum against other fungi, and this is associated with increased activity of antifungal enzyme, β-1,3-glucanase. It is necessary to further analyze how this partitivirus infection regulates the activity of a specific antifungal enzyme. Li et al. present a comprehensive review on fungal viruses infecting members of the fungal genus Fusarium. Fusarium species are important phytopathogenic fungi that cause damage in a wide variety of host plant species and also produce mycotoxin. Highly diverse viruses are widely present in Fusarium spp., and some of them induce hypovirulence in their fungal hosts. This review provides valuable knowledge for understanding virus diversity and prospects for development of biocontrol methods against Fusarium spp.In this topic, nine research papers report the characterization of novel fungal viruses from various fungi. Neupane et al. revealed that the Argonaute-like (agl)-2 gene of S. sclerotiorum is important for virulence and host tolerance against virus infection. Intriguingly, deletion of agl-2, agl-4, Dicer-like (dcl)-1, and dcl-2, which are key components of RNA silencing, does not affect the production of the most abundant endogenous small RNAs in S. sclerotiorum. This suggests the existence of alternative enzymes/pathways for small RNA biogenesis in S. sclerotiorum. Autophagy is a highly conserved degradation mechanism of cellular constituents in eukaryotes. Shi et al. observed that infection by a hypovirus in Cryphonectria parasitica induces proliferation of autophagic-like vesicles. Furthermore, they demonstrated that C. parasitica cpatg8, a gene encoding a homolog of a key autophagy component, Atg8, of Saccharomyces cerevisiae, is important for fungal virulence and sporulation. Interestingly, cpatg8 was also shown to play a positive role in hypovirus replication. Recently, autophagy is shown to have both anti- and pro-viral functions in other eukaryotic systems. This is a pioneering study that explores the involvement of autophagy in viral infection in filamentous fungi.Two papers report studies on the roles of RNA silencing and autophagy pathways in fungal virulence and defense against virus infection in fungi. Fusarium oxysporum, Torres-Trenas et al. demonstrated that a hyphovirulence-inducing chrysovirus affects the speed and spatial distribution of fungal colonization into plant roots. This is the first study that clearly provides microscopic evidence that a mycovirus can influence the pattern of plant colonization by its fungal host. Kashif et al. examined the reciprocal effects of partitiviruses on the horizontal transmission efficiency through hyphal anastomosis in Heterobasidion spp. They observed that two viruses could synergistically enhance the overall transmission rate or antagonistically affect their transmission depending on specific virus combinations. Therefore, the interaction between co-infecting viruses and their host is complex, and the underlying mechanism remains to be elucidated. Thapa and Roossinck discussed determining factors for fungal-virus co-infection. Co-infections of natural fungal isolates by fungal viruses are commonly observed. The authors present interesting perspectives of how suppression of fungal non-self-recognition and RNA silencing, and nutritional/chemical compounds could affect the outcome of virus co-infection in fungi.Using GFP-labeled strains of Overall, these works contain rich novel information from diverse viruses and fungal species regarding various topics of fungal virology. Indeed, they provide valuable research materials and interesting biological findings that warrant further detailed mechanistic studies. Mycovirus research will contribute to further advances of virology in general as well as lay a solid foundation for research development of control methods of many fungal diseases. Lastly, the editors of this topic greatly appreciate all of the authors and reviewers for their contributions to the collection.LS, NS, DJ, MT, and JX edited the Research Topic of Frontier in Fungal virus Research and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Targeting efforts aimed at increasing access to care for the poorest by reducing to a minimum or completely eliminating payments at point of use are increasingly being adopted across low and middle income countries, within the framework of Universal Health Coverage policies. No evidence, however, is available on the real cost of designing and implementing these efforts. Our study aimed to fill this gap in knowledge through the systematic assessment of both the financial and economic costs associated with designing and implementing a pro-poor community-based targeting intervention across eight districts in rural Burkina Faso.We conducted a partial retrospective economic evaluation  associated with the abovementioned targeting intervention. We adopted a health system perspective, including all costs incurred by the government and its