Behçet’s Disease (BD) is a chronic auto-inflammatory, multisystem relapsing/remitting disorder of unknown aetiology. Oro-genital ulceration is a key feature of the disease and has a major impact on the patients’ quality of life. Other clinical manifestations include ocular inflammation, rheumatologic and skin involvement, while CNS and vascular complications can lead to considerable morbidity. The availability of a valid monitoring tool for BD activity is crucial in evaluating the impact of the disease on daily life activity. The aims of this study were to validate a novel tool for monitoring genital ulceration severity in BD and to assess the impact of genital ulcers on the Genital Health Quality of Life (GHQoL).Genital Ulcer Severity Score (GUSS) was developed using six genital ulcer characteristics: number, size, duration, ulcer-free period, pain and site. A total of 207 BD patients were examined,  from the multidisciplinary Behçet’s Centre of Excellence at Barts Health NHS Trust. GUSS was used in conjunction with Behçet’s Disease Current Activity Form (BDCAF).P < 0.0001). Ulcer average size and ulcer pain were the major predicting factors in GUSS  respectively, and P-values were significant. Multivariate regression analysis indicated that the ulcer pain, size and site are the main ulcer characteristics having an influence on the GHQoL .The over-all score of GUSS showed a strong correlation with all genital ulcer characteristics, and the strongest correlation was with the pain domain  contains supplementary material, which is available to authorized users. Behçet’s Disease (BD) is a chronic auto-inflammatory, multisystem, peri-vasculitis disorder , 2, firsThe prevalence of BD is highest in Middle Eastern countries such as Turkey where it occurs in approximately 370/100,000 inhabitants and in Iran with a prevalence of 80/100,000 . BD alsoBD disease course, severity, and systemic involvement between patients is variable, and the treatment depends on gender, age and weight  at presentation, therefore, it is challenging to determine a single management strategy . ColchicGenital ulcers caused by BD are the second most common manifestation of BD , occurriMucocutaneous lesions in BD are important in presentation and diagnosis, and are considered hallmarks of BD . MonitorIn this clinical prospective study, we have designed and validated a tool for measuring genital ulcerations that will benefit the assessment BD patients’ disease status with respect to current and prospective therapies. This study also adds to the previous data by analysing the impact of GUSS on daily activity. The four factors are; siting, walking, passing urine and sexual activity, that interfere with their GHQoL at the time of genital ulceration.This prospective study is a part of The City Research Ethical Committee (COREC) approved study “Immune-regulation at the mucosal barrier” P/03/122) and was carried out at Barts Health NHS Trust in full compliance with the Helsinki Declaration . A cohor22 and waThe exclusion criteria for the BD cohort were as follows; patients not fully diagnosed according to the ISG 1990; pregnant and lactating BD mothers were also excluded.The GUSS form has been developed as a modification of the OUSS tool established by Tappuni et al. . Six OUSTo complete the GUSS form, the ulcers’ characteristics were converted into numerical values, in order to assess the genital ulcers in BD patients for the preceding 4 weeks.Average number of ulcers: the score corresponds to the average number of ulcers per episode. i.e. Score 1 if there was one ulcer and score 2 if there were two ulcers etc. The maximum number is 20.Average size of ulcers: the score is the average of the ulcer diameter in millimetres. A size diagram was provided as circles at the bottom of the GUSS form. The maximum score is 20 mm. i.e. score 2 for the ulcer’s size of 2 mm etc. If the patient was uncertain of the size (between two different size circles) then an average of the ulcers’ size were recorded.Duration of ulcers: the score corresponds to the average duration of the ulcers and was calculated in ½ week units; i.e. Half week (3 days) = 1, one week and half = 3 etc. The maximum score is 10.Ulcer-free period: the score corresponds to the time free of ulcers in a period of 4 weeks; i.e. if the patient was free from ulcers for 1 week, the score = 3. The maximum score is 4 when the patient is never free from ulcers.Pain: The use of the validated pain visual analogue scale in the GUSS form allows the patients to quantify their pain during the time of ulceration. The minimum score is 0 (no pain) and the maximum score is 10 (severe pain).Ulcer site: this estimates the most frequent sites which are affected by ulcers in the genital skin/mucosa in males and females. Score 1 each for the most common affected sites and score 2 each for the less common affected sites in genital.Discharge: an evaluation by the patient answering if there was a fluid discharge or not at the time of ulceration.Evidence of scarring: evaluated by the clinician at the time of assessment.Genital Health Quality of Life (GHQoL): A scale from 0 (does not interfere with the GHQoL) to 10 (excruciating ulcers interfering with GHQoL) was included to correlate the GUSS with the patients’ GHQoL at the time of episodes in terms of .The guidelines for GUSS domains calculation were as follows:The BDCAF form  is a welTreatment decisions varied, depending on the patients’ disease activity and symptoms and were based on the European League Against Rheumatism (EULAR) guidance for Behçet’s disease , and Behvalidity is defined as the degree to which a scale correlates with a theoretic concept [The  concept . To asset-test and one-way ANOVA test were done to compare gender and age range groups with the GHQoL. The relationship between the variables was assessed by Pearson coefficient analysis. Multivariate regression analysis was performed for assessing the influence of the six characteristics of genital ulcers on the GUSS and the GHQoL, as the outcome measure resembled the normal distribution. The calculated P value < 0.05 was considered statistically significant. The correlation between the variables were ranked as “weak” or no association with values between 0 and 0.29 or (0 and −0.29), “moderate” with values between 0.3 and 0.69 or (−0.3 and −0.69), and “strong” if they were between 0.7 and 1 or (−0.7 and −1). The R-squared is the proportion of variance in the dependent variable that is explained by the additive combination of effects of the independent variables, and The ANOVA results indicate that the regression is significant or not significant. Beta value (standardised regression coefficients) is a measure of how strongly each predictor variables influences the dependent variable. The higher the beta value the greater the impact of the predictor variables on the dependent variable.The descriptive analysis was performed for mean and standard deviation values. The results were analysed by using the IBM SPSS Statistics software . Independent The genital ulcer severity of 137 females (mean age ± SD: 39.83 ± 13.42) and 70 males (mean age ± SD: 39.98 ± 11.95) was recorded. The frequency of genital ulceration in BD patients per a year was (mean of frequency ± SD: 7.13 ± 6.83), from the Multidisciplinary Behçet’s Centre of Excellence at Barts Health NHS Trust were monitored and calculated.n = 127) were active, whereas 33.8 % were inactive (n = 70). From the active disease group, a total of 69/207 (33.3 %) had oral ulceration, while  had genital ulcers and most of the genital ulcers were herpetiform and minor in size. Almost 26 % of patients had a fluid discharge during the ulceration period, and 18 % had scarring in their genital area. In term of harmful habits, 2 patients had a tattoo, 1 patient had metal studs and 5 patients was used excessive soap to clean the genital area. Only 19/207 (9.18 %) patients had oral and genital ulceration at the same time. 21.7 % (45/207) of patients presented with skin manifestations such as erythema nodosum, pseudofolliculitis and papulopustular lesions, while joint and central nervous system manifestations, commonly presented as headache, were 73/207 (35.2 %) and 42/207 (20.2 %) respectively. Ocular manifestations occurred in 33/207 (15.9 %) patients  were treated by multiple (more than 2) immunomodulatory medicines, 30 % (62/207) patients received two immunomodulatory medications. 51 received one immunomodulatory medication and 24 patients out of 207 required no medication at the time of presentation.The most common immunomodulatory medications were as follows: Prednisolone 41 % 85/207) used predominantly during relapsed episodes; Colchicine, 39.6 % (82/207); Azathioprine, 30.4 % (63/207); Mycophenolate Mofetil (MMF) 9.6 % (20/207) and Methotrexate, 3.4 % (7/207). Infliximab was prescribed to 7.2 % (15/207); Humira 5.7 %. Topical corticosteroids for the oral ulcers were used in 58.5 % (121/207) of patients who used either Betamethasone mouthwash or Triple Therapy Mouthwash (1 tablet Betamethasone 500 mcg + 1 tablet Doxycycline 100 mg + 1 ml Nystatin 100.000 unit/ml dissolved in 10 ml of water). The patients are required to keep this solution in their mouth for 3 min and no food is to be taken for 1 h after use. These were prescribed for the patients with high OUSS score for use up to 4 times a day. 17.4 % (36/207) of patients used topical steroids for genital ulcers. However, only 5 patients were using the topical steroid medication for their skin manifestations (Table 07 used pn = 10) followed by the penis shaft  and the tip of the penis . Ulcers in perianal and perineal area in males occurred in 8 %, (n = 2) each. In females the genital ulcers were most frequently recorded in the labia minora , followed by the labia majora at . Vulvar ulcers occurred in 18 % of patients (n = 17) while only 9 % presented with intravaginal ulcers. Perineal and perianal ulcers were recorded in 8 % and 4 % respectively, while ulcers in the cervical and groin area were rare . Women had worse GHQoL than men. However, due to the unequal number of male and female participants, this finding cannot be substantiated in this study  was shown in  and (31–43), within these age ranges males were higher in their GUSS than females Fig. , this wa in Fig. .Fig. 3GUThe correlation coefficient between genital ulcer characteristics and over-all GUSS showed a strong positive correlation and P values were significant with all ulcer domains Table .Table 3C2), and in this case the value was 0.998, this suggests that our model (which includes the six ulcer characteristics) explains 99.8 % of the over-all GUSS, and the regression analysis of GUSS with the six ulcer characteristics is statistically significant (P < 0.0001).The strength of the correlation and the influence of the variables on GUSS are explained by , with ulcer average size , and ulcer site . The relationship between genital ulcer domains and BDCAF showed a positive moderate correlation; with ulcer duration , then with ulcer average number  and have positive weak correlations with the rest of the ulcer characteristics . The beta values indicate that most of the contributions to difficulties in sexual activity, walking and sitting were due to ulcer pain and duration of ulceration, respectively. Ulcer pain was the only factor making a statistical significant contribution to passing urine.The multivariate linear regression analysis Tables  and 8, iThe present study is the first to use GUSS to assess genital ulcer severity, monitor disease progression and evaluate the impact of genital ulceration on Genital Health Quality of Life (GHQoL) in BD.QoL is a multidimensional measurement relating to all areas of human behaviour, which has been difficult to define and to measure because cultural, ethnic, religious and personal values influence the way that an individual responds to changes in QoL. Health Quality of Life (HQoL) attempts to measure how disease affects the quality of life. .BD is a chronic inflammatory multisystem disease with periods of exacerbation and remission that negatively impacts on the patients’ QoL, both due to disease itself or the impact of its symptoms .Our results demonstrate that genital ulcers are common in females and can occur on the labia, vulva and intravaginally. They present most commonly on the labia minora followed by labia majora. Genital ulcers are less common in males and are found most frequently on the scrotum. The frequent occurrence of genital ulcers in adults between 20 to 40 years may be related to a combination of environmental and hormonal factors . Most ofThe multivariate regression analysis demonstrates a strong positive correlation between genital ulcer characteristics and the over-all GUSS as well as indicating that the ulcer average size and ulcer pain were major predictive factors on over-all GUSS.The pain score significantly correlated with the average ulcer size particularly in patients with the herpetiform ulceration. This is an important observation as these ulcers are frequently missed on examination by clinicians, leaving patients unable to carry out basic activities such as sitting, walking, passing urine and sexual intercourse during periods of genital ulceration.Not surprisingly, the correlation between genital ulcer characteristics and BDCAF was moderate since not all patients had active systemic disease and genital ulcers concomitantly. A finding is supported by previous clinical observation and the BD epidemiology literature.In patients with active genital ulcers, the pain, size and site are the main ulcer characteristics which correlated with GHQoL [see Additional file Sexual activity was one of the major factors affecting the GHQoL of BD patients which may be linked to 1) constant pain before and after sexual intercourse, 2) the expectation of developing ulcers subsequent to sexual intercourse and 3) the sexual partner may have a large psychological component.Psychological support is needed in patients with genital ulceration in BD. The Behçet’s Centre of Excellence at Barts NHS Trust provides such support.However, to date there is little information in the literature regarding the negative impact of BD on the quality of patients’ sexual life . This suThe initial estimation of GUSS validity suggested that this instrument is a practicable and valid tool for assessing disease activity, disease progression and GHQoL.Further effectiveness of this scoring system will become more apparent over the next few years as the use of GUSS has now become an integral part of our routine patient clinical practice.