development partners as well as costs incurred by the community when working as volunteers on behalf of government structures. To trace both financial and economic costs, we combined Activity-Based Costing with Resource Consumption Accounting. To this purpose, we consulted and extracted information from all relevant design/implementation documents and conducted additional key informant structured interviews to assess the resource consumption that was not valued in the documents.For the entire community-based targeting intervention, we estimated a financial cost of USD 587,510 and an economic cost of USD 1,213,447. The difference was driven primarily by the value of the time contributed by the community. Communities carried the main economic burden. With a total of 102,609 ultra-poor identified, the financial cost and the economic cost per ultra-poor person were respectively USD 5,73 and USD 11,83.The study is first of its kind to accurately trace the financial and economic costs of a community-based targeting intervention aiming to identify the ultra-poor. The financial costs amounted to USD 5,73 and the economic costs to USD 11,83 per ultra-poor person identified. The financial costs of almost USD 6 represents 21% of the per capita government expenditure on health.The online version of this article (10.1186/s13561-018-0205-7) contains supplementary material, which is available to authorized users. This study fills an important knowledge gap on cost of community-based selection and targeting interventionsFinancial costs associated with community-based targeting in Burkina Faso amounted to USD 5,73 and the economic costs to USD 11,83 per ultra-poor person identifiedEconomic costs amounted to USD 1,213,447 including the value of the effort contributed by volunteers, exceeding by two-folds the financial costs of USD 587,510Universal Health Coverage (UHC) aspires to provide affordable and quality health care to all . HoweverIn the absence of universal criteria to define poverty and identify the poorest, countries have traditionally adopted various targeting methods. In the literature, the identification of beneficiaries is presented as the process of selecting the persons that benefit from a certain intervention. Targeting, on the other hand, is commonly used as a broader term which includes the identification as well as the actual act of allocating scarce resources to the poorest with the aim of achieving certain policy objectives to preserve or improve health equity .In the health sector, most targeting experiences derive from those with the greatest health risks or the highest ability to benefit from the programs . Those aGenerally, high-income countries use a form of means-testing and identify ultra-poor based on a certain income threshold. On the contrary, targeting strategies without a direct observation of income, such as proxy means testing (PMT) and community targeting, are predominantly used in LMICs . While PAll targeting mechanism are prone to errors and generate costs . VariousThese arguments are mostly postulated in the light of theoretical assumptions on the costs and benefits associated with targeting procedures, but are not substantiated by any extensive empirical evidence . The litTowards this ends, our economic evaluation began to fill an important gap in knowledge by assessing both the financial and the economic costs associated with designing and implementing a pro-poor targeting project, with specific focus on the identification and targeting component integrated within the Performance Based Financing (PBF) intervention recently rolled out in Burkina Faso . We planThis study took place in Burkina Faso, where a PBF intervention was launched in 2014 under coordination of the Ministry of Health (MoH) with financial support from the Health Results Innovation Trust Fund (HRITF), administered by the World Bank (WB). The Programme d’Appui Au Développement (PADS), Burkina Faso’s initiative to better coordinate and manage resources for the implementation of the National Health Development Plan, channeled the funding provided by the HRITF to the CBT intervention. The Society for Studies and Public Health Research (SERSAP), a for-profit consultancy firm, led the implementation of the CBT intervention. The objective of the intervention is to combine a supply-side (PBF) with a demand side (subsidy for the poorest) component to maximize health gains for the poorest.  