Thioureas, often contained within neoprene to provide water resistance, are an important cause of allergic contact dermatitis (ACD) in those who use neoprene products. We wish to present three cases of thiourea-induced ACD from three different orthopaedic supports containing neoprene. The first case was a 67-year-old woman who developed an itchy rash on her heel three weeks after using a neoprene insole for plantar fasciitis. The second case was a 47-year-old man who developed an itchy rash on his wrist after wearing neoprene wrist splints for psoriatic arthropathy. The third case was a 77-year-old woman who experienced a severe erythematous rash with blistering from a neoprene elbow brace she received following a humeral fracture. All patients were patch tested to the British Society of Cutaneous Allergy Standard and rubber series and a cut piece from all the relevant supports. At 96 hours, all patients had a + reaction to mixed dialkylthiourea, diethylthiourea, and the supports' material. No other positive patch test reactions were identified. As neoprene is fast becoming one of the most popular materials used for orthopaedic supports, awareness of this reaction and close liaison between dermatologists and orthopaedic surgeons are therefore essential to allow for early recognition of this complication. Neoprene is a popular material widely used in a variety of products including wet suits and computer mouse pads . Due to The first case was a 67-year-old lady who presented with an itchy dermatitis on her heel three weeks after she used a neoprene insole as treatment for her plantar fasciitis. The second case was a 47-year-old man who developed an itchy rash on his wrist after wearing three different types of neoprene wrist splints for his psoriatic arthropathy . The rasNeoprene, developed in 1931, is one of the earliest synthetic rubbers. Thioureas are a group of chemicals used to accelerate the curing process that creates neoprene rubber and helps provide its water resistant properties . The firThe general prevalence of positive patch test reactions to mixed dialkylthiourea has been reported as high as 2.4% , and theAlthough each patient described an adverse skin reaction to their support and had positive patch tests to the supports material and to thioureas, we were not able to prove directly that thioureas were contained within each of the orthopaedic supports. We had written to each of the companies who supplied the supports but two of these did not reply, and one company stated they were not able to provide information regarding the material used. Nevertheless, the combination of these three reactions described above makes a causal connection highly likely in these patients.Our cases highlight an important complication of neoprene support use in a variety of orthopaedic treatment modalities. Awareness of this potential reaction and close liaison between dermatologists, orthopaedic surgeons, rheumatologists, occupational therapists, and physiotherapists are therefore essential so as to allow for early recognition and appropriate investigation to be undertaken.


Salvia hispanica), an herbaceous plant native to Latin America, has become important in the last 20 years due to its beneficial effects on health. Here, we present the first record and identification of two viruses in chia plants. The comparison of the complete nucleotide sequences showed the presence of two viral species with the typical genome organization of bipartite New World begomovirus, identified as Sida mosaic Bolivia virus 2 and Tomato yellow spot virus, according to the ICTV taxonomic criteria for begomovirus classification. DNA-A from Sida mosaic Bolivia virus 2 exhibited 96.1% nucleotide identity with a Bolivian isolate of Sida micrantha, and Tomato yellow spot virus showed 95.3% nucleotide identity with an Argentine bean isolate. This is the first report of begomoviruses infecting chia as well as of the occurrence of Sida mosaic Bolivia virus 2 in Argentina.Chia ( Salvia hispanica) is an herbaceous plant of the Lamiaceae family, native to mountain areas of South America, from Mexico to Guatemala. Chia, along with corn and bean, was the basis of the diet of indigenous people and was third in economic importance [Chia  in S. uliginosa [S. splendens [Broad bean wilt virus 2 in S. officinalis [Mung bean yellow mosaic virus and Tomato yellow leaf curl virus) in S. splendens [Clerodendron golden mosaic China virus [To date, several viruses naturally infecting the genus liginosa  and S. splendens , Broad bicinalis , two begplendens  and anotna virus .Up to the present, there has been no information of viruses affecting chia crop. In Argentina, a high percentage of plants with typical symptoms of viral infections, such as mosaic, deformed and stunted leaves and chlorosis, which notably affected crop production, have ben detected in recent crop seasons. The aim of this work was to detect and identify the infections produced by virus in chia plants collected from production areas.In 2013, samples of chia plants showing leaf deformation, chlorosis and dwarfing  were colAlfalfa mosaic virus, Cucumber mosaic virus, Potyvirus genus, Tobacco mosaic virus and Tospovirus group  by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) [Rep gene, the entire common region and the 5’end of the CP gene of begomoviruses.The symptomatic chia plants were screened for the presence of S-ELISA)  using coS-ELISA) , which a2Cl, 0.25 mM of dNTP mix, 0.75 µM of each primer, 1 unit of Taq DNA polymerase  and 1 µL of DNA. PCR cycle parameters were as follows: 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 55 °C for 90 s, 72 °C for 90 s, and a final extension of 72 °C for10 min. PCR products were analyzed by electrophoresis in 1.5% agarose gels and visualized with UV light after ethidium bromide staining.Total DNA from chia plants was extracted using ZR-96 Plant/Seed DNA Kit™ . PCR reactions were prepared in a 25 µL volume containing 5X buffer, 3.5 mM MgEcoRI, EcoRV, XbaI, PstI, KpnI, HindIII and BamHI). Linearized fragments were cloned in pBluescript SK+ [Escherichia coli DH5α and sequenced at the Genomics Unit of the Biotechnology Institute-INTA (Argentina).Total DNA was subsequently used as template to amplify the putative full-length begomovirus genomes by rolling circle amplification (RCA) with Φ 29 DNA polymerase  as previously described . To seleript SK+  digestedThe nucleotide and deduced amino acid sequences were compared with those of other begomoviruses available in the GenBank . DatabasA phylogenetic analysis of the sequences was performed with Mega 5.2 software  using thNone of the tested samples reacted with any of the used antisera. PCR products of the expected size (1250 bp) were obtained for two of the five chia plants analyzed, confirming the presence of a begomovirus infection.XbaI and KpnI restriction enzymes for SM1 and SM2 clones, respectively, and with BamHI for TO1 and TO2 clones. The sequence analysis of clones SM1 (KJ742421) and TO1 (KJ742419) showed the typical genome organization of DNA-A of bipartite New World begomoviruses, with five open reading frames (ORFs), encoding replication-associated protein (Rep), transcriptional activator protein (TrAP), replication enhancer protein (REn), AC4, and coat protein (CP). Clones SM2 (KJ742422) and TO2 (KJ742420) encoded the movement proteins (MP) and nuclear shuttle proteins (NSP), included in the DNA-B of begomoviruses.Circular DNA genomes amplified by RCA were linearized with the Sida mosaic Bolivia virus 2  previously detected infecting plants of Sida micrantha in Bolivia [According to the ICTV criteria for differentiation of begomovirus species (89% nucleotide identity) , the com Bolivia , therefoTomato yellow spot virus  infecting bean (Phaseolus vulgaris) in Argentina [Leonurus mosaic virus (LeMV), and one of Sida micrantha mosaic virus (SimMV), whereas the isolates of Okra mottle virus (OMoV), Sida yellow mosaic virus (SiYMV), Sida mosaic virus (SiMoV) and Sida yellow net virus (SiYNV) showed identity between 80% and 89% (Leonurus sibiricus isolate (JX513953) [Lycopersicon esculentum isolate (DQ336351) [Leonurus mosaic virus might be a strain of ToYSV [The clone TO1 showed the highest nucleotide identity (95.3%) with DNA-A of an isolate of rgentina , but als and 89% . The cloX513953)  and 90.1Q336351) . Consideof ToYSV  and thatThe phylogenetic tree  was consSalvia hispanica associated with severe disease symptoms in chia. Possibly, it was transmitted by whiteflies and the presence of these begomovirus in Argentina must be considered as a threat to the chia crop, since whitefly populations are present in the north-western Argentina [Here, we demonstrate the presence of two bipartite begomovirus infecting rgentina . This inrgentina , more stSida mosaic Bolivia virus 2 in Argentina. The latter was previously reported to infect the weed S. micrantha [To the best of our knowledge, this is the first report of viral infection in chia plants, as well as of the presence of icrantha , and the


Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile immunity and is therefore proposed to be a Disabled Infectious Single Animal (DISA) vaccine candidate. In the absence of viremia, uptake of this vaccine strain by blood-feeding midges would be highly unlikely. Nevertheless, unintended replication of vaccine strains within vectors, and subsequent recombination or re-assortment resulting in virulent phenotypes and transmission is a safety concern of modified-live vaccines.Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides sonorensis midges was investigated. Virus strains were generated using reverse genetics and their growth was examined in vitro. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV with or without expressing NS3/NS3a and replication in the midge was examined using RT PCR. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies.The role of NS3/NS3a in replication and dissemination of BTV1, expressing VP2 of serotype 2 within colonized C. sonorensis after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within Culicoides.Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges. Reoviridae, genus Orbivirus) .–34.Culicorensis) , a speciorensis) .Fig. 1C−1 Penicillin, 100 μg ml−1 Streptomycin and 2.5 μg ml−1 Amphotericin B, at 37 °C with 5 % CO2. KC cells 2 virus stock was 106.4 TCID50 ml−1, DISA 2 had a titer of 105.8 TCID50 ml−1(intended dose for insect experiments was 106 TCID50/ml). Wild type BTV11, used as a positive control, had a titer of 108.2 TCID50ml−1.Virus titers were determined by endpoint dilution on BSR cells and expressed as 50 % tissue culture infectious dose per ml  using standard procedures .6) or BSR cells (5 × 105) in 2 cm2 wells were infected with an MOI of 0.01. Virus was adsorbed for 1.5 h at 27 °C or 37 °C for KC and BSR cells respectively. Unattached virus was removed by washing with PBS, and fresh medium was added. This time point was set as 0 h post-infection (hpi). Incubation was continued and cells and culture media were harvested at indicated time points. Cells were lysed to be able to study the intracellular BTV fraction, by freeze thawing at −80 °C. Virus titers were determined and growth experiments were independently repeated.Monolayers of KC cells . Engorged females were put in cardboard cages with cotton-plugged vials containing 10 % sucrose and held at 26 °C. At 0, 7, 10 (BTV11 control only) and 14 days post feeding (dpf), 50 midges were anesthetized with CO2and heads were separated from bodies using ultra-fine tweezers  and a dissecting microscope . Heads and bodies were separately placed in 100 μl RNAlater , and stored at 4 °C.Colonized 3–4 day old female  KS, USA  were off KS, USA . Midges C. sonorensis midges were injected intrathoracically with 46 nl of virus stock using the Nanoject II microinjector  under a dissecting microscope (Nikon Instruments). This volume was based on hemocoel injection capacity of the midges, highest rate of consistency, complete fluid retention, and maximum post inoculation survival. Total virus injected per midge, based on highest starting virus stock titers possible, was 101.5 TCID50 of DISA 2 and 102.1 TCID50 of BTV1[VP2]2 virus. Inoculated midges were placed in cardboard cages with cotton-plugged vials containing 10 % sucrose and held at 26 °C. At 0, 7, and 10 days post inoculation, 25 midges were anesthetized, decapitated and stored in RNA later (Qiagen) at 4 °C as described above. Time 0 samples were taken from 1 to 4 h post-injection. This variation was due to the time-intensive nature of microinjecting large numbers of midges needed to ensure adequate numbers of surviving midges for each time point.Colonized 3–4 day old female Cp) values of each PCR were calculated, and negative results were arbitrarily set as 45 to allow these to be included in the analysis. Due to the maximum of 45 cycles, the highest Cp value that could still be calculated was 40. Statistical differences in Cp values were calculated using a one way ANOVA and subsequent Tukey’s multiple comparison test, with p < 0.001 indicating significant differences between days or groups.PBS (400 μl) and one 5 mm stainless steel ball (Qiagen) were added to midge bodies and heads in RNA later in micronic tubes. Tubes were shaken for 3 min at 50 Hz in a tissue lyser . After centrifugation, 200 μl of supernatant was used for RNA isolation using the MagNApure 96 DNA and viral NA Small Volume kit (Roche) by the MagNApure isolation robot (Roche) according to the manufacturer’s protocol . The rea7.2 TCID50 ml−1) compared to DISA 2 (104.9TCID50 ml−1) at the end of the experiment at 72 h post infection. Release of virus was also reduced for DISA 2 (103.1 TCID50 ml−1) compared to the parental virus (106.5TCID50 ml−1). Growth in KC cells was highly attenuated, with ten-fold lower virus titer in both the cell fraction and the released fraction for DISA 2 at the end of the experiment. DISA 2 virus release was diminished, with 102.1 TCID50 ml−1 at 0 dpi and still only 102.6 TCID50 ml−1 at 120 dpi, the end of the experiment , whereas heads were negative (13 out of 25) or had very high Cp values (mean Cp:39.5). As blood meals are digested after 3 dpf, the Cp value at 7dpf in the bodies , was slightly higher, but decreased significantly until day 10  (p < 0.001), indicating virus replication in the midge. From day 10 to 14 , no significant difference was found, suggesting no further increase of the virus titer in midge bodies. To study the ability of BTV11 to cross the midgut barrier and to replicate in the midge, viral RNA in the heads was determined. Cp values were significantly lower at day 7 , day 10;and day 14  compared to day 0 (p < 0.001) and were declining in time. This clearly demonstrated midgut escape and virus replication in colonized C. sonorensis by BTV11.BTV11-fed midges were harvested at day 0, 7, 10 and 14 dpf. For each day of sampling, heads and bodies of 25 midges were harvested and were tested individually. Viral RNA was isolated for PCR testing and semi-quantitated by ues Fig. . DirectlCp values  not significantly different from that of bodies of BTV11 fed midges. At day 7, 40 out of 50 bodies were PCR negative, and at day 14 post feeding only 6 out of 50 bodies had a Cp value of 40. All heads were PCR negative at day 0, 7 and 10, except for one head at day 0 and 7 (Cp:40). These results indicated that BTV1[VP2]2 was unable to infect the midgut epithelium and was unable to escape the midgut barrier in C. sonorensis. Feeding with DISA 2 virus was also performed, but harvested heads and bodies were not further processed because of the negative results of the BTV1[VP2]2 parental strain.Similarly, 50 BTV1[VP2]2 fed midges were harvested at day 0, 7 and 14, and tested by PCR. At day 0, directly after feeding, bodies showed Intrathoracic injection of virus into the hemocoel of midges can lead to replication of virus unable to naturally escape the midgut following oral uptake , 47. TheCp: 31.3, range 40–30) and DISA 2 . There was no significant difference between both groups. Injected virus was also detected in the heads of 23 out of 25 midges on day 0 due to dissemination throughout the hemocoel. At both day 7 and 10 post injection of BTV1[VP2]2, mean Cp values in the bodies were 23.7 and 23.2 (range: 26.5-21.9 and 24.4-20.3 respectively), indicating significantly higher RNA levels than that of input virus. In addition, Cp values of tested heads on these days were also significantly lower (p < 0.001)  compared to day 0 (mean Cp: 36.1). These results clearly indicate virus propagation of BTV1[VP2]2 throughout injected C. sonorensis midges.On day 0, RNA was detected in all bodies for BTV1[VP2]2  at day 7  compared to day 0 (mean Cp: 40.5 range 45–35.6). However, at day 10, the mean Cp value was not significantly different from day 0 . Remarkably, even at the day 0 time point (1–4 hpi), DISA 2 injected midges had a higher mean Cp value of 40.5 (range: 45–35.6) in their heads compared to BTV1[VP2]2 . Unlike the parent BTV1[VP2]2 virus strain, DISA 2 was unable to propagate after injection of colonized C. sonorensis. Because these viruses only differ in NS3/NS3a expression, we conclude that NS3/NS3a proteins play a crucial role in virus propagation in the insect vector.In contrast, DISA 2 injected midges did not show significantly lower in vitro 2 and an American BTV11 isolate. This BTV11 isolate was included to confirm ‘normal’ feeding behaviour of this specific midge colony and was fed with a very high dose of 108.2 TCID50 ml−1, since this dose led to virus replication in numerous previous experiments (not shown). Unfortunately, the BTV1[VP2]2 strain was not able to escape the midgut barrier as evidenced by the lack of viral RNA in the heads. The results of day 0 post feeding suggests a similar starting dose in the midguts of both groups by PCR testing. However, the blood meal for BTV1[VP2]2 contained 100 times less infectious virus than that of BTV11 as determined by titration. This might be explained by the presence of non-infectious particles in the BTV1[VP2]2 virus stock, although this needs further investigation. The lower dose could explain the negative results for BTV1[VP2]2, but a similar dose of about 106 TCID50 ml−1 of BTV1, as used in our study for BTV1[VP2]2,has been used already successfully in several vector competence studies using C. sonorensis 2 and DISA 2 to propagate within Culicoides.Only a very few s of BTV , 50–53. s of BTV , 54–57. p < 0.001) (higher Cp), compared to BTV1[VP2]2 at day 0. This might be due to possible variations in exact sampling times post inoculation for the day 0 time point (1–4 hpi) or differences in amount of virus delivered in the 46 nl injection (101.5 vs. 102.1). Less time between injection and decapitation could lead to incomplete dissemination of virus to the head via the hemolymph. Significantly higher DISA 2 RNA levels  were measured in the heads at day 7 compared to day 0, which is likely due to full dissemination of injected virus at this time point. Indeed, the Cp value at day 10 was higher again compared to day 7, suggesting degradation of viral RNA. A few individual bodies and heads of DISA 2 fed midges showed a Cp value lower than the average at day 7 and 14. This might indicate a low level of replication in these midges. However, compared to BTV1[VP2]2 fed midges, Cp values are still high, and spread via the salivary glands to the ruminant host is highly unlikely.Replication of BTV is likely after intrathoracic injection, even for viruses that do not replicate in the midge after oral uptake . Indeed,Culicoides cells. It is also possible that the delayed release of DISA 2 from the cell results in faster virus clearance by intracellular innate immunity such as RNAi [In conclusion, NS3/NS3a knockout strongly reduces virus release and propagation in the midge after injection, once the midgut barrier has been passed. This is likely due to the strongly reduced virus release, also seen  as RNAi , 59, Tol as RNAi .C. sonorensis.The virus backbone used in DISA vaccines is based on non-virulent BTV and is safe with respect to fever and clinical signs . CurrentLive-modified vaccines for orbiviruses potentially imply several risks, such as reversion to virulence, re-assortment events and uncontrolled spread of vaccine virus. An elegant method to combat these major disadvantages is to target functions that are specific and essential for virus propagation in the insect vector, and for virus transmission between host and vector. Most likely, similar NS3/NS3a deletion mutants for other orbiviruses will have similar characteristics , whereas


Fabavirus. Polyproteins RNA-1 and RNA-2 of the virus tentatively named grapevine fabavirus (GFabV) shared 34 to 23% sequence identities with Broad bean wilt virus 2 (BBWV2), respectively. GFabV was successfully graft transmitted to Vitis vinifera cv. Cabernet Franc.A novel virus-like sequence from grapevine was identified by Illumina sequencing. The genomic organization was most similar to that of members of the genus  Fabavirus, family Secoviridae, consists of ﬁve viral species: Broad bean wilt virus 1 (BBWV1), BBWV2, Gentian mosaic virus, Cucurbit mild mosaic virus, and Lamium mild mosaic virus. Fabaviruses infect a wide range of monocotyledonous and dicotyledonous host plants, and they are nonpersistently transmitted by aphids (Vitis vinifera cv. Black Beet (BB) and Nagano Purple (NP) introduced to the Foundation Plant Services (FPS), Davis, CA, from South Korea in 2013. To explore the virome of the NP and BB selections, total RNA was extracted from leaf petiole tissue using the MagMAX-96 viral RNA isolation kit, according to the manufacturer’s instructions, and analyzed by high-throughput sequencing (HTS) using the Illumina NextSeq 500 platform. The analysis generated approximately 32 and 34 million Illumina reads (~50 nucleotides [nt] in length) for NP and BB, respectively. Bioinformatics analysis was performed as described by Al Rwahnih et al. , thus indicating that it is a distinct member of the genus Fabavirus based on the established criteria (Vitis vinifera cv. Cabernet Franc test plants, as described by Al Rwahnih et al. (Fabavirus in North America and the first sequence evidence of its occurrence in grapevines. Field surveys and biological studies are under way to determine the prevalence of GFabV in California, evaluate its potential natural spread, and assess its effect on vine performances and wine quality.The genus KX241482 to KX241485.The GenBank accession numbers for the sequences of this virus are