In eightThe process of selecting and identifying the ultra-poor by community members, including the design and implementation of the structures, lasted from May 2014–January 2016. Across the eight districts, it covered 1,745,789 individuals, i.e. approximately 10% of the country’s total population (18.1 million) . AdditioThe lists of the ultra-poor persons were verified by two entities, the Local Implementation Teams (ELMEO) and the Local Validation Groups (GVL). For this validation process, the respective teams used a list of 20 ultra-poor criteria which were initially developed in the context of the action research done in one district in Burkina (Ouargaye) . The ELMMonitoring and Evaluation (M&E) was secured by the ELMEO, the Regional Directorate for Health and Social Action and the Regional Technical Assistants (ATR). Originally, it was envisioned that 15 to 20% of the population will be targeted and exempted from user fee payment. On average 6% of the total population were finally identified as the ultra-poor .We conducted a retrospective partial economic evaluation  with theAs financial costs (accounting costs), we defined all explicit financing transactions made by any of the concerned actors to carry out any activity related to either the design or the implementation of the intervention. These costs can be verified retrospectively on the accounting systems of the implementing agencies. As economic costs, we defined the real value of the resources consumed by the intervention.We adopted a health system perspective, meaning that we aimed at tracing all costs incurred by the MoH and its partners, including development partners and implementing agencies. Costs incurred at the community level were included in the analysis insofar as the community was engaged as a volunteer agent to act on behalf of the Ministry as a formal implementing partner, effectively carrying out/substituting key intervention functions.To trace both financial and economic costs, we combined Activity-Based Costing (ABC-approach) with Resource Consumption Accounting. ABC aggregates costs by activity, attributing indirect and support expenses to the individual activities . ResourcWe carried out data collection over a period of six months from October 2016 to March 2017. We computed all costs in US dollars (USD), adjusting for inflation from the year in which the costs were incurred to the year 2015. We used the average exchange rate for the period May 2014 to January 2016 to convert values from FCFA  to USD .In line with the conceptual approach described earlier, data collection reflected three steps: identification, measurement and valuation.Step 1: IdentificationStep 2: MeasurementWe started the data collection by asking the main stakeholders (MoH and WB) and implementing actors from the central level to share planning and implementation documents with us, including financial statements , that would allow us to reconstruct all activities carried out from the moment the intervention was conceived to the moment the identification cards were distributed to the ultra-poor. We examined all project documents in order to generate a detailed list of activities, including those of the main stakeholder as well as of the different implementation actors involved at each and every stage of the design and implementation phase Table . This prStep 3: ValuationTo estimate resource use, we triangulated information across data sources and filled gaps that emerged as we progressed through the data by conducting a series of face-to-face key informant interviews. We interviewed the two central level coordinators employed at SERSAP and the four regional coordinators. We used structured interview forms to ask respondents to recall the time spent by the various actors, including communities, on design and implementation activities  and by the Comité National d’Éthique pour la Recherche en Santé (CNERS) in Burkina Faso (protocol number 2013–7-066 and 2017–9-138). All parties linked to the intervention agreed on processing the data for this study. Information from informants was obtained anonymously.Table With a total of 102.609 identified ultra-poor persons, the financial costs amounted to USD 5,73 and the total economic costs to USD 11,83 per identified ultra-poor person.Sixty percent of the total financial costs  were incurred by activities carried out by the implementation agency. Forty percent of the financial costs  came about due to activities performed by the WB.Figure Table Table Figures The sensitivity analysis revealed that varying the wage for informal workers from minimum to average wage did not have a major impact on the total economic costs. By applying this variation, the total economic costs only changed by USD 50,506 (= 4.2%) and amounted to USD 1,263,953  accounted for less than one sixth of all financial costs, but for one third of all economic costs. Moreover, the high cost associated with preparing for and producing identification cards may be surprising for those unfamiliar with the intervention, since the two activities combined accounted for nearly 50% of all financial costs. While we have been reassured by key stakeholders  that the cost of card production was much lower in Vietnam compared to what it would have been in Burkina Faso, our appraisal of this specific set of findings inevitably questions the need for sophisticated individual digitalized cards vs. simpler household traditional