Slave trading of Africans to the Americas, during the 16th to the 19th century was responsible for the first recorded emergence in the New World of two arthropod-borne viruses (arboviruses), yellow fever virus and dengue virus. Many other arboviruses have since emerged from their sylvatic reservoirs and dispersed globally due to evolving factors that include anthropological behaviour, commercial transportation and land-remediation. Here, we outline some characteristics of these highly divergent arboviruses, including the variety of life cycles they have developed and the mechanisms by which they have adapted to evolving changes in habitat and host availability. We cite recent examples of virus emergence that exemplify how arboviruses have exploited the consequences of the modern human lifestyle. Using our current understanding of these viruses, we also attempt to demonstrate some of the limitations encountered in developing control strategies to reduce the impact of future emerging arbovirus diseases. Finally, we present recommendations for development by an international panel of experts reporting directly to World Health Organization, with the intention of providing internationally acceptable guidelines for improving emerging arbovirus disease control strategies. Success in these aims should alleviate the suffering and costs encountered during recent decades when arboviruses have emerged from their sylvatic environment. However, many other RNA viruses have emerged or reemerged and dispersed globally despite being considered to be neglected diseases.3 Chikungunya virus (CHIKV), West Nile virus (WNV) and dengue virus (DENV) are three of a large number of neglected human pathogenic arthropod-borne viruses (arboviruses) whose combined figures for morbidity and mortality far exceed those for Ebola, severe acute respiratory syndrome and Middle East respiratory syndrome viruses. For instance, for DENV, the number of cases of dengue fever/hemorrhagic fever is between 300–400 million annually, of which an estimated 22 000 humans die.4 Moreover, in the New World, within 12 months of its introduction, CHIKV caused more than a million cases of chikungunya fever according to Pan American Health Organization/World Health Organization, with sequelae that include persistent arthralgia, rheumatoid arthritis and lifelong chronic pain.5 Likewise, within two months of its introduction, to Polynesia, the number of reported cases exceeded 40 0006 and is currently believed to be approaching 200000 cases. Alarmingly, this rapid dispersion and epidemicity of CHIKV (and DENV or Zika virus in Oceania) is now threatening Europe and parts of Asia through infected individuals returning from these newly endemic regions. This is an increasingly worrying trend. For example, in France, from 1 May to 30 November, 2014, 1492 suspected cases of dengue or chikungunya fever were reported.7 Accordingly, this review focuses on the emergence or reemergence of arboviruses and their requirements and limitations for controlling these viruses in the future.Despite the announcement of the successful eradication of smallpox in 1979, the last case of rinderpest in 2008 and the current campaigns to eradicate poliomyelitis and measles through mass-immunization programmes, we still face the prospect of emerging or reemerging viral pathogens that exploit changing anthropological behavioural patterns. These include intravenous drug abuse, unregulated marketing of domestic and wild animals, expanding human population densities, increasing human mobility, and dispersion of livestock, arthropods and commercial goods via expanding transportation systems. Consequently, the World Health Organization concluded that acquired immune deficiency syndrome, tuberculosis, malaria, and neglected tropical diseases will remain challenges for the foreseeable future.8 Many arboviruses are zoonotic, i.e., transmissible from animals to humans.10 As far as we are aware, there are no confirmed examples of anthroponosis, i.e., transmission of arboviruses from humans to animals.10 The term arbovirus is not a taxonomic indicator; it describes their requirement for a vector in their transmission cycle.12 Humans and animals infected by arboviruses, may suffer diseases ranging from sub-clinical or mild through febrile to encephalitic or hemorrhagic with a significant proportion of fatalities. In contrast, arthropods infected by arboviruses do not show detectable signs of sickness, even though the virus may remain in the arthropod for life. As of 1992, 535 species belonging to 14 virus families were registered in the International Catalogue of Arboviruses.12 However, this estimate is continuously increasing as advances in virus isolation procedures and sequencing methods impact on virus studies. Whilst many current arboviruses do not appear to be human or animal pathogens, this large number of widely different and highly adaptable arboviruses provides an immense resource for the emergence of new pathogens in the future.Arboviruses are transmitted between arthropods  and vertebrates during the life cycle of the virus.Aedes and Culex mosquitoes are the species most frequently associated with arbovirus transmission .12 Ticks are also prevalent vectors, 116 different species are currently known to transmit arboviruses. In addition, 25 midge species have been shown to transmit arboviruses, mainly Culicoides (24 types) and Lasiohelea. Sandflies, blackflies, stinkbugs, lice, mites, gadfly, and bedbugs can also transmit arboviruses.13 This diversity of species and the wide distribution of these transmission vectors explain why arboviruses are so successful in dispersing globally via the mechanisms highlighted earlier.12 Arboviral diseases are primarily associated with specific vectors. However, many other arthropod species, in which viruses have been identified, may be involved in perpetuating the virus life cycle without having been associated with overt disease in humans or animals. For example, WNV is typically mosquito-borne but can be vectored by many different mosquito species and also by ticks and other arthropods.12 Moreover, Japanese encephalitis virus appears to be transmissible by Culex, Anopheles, and other mosquito species, as well as midges, sandflies and ticks. As a general rule, a specific arthropod species, is likely to predominate during an epidemic. However, if the availability of the vertebrate host, for example birds, becomes limited towards the end of the summer as the birds migrate to warmer countries, the vector might switch its preference to a different vertebrate host. This is consistent with the observation that outbreaks of West Nile fever/encephalitis in North America often appear to occur in the late summer, following peak incidence in birds, after they commence their migration to warmer regions.14 Alternatively, Aedes aegypti has adapted to the urban environment whereas Aedes albopictus occurs more commonly in semi-urban or rural areas. However, these are not hard and fast rules. For example, on the French island of La Reunion, where Aedes aegypti was not recognizably present, CHIKV was simultaneously epidemic in both rural and urban environments, and was primarily associated with Aedes albopictus.15In addition to evolving strategies for long-term survival, arboviruses have an enormous choice of arthropod species potentially capable of being infected the predominant species of which appear to be mosquitoes and ticks. Approximately 300 types of mosquito can transmit arboviruses. Culex tritaeniorhynchus mosquito species and intensely farmed pigs which act as amplifying hosts for the virus and, importantly, the mosquitoes.17 In Southeast Asia, rice cultivation with enormous areas of paddy fields also attracts migratory birds and provides ideal breeding areas for the mosquitoes which transmit the virus to the birds, ensuring virus dispersion over large areas of Asia.18 In contrast, tick-borne encephalitis virus occurs mainly in northern temperate regions which are the primary habitats of Ixodes species ticks (forests etc.).19 Nevertheless, even within this relatively localized distribution of arboviruses, dispersion to distant locations occurs via animal or vector migration. As an example, Powassan virus, a close relative of tick-borne encephalitis virus, is found in Far East Asia and also in Canada.19 In contrast, Rift Valley fever virus (RVFV) was localized in Africa but in 1977, it was introduced to the Middle East, where it caused thousands of human infections, with an estimated 598 deaths, during a single epidemic.20A high proportion of arboviruses associated with human and animal disease circulate in tropical, and subtropical regions, where mosquitoes, and other flying insects, tend to be abundant. However, many arboviruses also circulate amongst wildlife species in temperate regions of the world. Despite the global distribution of viruses such as WNV, dengue virus, bluetongue virus and now CHIKV, most other arboviruses are generally endemic to specific regions of the world. For example, mosquito-borne Japanese encephalitis virus is prevalent in India, Central and Southeast Asia, largely due to the prevalence of highly competent 12 Four virus families, Togaviridae, Flaviviridae, Bunyaviridae and Reoviridae, contain most of the arboviruses that cause human/animal diseases.12 Arbovirus infections are not always clinically obvious and often resolve spontaneously after 1–2 weeks. However, some arboviral infections result in high fever, hemorrhage, meningitis, encephalitis, other serious clinical symptoms, and even death. Therefore, they cause a large social and economic burden. A summary listing the arboviruses associated with human diseases and their geographic distributions was published previously.21More than 100 species of arbovirus that cause human/animal or zoonotic diseases have been identified.21 Eggs can also provide a long term reservoir for tick-borne arboviruses but in many cases the viruses exploit the extended life cycle of some tick species, surviving for years through the transstadial stages, reproducing at low levels.21 This long-term survival strategy is also enhanced by non-viraemic transmission during which infected and non-infected ticks co-feed on small animals in the forests. Non-viraemic transmission provides an efficient mechanism for transmission of the virus directly between ticks, without necessarily infecting the vertebrate host. Whilst co-feeding is taking place, virus infectivity in the tick salivary glands may increase by orders of magnitude presumably increasing virus transmission efficiency between the ticks.22 A similar non-viraemic co-feeding transmission process involving mosquitoes and blackflies, has also been described for WNV and vesicular stomatitis virus.23 Alternatively some insect-specific flaviviruses exist in the form of DNA when they infect insect cells.24 Diverse sequences have been discovered that appear to be related to insect-specific flaviviruses, amplified from mosquitoes, belonging to the culicine genera Culex, Aedimorphus, Ochlerotatus and/or Stegomyia. Many of these sequences may represent DNA integrations into mosquito genomes.34 Moreover, sRNAs related to WNV in apparently uninfected Culex mosquitoes have also been identified.35 Whilst there is no current evidence that such forms of DNA, provide a long-term arbovirus survival strategy, the similarity with other viruses that use DNA intermediates and episomal DNA should not be ignored. These and other diverse survival strategies provide safe havens for their long-term survival from which they can reemerge to cause epidemics amongst human and animal populations.Arboviruses have evolved a wide variety of strategies to ensure their long-term success, dispersal and survival. They associate with specific arthropods and exhibit distribution characteristics that reflect the environmental preferences of these particular species. However, once an epidemic has run its course, the virus survives via its sylvatic life cycle which may involve a wide variety of species currently not identified. Alternatively, arboviruses can be maintained for months or maybe even years in mosquito eggs which remain dormant until the rainy season triggers hatching of the new, healthy but infected mosquito larvae.Aedes albopictus.36 Coincidentally this mosquito species has gradually dispersed westwards and CHIKV is now a major global human epidemic pathogen throughout Asia.5 Moreover, on 6 December 2013, it was reported to have crossed the Atlantic Ocean, reaching the French Caribbean island of Saint Martin from where it dispersed to the Americas.37 It also dispersed eastwards from southern Asia, reaching Polynesia by October 2014.6 Worryingly, CHIKV is now frequently being introduced into non-endemic Europe and northern Asia by incoming humans infected in the Americas and Polynesia. In the Americas, WNV, was first reported in New York in August 1999,38 following a hot and humid summer and many publications describe its emergence and dispersal.41 In contrast with CHIKV, the major determinant for the dispersal of WNV, throughout North America, over a period of five years was primarily birds and their associated Culex mosquito species.36 The dispersal and increasing epidemicity of dengue fever/dengue hemorrhagic fever which is confined to humans in the tropics, sub-tropics and southern temperate regions, can generally be attributed to human and Aedes aegypti population density increase during the past century, resulting from intensive urbanization and the influence of increased transportation of humans, commercial goods, livestock and major military movements across the oceans.42 On the other hand, RVFV is enzootic and confined to a wide range of animals, mosquitoes and sandflies throughout Africa and the Arabian Peninsula. Generally, the virus circulates without causing major disease outbreaks which usually arise following periods of rainfall when herded ruminants are introduced to RVFV-endemic areas. Two important factors are believed to have influenced the epidemiology of RVFV, firstly, major irrigation projects and secondly, the El Niño effect.43 In each case, cited above, a combination of two or more of the factors identified earlier have had an important influence on the appearance of these emerging arboviruses in new territories and/or old territories. Other recently emerging arboviruses, not included in this brief outline, include, Zika virus in Oceania, bluetongue virus and Schmallenberg virus in northern Europe and Bagaza virus in Spain.47Changing anthropological behaviour, climate change and high mutation frequency are important determinants of arbovirus emergence. Arboviruses adapt readily to new susceptible hosts by alteration of receptor specificity, transmission efficiency, antigenicity, and ecological and environmental conditions. Humans, livestock and/or domestic animals are not an essential part of this arbovirus life cycle. Therefore, unlike, smallpox virus, measles virus or poliovirus, arbovirus disease control based on humans, livestock and/or domestic animals cannot eradicate the arbovirus. Consequently, the reservoir for arboviruses in wild species places a limitation on our ability to control disease emergence. For instance, CHIKV was a zoonotic arbovirus that cycled harmlessly between simians and mosquitoes in the African tropical forests causing localized outbreaks of polyarthralgia in humans. It also occasionally “escaped” to Asia gradually becoming zoonotic. However, prior to 2005, CHIKV was rarely an epidemic arbovirus until a mutation occurred in the gene encoding the surface protein of the African strain which increased its capacity to infect, reproduce and be transmitted by the striped Asian “Tiger” mosquito 48 and 40 years later developed by Carlos Finlay49 who proposed mosquitoes as the agents for transmission of yellow fever. Subsequently, empirical methods, such as mosquito eradication, which was used very successfully in Cuba,50 and the development of a yellow fever vaccine has protected millions of humans from potentially fatal infection by yellow fever virus.53 Now, in the 21st century genetically engineered live attenuated vaccines can be manufactured within months, to protect humans against the ravages of pandemic influenza and other virus diseases. Moreover, a spectrum of antiviral molecules has been developed to treat humans dying from infection with human immunodeficiency virus and several antiviral drugs have also been developed against other viruses. Does this mean that if we develop vaccines and antiviral drugs to prevent or treat humans against infection by pathogenic arboviruses we will resolve the challenges associated with emerging arboviruses? Regrettably it is not that simple! It is a remarkable fact that in the future, because of their high mutation rates, many new pathogenic arboviruses will emerge even though they do not currently exist as epidemic strains in the sylvatic environment. It is also becoming clear, from early results of genome sequencing, that mosquitoes carry large numbers of known and unknown viruses that infect humans, primates, mammals, birds, insects, and plants.54 Therefore, should we attempt global eradication of arthropods? The answer is a definite no. This would have a catastrophic impact on the survival of many wildlife species, as first became apparent when dichlorodiphenyltrichloroethane was used widely as a general insect control agent rather than being precisely targeted to relevant mosquito species.55 However, implementation of temporary localized arthropod control measures during epidemics, for example in high density urbanized areas, can still play an important but transient role in reducing the impact on humans and animals of emerging arboviruses.The concept of arthropod-borne disease transmission was born out of the studies of a physician, Josiah Clark,The breadth and depth of arbovirus surveillance differs regionally, and several areas lack surveillance altogether. There is also a lack of inter-disciplinary expertise on arbovirus diseases and understanding their vectors, and epidemiology. In addition, only a small number of arboviral diseases can be prevented using vaccines or specific antiviral drugs, and there are few validated diagnostic reagents, with which to monitor disease progress and control. Until such disease control reagents become available, the most effective alternative is to focus on practical procedures to reduce risks of exposure to arthropods.It is clear that we are now entering a period in virological discovery that will reveal a “Pandora's box” of new viruses with unique characteristics, which circulate in the ecosystem and will potentially lead to the evolution of novel pathogenic arboviruses. Future disease control strategies must therefore recognize this and be planned accordingly. What are the major objectives that need to be addressed if we are to win the war against these versatile viruses that appear to have an infinitesimal variety of strategies to confuse and disorganize our ability to bring them under control? (i) Develop vaccines to reduce the incidence of disease caused by known viruses; (ii) develop therapeutic drugs to treat clinical diseases caused by known viruses; (iii) develop unified vector-controlled strategies that will not unduly threaten the survival of wildlife species but locally will reduce the risk of disease in humans and animals; (iv) develop universal teaching/training courses to be taught worldwide, to provide cores of expertise to implement these policies; (v) encourage the strengthening of levels of cooperation between academia and drug and vaccine development companies; (vi) encourage the development of research programmes to understand the underlying mechanisms of arboviral pathogenicity, evolution, emergence and dispersal; (vii) develop, at the international level, public health measures to inform and educate citizens in local arboviral disease control measures, including monitoring and reporting; (viii) implement measures to improve monitoring procedures at borders, harbours, airports to reduce the influx of arthropods to new countries; (ix) develop unified public health strategies for arboviral disease control; (x) simplify the procedures for establishing safety and efficacy of antiviral drugs; (xi) establish an international committee of experts charged with the objective of reviewing global arthropod control strategies; (xii) develop and implement internationally acceptable and user-friendly guidelines for avoiding exposure to the different types of arthropod likely to carry human pathogens. This is undoubtedly an incomplete list of recommendations and probably some recommendations cannot easily be implemented. Nevertheless, the list provides the building blocks for a unified strategy on which to develop methods that will reduce the high morbidity and mortality rates due to human or animal arbovirus infections. Finally, we also have to recognise that arboviruses and arbovirus-related viruses infect more than just humans, invertebrates and land-based animals. They also infect plants, fish and marine animals. These relatively new discoveries also need to be accommodated if arboviral disease strategies are to be developed in a rational and unified manner. The lesson of the past is that human endeavour is at its best in the face of adversity. Let this be the mantra for arbovirus disease control in the future!