paper-based cards. It is beyond the scope of our work to assess the comparative advantage of a system based on digitalized vs. traditional paper-based identification cards, albeit we do imagine this to be a relevant area for further research, estimating cost-effectiveness of one system compared to the other. Likewise, the government and its partners, in Burkina as elsewhere, could test the possibility of lowering CBT costs by integrating activities related to the database establishment into the initial identification effort.Furthermore, when assessing both the financial and the economic value of targeting interventions, one must consider the extent to which these are driven by national governments or by development partners . In the We also faced some challenges during data collection and analysis, which are worthwhile discussing here as potential limitations of our study. First, we assessed the value of resources consumed by SERSAP activities on the basis of the unit costs and the units consumed as indicated in the initial implementation budgets rather than in the closing financial statements, to which we could not gain access. Hence, we cannot fully exclude having over or underestimated unit costs or resource consumption for some items. Nevertheless, given that we triangulated information coming from SERSAP documentation with information obtained during the key informant interviews, we are quite confident that we have not largely misestimated these values. Second, our analysis did not account for any fixed costs incurred at SERSAP as the agency was already operative and the targeting process only built upon it. This might have led to an underestimation of the full cost of CBT. Third, we must also mention that tracing all activities and all related resource consumption was difficult due to the retrospective nature of our study. It would have been desirable to conduct a prospective study which could have generated more accurate data  by applyUniversal Health Coverage can only be achieved by ensuring that the poorest are not left behind. Thus, it is necessary that once implemented, measures to identify and address health inequity are systematically evaluated. With our study, we set the first example of how to systematically assess both the financial and economic costs of a community-based targeting program in a LMIC setting. We view the contribution of this costing study as meaningful, particularly because pro-poor targeting programs continue to expand, within and beyond the health sector. Future research should not only replicate this approach in other settings to generate additional and comparable evidence to better inform policy, but should also reach beyond the mere estimation of the costs to assess the costs of targeting in relation to the benefits accrued, within the framework of a comprehensive economic evaluation, such as a cost-effectiveness analysis.Additional file 1:Implementing actors with level, composition and responsibilities. (DOCX 16 kb)Additional file 2:Data Collection Form SERSAP. (DOCX 1872 kb)Additional file 3:Data Collection Form Regional Technical Assistants (ATR’s). (DOCX 2353 kb)Additional file 4:Self-administered Questionnaire WB. (DOCX 288 kb)Additional file 5:Interview Guide. (DOCX 519 kb)Additional file 6:Financial and economic costs in USD differentiated between fixed and variable costs and organizational level. (DOCX 24 kb)Additional file 7:Sensitivity Analysis: Wage Informal Workers (in USD). (DOCX 14 kb)Additional file 8:Sensitivity Analysis: Increase and Decrease of overhead costs (in USD). (DOCX 16 kb)Additional file 9:Sensitivity Analysis: Increase and decrease SERSAP Budget (in USD). (DOCX 15 kb)


West Nile virus is an arbovirus responsible for an infection that tends to peak during the late summer and early fall. Tools monitoring Web searches are emerging as powerful sources of data, especially concerning infectious diseases such as West Nile virus.This study aimed at exploring the potential predictive power of West Nile virus–related Web searches.Different novel data streams, including Google Trends, WikiTrends, YouTube, and Google News, were used to extract search trends. Data regarding West Nile virus cases were obtained from the Centers for Disease Control and Prevention. Data were analyzed using regression, times series analysis, structural equation modeling, and clustering analysis.In the regression analysis, an association between Web searches and “real-world” epidemiological figures was found. The best seasonal autoregressive integrated moving average model with explicative variable (SARIMAX) was found to be x4. Using data from 2004 to 2015, we were able to predict data for 2016. From the structural equation modeling, the consumption of West Nile virus–related news fully mediated the relation between Google Trends and the consumption of YouTube videos, as well as the relation between the latter variable and the number of West