Dicistroviridae; however, it shares <33% translated amino acid sequence within the nonstructural open reading frame (ORF) from aparavirus or cripavirus.We report the draft genome sequence of goose dicistrovirus assembled from the filtered feces of a Canadian goose from South Lake Union in Seattle, Washington. The 9.1-kb dicistronic RNA virus falls within the family  Branta canadensis) are well known for their prolific production of feces, depositing up to one dropping per minute and up to 1.5 lb per day  in phosphate-buffered saline (PBS), vortexed, and syringe-filtered through a 0.8-µm filter (Millipore). RNA from 1 mL of filtrate was extracted using the Zymo RNA miniprep kit using on-column DNase treatment. Sequencing library preparation from cDNA was performed using Nextera XT with 15 cycles of PCR amplification as described previously , 8. A tong Abyss .Dicistroviridae family. The contig had 55× coverage and contained two large open reading frames (ORFs) of 5,481 and 2,577 nucleotides, along with a 352-nucleotide intergenic region. The first ORF contained conserved domains for RNA helicase, C3G peptidase, and RNA-dependent RNA polymerase, while the second ORF contained conserved domains for picornavirus and cricket paralysis virus (CRPV) capsid proteins. The putative translated nonstructural protein gene sequence best aligned with 33% amino acid identity to cricket paralysis virus (AKA63263) while the capsid protein best aligned with 35% amino acid identity to Drosophila C virus (NP_044946). The translated nonstructural gene was also 32% identical by amino acid to the acute bee paralysis virus polymerase protein, indicating substantial divergence from both cripavirus and asparaviruses of the Discistroviridae family.A 9,130-nucleotide contig was assembled that aligned by blastx to members of the The genome likely encodes an insect virus that is only found associated with goose feces, rather than a bona fide goose virus. Of note, a 4,177-nucleotide contig was also assembled that aligned with 96% identity by nucleotide to subterranean clover mottle virus isolate MJ (AY376453) . FurtherKT873140.The GenBank accession number for goose dicistrovirus is


Road Traffic Injuries (RTIs) as a result of road traffic crashes (RTCs) rank as the leading cause of death, disability and property loss worldwide, especially in low and middle-income countries. This study aims to analyze the costs of RTCs in Iran.A standard human capital approach was used to estimate the costs. Costs included medical, administrative and funeral costs, property damage, production lost and intangible costs. Data about the number of deaths and injuries resulting from RTIs between 20 March 2009 and 20 March 2010 was obtained from two national databases designed at the Center for Disaster Management and Medical Emergencies (CDMME) and the Legal Medicine Organization (LMO), respectively. The severity and medical costs of injuries were identified by reviewing 400 medical records that were selected randomly from patients who were admitted to two large trauma centers in Shariati and Sina hospitals in Tehran province. Moreover, information about production lost, property damage, rehabilitation cost, intangible costs and administration costs were collected by review of current evidence and consulting with expert opinion.In total 806,922 RTIs and 22,974 deaths resulted from the RTCs in the study period. The total cost of RTCs was about 72,465 billion Rials (7.2 billion US Dollars), which amounts to 2.19% of Iran’s Gross Domestic Production (GDP). Direct costs were 3,516 billion Rials , following by 24,785 billion Rials  for production lost and 12,513 billion Rials  for intangible costs. This study indicated that the burden of both RTCs and RTIs in Iran is substantial. Moreover, RTCs have significant economic consequences and are a large drain on healthcare resources. In low and middle-income countries (LMICs) they are responsible for around 90% of injury-related mortalities.4 Each year approximately 1.2 million people die and an extra 20 to 50 million people are injured or become disabled from the RTCs in the world.1 RTCs are responsible for approximately 23% of deaths due to injuries, and about 90% of fatal RTIs occur in the LMICs.1 Recent studies have shown an increasing trend of fatalities caused by RTIs in several LMICs including Pakistan, Bangladesh and Nepal.8Road traffic crashes (RTCs) and thus road traffic injuries (RTIs) are among the leading causes of death, disability and property loss worldwide.9 Focusing on the cost of injury by means of gross national product (GNP) and based on a WHO report on RTIs, RTIs’ cost approximately is 1% of GNP in low-income countries, and around 1.5% in middle-income countries, followed by 2% in high-income countries.1The UK Transport Research Laboratory Report, published in 2000, estimated that the global cost of RTCs was around 518 billion US dollars ($US) annually, and 65 billion dollars of these are attributable to LMICs, which is more than the financial support they normally receive from high-income countries.11 Several studies have been conducted in Iran, but the main focus has been on the epidemiological pattern or advocacy of RTIs as well as post-crash events.1 One study in Iran showed that they account for around 5% of the GDP, which is actually more than the GDP of some countries. In recent years the Iranian government has decided to reduce the number of fatal RTIs by 50% in the whole country,12 by means of a new national policy. Evaluating the extent, environmental costs and consequences of RTCs is an important step in planning for their prevention and reducing their costs. Moreover, WHO emphasize that all activities for prevention of RTIs, should be based on valid and reliable evidence. To our best knowledge there are limited studies about the cost of RTIs in Iran. Accordingly the aim of this study is to analyze these costs using a standard human capital (HC) approach.In Iran RTIs is the second largest cause of death after cardiovascular diseases.13 In the HC approach the cost of RTCs is estimated as the sum of resource costs  and other costs normally named as intangible costs .13 In this study a standard human capital approach was used17 to estimate the costs of RTCs in Iran. This is a cross sectional study carried out between 20 March 2009 and 20 March 2010. The Human Capital approach is one of the main methods for estimating the costs of RTCs.In this study the costs were divided into three domains: direct, indirect and intangible costs. Direct costs included pre-hospital, hospital, post-hospital , administrative and funeral costs and the cost of property damages. Indirect costs were considered as production lost. Finally intangible costs were considered as cost of pain, grief and suffering. The costs were estimated according to the severity and the level of injuries resulted from the RTCs. All the costs were calculated in Iranian Rials (IRR). At the time of the study, the exchange rate for a US Dollar was about 10.000 IRR.15 The medical costs, administrative costs, funeral costs, intangible costs and production lost were put in one category of costs and these costs have calculated by the severity of injuries. Finally the costs of property damage were calculated separately.The severity of injuries was classified into four groups including fatal injury, serious injury, slight injury and property damage only.18 Data about the number of fatalities due to RTIs were obtained from the database specially designed at Iran LMO.19According to current practice, for all the cases that are transferred to hospitals after RTIs, information should be reported to the CDMME every month. Therefore, we considered the CDMME database as the best available database for the number of RTIs. On the other hand, for all the cases of fatal RTIs, information is normally reported to the Legal Medicine Organization (LMO) database irrespective of whether death occurs at the RTC scene, at the pre-hospital phase or at hospital. Therefore, the LMO database was considered as the best available database for the number of fatal RTIs. The data about the number of RTCs and number of people that were injured in these accidents were collected from the database specially designed at the CDMME, Ministry of Health and Medical Education (MOHME).Pre-hospital costs18 therefore we estimated that 24% of the annual EMS budget was used to transport injured people from the RTC scene. We calculated that the remaining injured people made their own way to the hospital or by other means and estimated 10 US Dollars for each of these transportations. 53% of cases that are injured in RTC are transferred to the hospitals by the Emergency Medical Services (EMS) and the remaining 47% make their own way to hospitals or are transferred by members of the public like taxi drivers, commercial drivers, police officers or laypeople. In addition, around 24% of patients that are transported to hospital by the EMS are involved in RTCs;Hospital costs18 Therefore the total number of RTIs referred to hospitals was estimated as 799,514 people .Since studies in Iran have shown that 38% of deaths from fatal RTIs  occur at hospitals and the remaining 62% occur at the scene or during transportation to the hospital. In addition, 2%  of RTIs are minor injuries and treated at the accident scene and hence do not need any transfer to hospital.According to the current practice in Iran, any medical procedure has a relative weight or relative value unit (RVU) that ranges between 0 and 200. These tariffs are the basis for calculating the medical costs in Iranian hospitals. In order to identify the proportion of patients with slight (length of stay less than 24 hours) and serious (length of stay more than 24 hours) RTIs and the average hospital costs related to them, medical records of 400 patients admitted to two large trauma centers  were reviewed in the study period. Physiotherapy and rehabilitation costs1 The average cost of rehabilitation for each person was 45,000 IRR. This figure was estimated according to the tariff that is normally used for these services in Iran.Recent studies have shown that about half of the RTIs are orthopedic cases and 10% of them  need physiotherapy and rehabilitation services.Property damage costs21 According to inflation rate in the average price of vehicles, we added an annual rate of 10% to estimate the average price of each type of vehicle in 2009.20 According to this report, in each RTC on average, 1.89 vehicles were involved, therefore it was estimated that a total of 1,492,355 vehicles had been damaged in RTCs in 2009.21 The total cost of vehicle damages was estimated by multiplying the number of vehicles involved in RTCs by the average damage per vehicle (18.7% of the vehicle cost).a) Damage to vehicles: A recent study from 2004 was used to estimate the number and type of vehicles involved in the RTCs and the proportion and amount of damage to the vehicles. This study used the national insurance data and estimated that on average 18.7% of the value of each vehicle incurred damaged in the RTCs.20b) Damage to public property, fixed objects and reparation costs: 5% of the total vehicle costs was considered as the cost of damage to public property and fixed objects.Administrative costs13Administrative costs from RTCs are incurred by the police, the insurance companies and the legal services. These costs were estimated according to the Transportation Research Laboratory (TRL) as 0.2% of fatal RTI costs, 0.4% of serious injury costs, 14% of minor injury costs and 10% of property damage only.Funeral costsIn order to calculate this, 100 students at the Tehran University of Medical Sciences from 25 provinces of the country were asked to estimate the average cost of a funeral. The average cost of a funeral in the 25 provinces was estimated and used in this study. Production lost15Production lost was calculated for fatal, serious and minor injuries by the following formula.i / (1+r)i]Production lost of fatal injuries= [Ʃw (1+g)Where:W=average annual GDP per capita, r=discount rate (5%), g=growth rate of the economy , i=average number of years lost per crash (72-35=37 years). 