Nile virus cases. Web searches fully mediated the relation between epidemiological figures and the consumption of YouTube videos, as well as the relation between epidemiological data and the number of accesses to the West Nile virus–related Wikipedia page. In the clustering analysis, the consumption of news was most similar to the Web searches pattern, which was less close to the consumption of YouTube videos and least similar to the behavior of accessing West Nile virus–related Wikipedia pages.Our study demonstrated an association between epidemiological data and search patterns related to the West Nile virus. Based on this correlation, further studies are needed to examine the practicality of these findings. Flavivirus genus and to the Flaviviridae family that can cause West Nile fever. This mosquito-borne infection has a seasonal trend with peaks during summer and autumn. In 70% to 80% of West Nile fever cases, no or few symptoms are reported [West Nile virus, first isolated in Uganda in 1937, is a widely distributed arbovirus belonging to the reported .Corvus brachyrhynchos), Chilean flamingos (Phoenicopterus chilensis), and a snowy owl (Bubo scandiacus). New human cases were subsequently diagnosed in Brooklyn, the Bronx, and Manhattan. Since then, West Nile virus has spread to the contiguous states, from the Mississippi River to the Pacific Coast, causing further outbreaks such as those that occurred during the summer of 2002. Cases decreased from 2008 to 2011, but in 2012 a major outbreak took place with its epicenter in the Dallas-Fort Worth, Texas metroplex [West Nile virus was detected in North America for the first time in August 1999 during an outbreak that occurred in College Point, Queens, in New York City. A cluster of human encephalitis cases, all residing in the same 16-square-mile area, was identified by Drs Deborah Asnis , Marcelle Layton, and Annie Fine  . This cletroplex ,4. The TThe widespread resurgence of human West Nile virus disease in 2012 following several years of relatively low incidence has highlighted the continued public health hazard posed by West Nile virus, and has emphasized the need for more accurate predictive models of when and where new West Nile virus outbreaks will occur.Infodemiology (a portmanteau of information and epidemiology) and infoveillance (a portmanteau of information and surveillance) indicate the emerging “science of distribution and determinants of information in an electronic medium, specifically the internet, or in a population, with the ultimate aim to inform and improve public health and public policy” [Web-based tools are emerging as remarkable sources of data, especially for infectious diseases, by enabling Web search monitoring in real time and potentially capturing epidemiologically relevant information -7. Infod policy” . Systema policy” .Little is known about West Nile virus–related digital behavior. To the best of our knowledge, only a few authors have investigated this topic. Carneiro and Mylonakis  reportedr=.76 (P<.001) and r=.80 (P<.001) between Google searches and “real-world” epidemiological cases in the same study period on a monthly basis and a yearly basis, respectively. The presence of a regular pattern in West Nile virus–related Web queries was confirmed by the partial autocorrelation function analysis and by spectral analysis. From a geospatial point of view, correlation between digital behavior and epidemiological figures yielded r=.54 (P<.05).Bragazzi and collaborators  assessedHowever, the potential predictive power of West Nile virus–related Web searches has not yet been explored. To fill this gap in knowledge, we conducted this study.West Nile virus–related data were retrieved, downloaded, and analyzed from several novel data streams, including Google Trends, WikiTrends, YouTube, and Google News, as well as from epidemiological repositories.Google Trends (an open source tool) was mined from inception (2004) to 2015, by searching for West Nile virus in the United States and using the “search topic” option. This strategy enables one to systematically collect all the searches related to a given keyword or list of keywords , including synonyms and related terms, not just the precise string of characters typed by users . WikiTreMorbidity and Mortality Weekly Report, a publication of the CDC (data available on a trimester basis).Epidemiological data related to West Nile virus cases in the United States were obtained from the Centers for Disease Control and Prevention (CDC) website and the bulletins of the p , d (the degree of differencing of the model), q (the order of the moving average model), P , D , and Q  coefficients and s (lag parameter) were determined. The explicative variable was the number of West Nile virus cases. Different models were run, and the best one was chosen based on the Akaike information criterion (AIC), corrected AIC, and Schwartz Bayesian information criterion values. The best model was used to forecast Google Trends–based relative search volumes for 2016. Furthermore, structural equation modeling and clustering analysis were used to capture the complex interplay between the different novel data streams.Novel data streams-generated data were retrieved and downloaded from 2004 for Google Trends and 2008 for the other open source tools to 2015. All data were analyzed on a trimester basis. To detect a potential association with “real-world” epidemiological figures, regression analyses (with time as the confounding variable) were carried out. Furthermore, novel data streams-generated data were modeled as a time series and analyzed using time series analyses. In particular, a seasonal autoregressive integrated moving average model with explicative variable (SARIMAX) was used. By visually inspecting the autocorrelogram and the partial autocorrelogram based on the autocorrelation and partial autocorrelation function, respectively, P<.05 was considered statistically significant.Regression and clustering analyses were performed using SPSS version 24.0 , whereas the SARIMAX models and the structural equation modeling were carried out with XLSTAT . A Visual inspection of novel data streams–based data showed that each tool captured a specific digital behavior, generating specific curves which were not perfectly superimposable . Concerning temporal trends, only West Nile virus–related Web searches pattern well-reproduced the epidemiological trend, with most Google queries concentrated in August. For instance, the number of accesses to the West Nile virus–related Wikipedia page (as captured by WikiTrends) and the consumption of YouTube videos exhibited high search volumes also during winter months compared to Google Trends .P=.001) and of West Nile virus cases (P<.001) reaching statistical significance.Regression analyses showed a significant correlation between real-world epidemiological data and novel data streams-generated figures only for Google Trends data , with th4 , which was less close to the consumption of YouTube videos and least similar to accessing the West Nile virus–related Wikipedia page as captured by WikiTrends , which has so far not been used in predicting West Nile virus epidemiology. We explored different novel data streams  concerning seeking behavior and we were able to find a statistically significant association between epidemiological figures and digital behavior only in the case of Web searches as captured by Google Trends. Furthermore, we computed the best SARIMAX model for the period of 2004 to 2015, and we were able to forecast data related to 2016. Moreover, structural equation modeling and clustering analysis have enabled us to capture the complex interplay between the different novel data streams and the West Nile virus–related digital seeking behavior.Even if our experience suggests the usefulness of using Google Trends for predicting West Nile virus, this should be considered as a pilot study, calling for the need for making our model more accurate and reliable, and maybe incorporating other variables . This is of fundamental importance when designing and implementing a digital system for West Nile virus surveillance, which could complement the classical one or those actually under experimentation ,22. The Our study has some limitations that should be recognized. Some of the novel data streams used provide users with relative, normalized figures, and not with raw, absolute data, thus hindering further mathematical processing and statistical analysis. Another drawback is given by the fact that Google Trends captures only a portion of the entire population, namely the percentage of people using Google as their preferred search engine . Furthermore, we did not perform a content analysis of the West Nile virus–related material; from the existing literature, it is known to be of rather poor quality and to exhibit some degrees of inconsistencies ,24. For Statistically significant temporal correlations between West Nile virus epidemiological data and Google Trends suggest the feasibility of exploiting Google Trends as an internet-based monitoring tool. This is timely and of crucial importance given the recent re-emergence of arboviral infections. Workers in the field of public health and health authorities should be aware of the public interest and reaction to West Nile virus outbreaks in terms of Web searches. They could exploit the new information and communication technologies both for performing real-time monitoring of new population-based epidemic events and for carrying out a content analysis of the available online material, promptly replying to public concerns and correcting prejudices and inaccurate and misleading reports by disseminating high-quality information. However, based on the previously mentioned limitations of this paper, further studies are warranted to make our model more useful and practical.