22 and their life expectancy was 72 years.23 Therefore the average number of years lost by each fatality due to RTI was 37 years. According to the World Bank database, the average GDP per capita was 0.045 billion IRR.24 For serious and slight RTIs the production lost was 15 and 3 days, respectively and the wage lost was 87,000 IRR per day according to the wages in 2009.25A recent study has shown that the average age of people who died in RTIs was 35 yearsIntangible costs26The Asian Development Bank (ADB) recommendation was used to estimate the intangible costs as 28% of the total cost of fatal injuries, 50% of the total cost of serious injuries, 8% of the total costs of minor injuries and 0% for property damage only.The total cost of property damage was about 28,708 billion Rials, and 1,367 billion Rials (5%) of this was related to public property and fixed object damage. The total cost of vehicle damage was about 27,341 billion Rials. Medical costs from RTIs were 2,952 billion Rials , of which 2,574 billion Rials (87%) were related to hospital costs, 360.4 billion Rials (12.3%) were related to pre-hospital costs and 17.7 billion Rials (0.7%) were related to rehabilitation costs. The average cost of each fatal RTI was about 1.47 billion Rials, followed by 0.021, 0.013 and 00.02 billion Rials for property damage, serious and minor RTIs, respectively. The average production lost from a fatal RTI was 1.05 billion Rials, followed by 0.001 and 0.0002 billion Rials for serious and minor RTIs, respectively. The average administration costs per death, serious, minor injury and property damage only were 0.002, 0.000005, 0.00003 and 0.002 billion Rials; and the average intangible costs per death, serious and minor injuries were 0.412, 0.006 and 0.0001billion Rials, respectively .17 We also used the HC approach to estimate the cost of RTCs in Iran for the first time. This study, the first of its kind in Iran, indicated that the total cost of RTCs was 72,465 billion Rials in one Iranian calendar year, 48.5% of which were direct costs , 34.2% were related to production lost and 17.3% were intangible costs, employing HC approach. The choice of the HC approach normally depends on the research aim and data availability. Due to the lack of precise data in LMICs, the majority of studies conducted in countries like Egypt, Vietnam, Taiwan, the Philippines and Jordan have used the HC approach to estimate the cost of RTCs.1 The cost of RTCs in Egypt in 2008 was around 10 billion Egyptian Pounds, and about 1% of their GDP.14 Moreover, the cost of RTCs in Vietnam was around 0.45% of their GDP in 2004.15 However, the total cost of RTCs in Iran was 2.19% of GDP which is considerably more than the other countries. This shows that RTCs are a relatively large drain on the public resources in Iran compared to other countries in the region.According to a report by the World Health Organization, the annual cost of RTCs in LMICs is between 1 and 2 % of GDP.14 and the Philippines.16 Our findings indicated that the production lost related to RTCs was about 24,785 billion Rials , which is similar to other studies.16 This seems to be more likely to be related to the mean length of hospital stay for inpatients that was 6 days and similar to the figures reported by a study from Bangladesh (5.7 days). However, it is significantly lower than the 21.5 days reported by a study that was conducted in Bangkok.27 This might be because we did not follow up those patients that were referred to tertiary care for further treatment. In addition, the latter study included hospitalization days, rehabilitation and recovery days. The other reasons for the difference in length of hospital stay in this study compared with other countries might be due to difference in health care delivery systems and differences in the design of the studies. However, the differences in the costs of RTCs among the countries might be partly because of variations in their health care systems, service costs, pattern of injuries, the safety of vehicles and roads, the driving culture and a variety of other factors. It is therefore, important to identify the main factors contributing to the RTCs, to develop and implement strategies to reduce their occurrence and to mitigate their consequences. The intangible costs  and administration costs of RTCs were about 17.3% and 4.7% of the total costs, respectively. This is similar to recent studies published in EgyptLimitations and strengthsAlthough this study provides a valuable insight about the cost of RTCs in Iran, there are several limitations that should be considered in any future study. Using two different sources of data to estimate the number of deaths and injuries resulting from RTCs might be a limitation of this study. However we selected these databases, in particular the CDMME database, as they provide a more complete and accessible database for the number of injuries. Moreover another study has indicated that the LMO database has a better coverage and accuracy in fatal RTI registration. However, register databases usually suffer from underreporting and thus under-estimation, so we recommend caution when making interpretations.Moreover, some data about property damage, intangible costs, administration costs, the average age of fatal RTIs, physiotherapy costs, the average cost of transportation by EMS and the mean age of fatal RTIs were not available, so we had to use data from other studies and expert opinion to estimate the costs of RTCs. Therefore, some of the costs that were estimated in this study might differ to some extent from the real costs.This study indicated that the economic burden of RTCs in Iran is substantial and the cost imposed by RTCs as a proportion of GDP in Iran is greater. While data required for estimating the real cost of RTCs may need some of surveys on national level for increased precision in estimating the real costs of RTCs; however, the estimated cost of RTCs, at least rough data is available and on national level, this is useful for analysis and policy making in improving road safety and reduction cost of RTCs. This suggests that prevention of RTCs should be considered as a top priority for the policy makers and efforts should be made to identify and prioritize improvement strategies followed up by an evaluation of their effects.


Cell culture adaptation of very virulent infectious bursal disease virus (vvIBDV) was shown to be mainly associated with the VP2 capsid protein residues 253, 279, and 284. The single mutation A284T proved critical for cell culture tropism, but did not confer efficient virus replication, which at least required one additional mutation, Q253H or D279N. While the double mutation Q253H/A284T was unambiguously shown to confer both efficient replication in cell culture and attenuation in chickens, conflicting results have been reported regarding the replication efficiency of vvIBDV mutants bearing the D279N/A284T double mutation, and no data are hitherto available on their virulence in chickens.in vivo reverse genetics system to assess the impact of the D279N/A284T double mutation on the replication and attenuation of a chimeric IBDV virus, whose polyprotein derived from a non-culturable vvIBDV clinical isolate. We found that the D279N/A284T double mutation did indeed confer efficient replication in chicken embryo fibroblast (CEF) cell culture, but the mutant virus remained highly pathogenic to chickens.Here we used an The double mutation D279N/A284T of the VP2 major capsid protein of vvIBDV is sufficient to confer cell culture tropism and replication efficiency, but does not necessarily lead to virus attenuation. Birnaviridae, genus Avibirnavirus, is the causative agent of an avian restricted disease known as “Gumboro disease” [Infectious bursal disease virus (IBDV), a member of the family disease” . The virdisease” . These idisease” -5. IBDV disease” .The genome of IBDV consists of two segments of double-stranded RNA (dsRNA), referred to as segments A and B ,8. The sTwo different IBDV serotypes exist, serotypes 1 and 2 . Wild-tyEarly studies using site-directed mutagenesis coupled with reverse genetics showed that capsid VP2 amino acid residues at positions 253, 279, and 284, are involved in cell culture adaptation and/or virulence of vvIBDV -30. Morein vivo reverse genetics, we show that the double exchange D279N/A284T in the VP2 sequence of vvIBDV indeed confers efficient replication in CEF cells, but could not attenuate the rescued chimeric virus in chickens.Here using a chimeric virus generated by Primary Chicken Embryo Fibroblast (CEF) cells were freshly prepared from 9 to 11-day-old-embryonated specific pathogen-free (SPF) eggs. The cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37°C in a humidified 5% CO2 incubator. CEF cells were used for transfections, virus propagation and tittering.in vivo reverse strategy which has significantly improved the titer of rescued IBDV [Plasmid constructs pVAXSA.Rib and pVAXSB.Rib ,33 contaued IBDV .Taq polymerase mix . The resulting amplicon was cloned into kpnI/EcoRI-restricted pCR2.1 plasmid vector (Invitrogen), yielding the construct pCR.PO7poly.wt. This was next used as template to substitute the VP2 amino acid residues aspartic acid (D) to asparagine (N), and alanine (A) to threonine (T), at positions 279 and 284, respectively. Site-directed mutagenesis was carried out by overlap extension using PCR [KpnI/SalI and subcloned into the similarly digested pCR.PO7poly.wt plasmid, yielding pCR.PO7poly.mt. Effective incorporation of the nucleotide changes was confirmed by DNA sequencing.The full-length coding sequence of the polyprotein and the 3′ non-coding region of PO7, a Tunisian bursal-derived field isolate of vvIBDV , was ampsing PCR . BrieflyBspEI/EcoRI and subcloned into BspEI/EcoRI-digested pVAXSA.Rib. Since EcoRI digestion of pVAXSA.Rib eliminates the HDV ribozyme sequence, the latter was restored at the 3′ end of PO7 segment A, using the same previously described strategy [Next, pCR.PO7poly.wt was restricted with strategy . The resBspEI/SalI and inserted into the corresponding restriction sites of pVAXSA.PO7wt.Rib, yielding pVAXSA.PO7mt.Rib, which contains the double mutation D279N/A284T in the PO7 VP2 capsid sequence  was further subjected to two consecutive passages (P1 and P2). The titers of the original virus stock (P0) and the two passages (P1 and P2) were determined upon three independent assays on CEF cells and expressed as TCID50/ml (Tissue Culture Infectious Dose 50).To rescue chimeric IBDV virus expressing the wild-type or the D279N/A284T mutated PO7 polyprotein, 5 μg each of plasmid constructs pVAXSA.PO7wt.Rib or pVAXSA.PO7mt.Rib were cotransfected with the same amount of pVAXSB.Rib. For comparative purposes, pVAXSA.Rib and pVAXSB.Rib plasmid constructs were cotransfected in parallel in order to rescue the cell culture-adapted and attenuated P2 strain, as previously described . Co-tran0) virus stock (the supernatant of cotransfected CEF cells) of the rescued viruses was used. Chickens were inoculated with 105 TCID50 of the P0 stock of each rescued virus, or with phosphate-buffered saline (PBS), by eye drop.Three-week-old specific-pathogen-free (SPF) chicks were used. A group of 30 chickens were raised in isolators with filtered air-flow and randomly divided into three groups of 10 chicks each. Prior to inoculation, chickens were checked for seronegativity to IBDV. To minimize the risk that viruses used to infect chickens may contain undesired mutations in either genomic segment, only the unpassaged  as described in Nouen et al. . ChickenOrgans were split into two parts. One part was used to extract total RNA, and the second part was processed for histopathology.Bursas were cut into small pieces, ground with sterile sea sand in PBS, and the homogenates were centrifuged at full-speed in an eppendorf centrifuge for 10 min at +4°C. RNA was extracted using the QIAmp viral RNA mini kit (Qiagen) following the manufacturer’s instructions. RT-PCR amplifications were performed using the Qiagen OneStep RT-PCR-Kit (Qiagen), essentially as recommended by the supplier, using segment-specific primer pairs SAS1/SAR1 and SBS1/SBR1  of 0 , 1 (mild lymphoid depletion with absence of necrosis/oedema), 2 , 3 .t test was used. The level of significance was P <0.05.Statistics were calculated using Prism software (GraphPad Software Inc.). The data were presented as the means and evaluated by one-way ANOVA followed by Newman-Keuls multiple comparison test. For comparison of virus titers, the unpaired BspEI restriction site (nucleotide position 215) to the 3′ end ultimate nucleotide with its corresponding sequence of the wild-type Tunisian PO7 vvIBDV strain. With this cloning strategy, the entire polyprotein amino acid sequence specified by this chimeric P2/PO7 segment A is identical to that of the wild-type PO7 strain, and differs from the segment A cDNA infectious clone of the P2 strain by 19 amino acid residues    showed typical clinical symptoms of vvIBDV infection. Nine of ten chickens (90%) died in the first 3 days post-inoculation. The bursa/body and spleen/body weight ratios  were significantly lower than in normal chickens  (Table P >0.05).Chickens inoculated by eye drop with 10All bursae of dead chicks inoculated with the resuced P2/PO7mt IBDV virus showed severe necrosis, atrophy, and loss of the follicular architecture. By contrast, histopathologic sections of bursa derived from control (inoculated with PBS) or P2-infected chicks were similar and had normal follicles and follicular connective tissues Figure . These rSequence analysis of both genomic segments of the rescued P2/PO7mt virus from the bursa of Fabricius of dead chicks did not reveal any amino acid change (data not shown).Previously we reported the nucleotide sequence of the VP2 hypervariable region of bursal-derived Tunisian vvIBDV field isolates and the polyprotein coding sequence of PO7, a Tunisian vvIBDV representative isolate . This stin vivo reverse genetics approach that we have previously developed in our laboratory [Several attempts to adapt the PO7 isolate to cell cultures proved unsuccessful. This result was congruent with the nature of its VP2 amino acid residues at positions 253 (Q), 279 (D), and 284 (A). Using an boratory ,33, we cin vivo reverse genetics method, which could have contributed to rescue high-titer virus. We also used in vivo reverse genetics, but unlike the approach of Lim et al. [The finding that the double exchange D279N/A284T yielded a high-titer virus further confirmed the results of Lim et al. , in thatm et al. , our metm et al. , which iThe data obtained in this study demonstrate, for the first time, that acquisition of cell culture tropism by IBDV was not accompanied with its attenuation in chickens. The D279N/A284T double exchange in the VP2 capsid protein proved sufficient to confer cell culture adaptation and replication efficiency but did not lead to attenuation. This finding is in agreement with the study previously reported by Raue et al. . Indeed,However, the finding that the cell culture-adapted P2/PO7 mutant virus remained pathogenic contrasts with the attenuated nature of strain OKYMT , which lBased on the work described here, we conclude that the double mutation D279N/A284T of the VP2 major capsid protein of vvIBDV is sufficient to confer cell culture adaptability and replication efficiency, but it does not necessarily lead to virus attenuation.


Hippotragus niger variani), and the naturally sympatric roan antelope (H. equinus). Hybrids with intermediate phenotypes, including backcrosses with roan, were confirmed in one of the two remnant giant sable populations. Hybridization followed population depletion of both species due to severe wartime poaching. In the absence of mature sable males, a mixed herd of sable females and hybrids formed and grew progressively over time. To prevent further hybridization and recover this small population, all sable females were confined to a large enclosure, to which sables from the other remnant population were translocated. Given the large scale declines in many animal populations, hybridization and introgression associated with the scarcity of conspecific mates may be an increasing cause of biodiversity conservation concern. In these circumstances, the early detection of hybrids should be a priority in the conservation management of small populations.Population declines may promote interspecific hybridization due to the shortage of conspecific mates (Hubb’s ‘desperation’ hypothesis), thus greatly increasing the risk of species extinction. Yet, confirming this process in the wild has proved elusive. Here we combine camera-trapping and molecular surveys over seven years to document demographic processes associated with introgressive hybridization between the critically endangered giant sable antelope ( The intensive monitoring of this population over seven years provided a unique opportunity to test Hubb’s desperation hypothesis, by evaluating whether the putative hybridization events could be linked to the shortage of conspecific mates. Results from this study were also used to trigger emergency conservation action to avoid further hybridization and promote population recovery.Intensive camera-trapping revealed that shortly after the end of the Angolan civil war only one giant sable herd was present in Cangandala . In 2005Births of phenotypically pure sable calves were not observed after 2005 and were only recorded again in 2010, following the translocation to Cangandala of a sable bull captured in Luando. Births of putative hybrid calves were estimated to have occurred every year since 2002, but they were not detected again after 2010 when all phenotypically pure female sables were physically separated from putative hybrids and roan males. The number of putative hybrids recorded grew steadily over time, equalling sables in 2009 . Two indE = 0.329; N = 35), compared to Namibian sables , and particularly roan  , and between these and roan . In the biplot of a Principal Components Analysis , there were three clear clusters corresponding to Angolan and Namibian sables, and roan, whereas the nine putative hybrids genotyped from Cangandala showed an intermediate position between roan and giant sable (Genetic diversity based on microsatellites was significantly lower (p < 0.05) in giant sables from Angola  . Divergent sable .i>0.99. All morphologically intermediate individuals were partially assigned to both species, with individual qi ranging from 0.21 to 0.79. Bayesian clustering analysis to detail hybrids ancestry ascribed all putative hybrids to strict hybrid classes with posterior probabilities above 99%, including the confirmation of two individuals as backcrosses to roan  that were suspected to be visited by giant sables. Sites were identified with the help of local rangers, based on the presence of spoor and witness accounts of visual observations. Due to access and logistic difficulties, the number and type of cameras, as well as their operating period, varied greatly in space and over time. Until the end of 2007 we used six TrailMaster infrared trail monitors TM1500 and TM1550 connected to external photography film cameras or digital video. In October 2007 we introduced up to 12 passive digital cameras Stealth Prowler DVIR5. Due to variation in sampling effort and demographic changes, the number of photos recorded for sable and putative hybrids varied greatly from <25 (2005) to >9,000 (2010) . The larIn July and August of 2009 and 2011, we conducted extensive aerial surveys and capture sessions that involved darting animals from a helicopter, aiming to capture all sables and putative hybrids remaining in Cangandala. The aerial surveys were maintained for several days after the last animal had been spotted and captured in 2011. Although dense canopy cover in most of the area makes it impossible to guarantee that all animals were indeed captured, it is very unlikely that any giant sable or hybrid remaining outside enclosures would be missed, as the Cangandala region is relatively small and has been visited regularly during field work carried out to the present day.Each year, the minimum number of sable, roan and putative hybrids present in Cangandala was estimated from the number of individually recognizable animals detected in photos and videos obtained through camera-trapping. Assignment of individuals to gender and age class further allowed the estimate of sex-ratio, age structure and annual number of births. To obtain these estimates, we first screened all photos and videos for the presence of sable, roan and putative hybrids. Each animal recorded in a photo or video was then identified to species-level, and ascribed to gender and age class. Species identification was straightforward, given the marked morphological differences between sable and roan. The individuals with intermediate morphology, or showing unusual phenotypes, were classified as putative hybrids. . Gender The annual number of births of sable and putative hybrids was estimated by combining information on females with evidence of pregnancy, and the number and estimated age of calves, juveniles and young adults. Specifically, calves were assumed to be born either in the year when they were first observed or in the previous year, based on the timing of observations, body size and horn developmentThe information obtained through camera trapping was complemented by aerial surveys and the capture sessions carried out in 2009 and 2011. Aerial surveys confirmed that a single sable herd was present in Cangandala during the entire study period, and the number of individuals spotted was compatible with camera-trapping data. Also, we never recorded a mature sable male during the aerial surveys, which matches the results from camera trapping. This was in contrast with similar surveys carried out in Luando, where a comparable sampling effort allowed the detection of 16 mature males and at least 4 herds. The animals captured in 2009 and 2011 in Cangandala, totalling nine giant sable females and nine hybrids, were the same recorded through camera trapping in previous years. No new animals were found, and we confirmed the individual identifications previously made using facial mask patterns and other unique features. Overall, close matching between data obtained through camera trapping, aerial surveys and capture sessions suggest that population parameters were accurately estimated.During the capture operations carried out in 2009 and 2011, we collected 18 ear tissue samples, representing the whole lot of giant sable and putative hybrids surviving in Cangandala. During the concurrent capture operations carried out in Luando we collected ear tissue samples from another 26 giant sables and four roans. To allow comparisons with other sable and roan populations, we obtained 22 sable samples collected in Etosha National Park and Waterberg National Park in Namibia, both descendent from an original population translocated from the Caprivi Strip of Namibiaet al.http://dx.doi.org/10.5061/dryad.g615h.Genomic DNA was extracted from tissue samples using the DNeasy Blood & Tissue Kit (QIAGEN), following manufacturer instructions. Individual multilocus genotypes were scored using a set of 51 sable-specific microsatellites, which all proved to be polymorphic in one or both speciesHippotragus niger complete mitochondrial DNA  lineage using primers LS-1 (5′-AATATACTGGTCTTGTAAACC [15305]) and HS-3 (5′-AGGCATTTTCAGTGCCTTGC  KM245339. AdditioO) and expected (HE) heterozygosities for each locus/population using GENETIX 4.05ST  and observed .