Melhor em Casa program can actually reduce hospitalization costs. To verify if the  We use as an empirical strategy a Regression Discontinuity Design, which reduces endogeneity problems of our model. We also performed tests of heterogeneous responses and robustness. Data on the dependent variable, namely hospitalization costs, were collected in the Department of Informatics of the Unified Health System (DATASUS), using the microdata set from the Hospital Admissions System of the Unified Health System (SUS) from 2010 to 2013, totaling 3,609,384 observations. The covariates or control variables used were age and costs with patients in the intensive care unit, also from DATASUS.Melhor em Casa program effectively reduced hospitalization costs by approximately 4.7% in 2011, 5.8% in 2012 and 10.2% in 2013. The results point out that the  Based on the analyses, we observed that maintaining the program can effectively improve the management of public resources, since it reduced the hospitalization costs in the three years studied. The program reduced hospitalization costs of risk groups and also in situations that usually increase hospital costs such as lack of equipment and elective hospitalizations. Thus, it can be affirmed that the program can reduce hospitalization costs, especially in risk and more vulnerable groups, showing efficiency as a public policy. Melhor em Casa program (MemC) can actually reduce hospitalization costs. Hospital care is understood as an important form of social policy for life maintenance and a mandatory form of restoring constitutional principles to the Brazilian population. Hospital care should be effectively provided to avoid a system collapse, especially with a limited budget.Our study verified if the 2 . The program aims to articulate with home care (HC) – that is, the patients admitted to the health unit and ready to complete their recovery in the comfort of their household, with the relatives, can be transferred. Thus, hospitalization costs reduce and hospital mortality decreases.In 2011, the federal government created the MemC to control hospitalization problems and reduce unnecessary costs for the population3 .However, we could not see the commitment of health managers. We observed cases of maladministration and poor technical and structural conditions instead, which reduce the efficiency of hospital services. The consequences are unrecoverable economic and life-related costs9 . In addition to verify the effect of MemC, our article also contributes to the application of the empirical strategy of the regression discontinuity design (RD), which guarantees responses without endogenous bias, confirming or not the program efficiency.One of the major problems of hospital care is the cost of services provided in the health unit, with high waste, which reaches R$ 3.6 billion a year with consultations, hospitalizations and unnecessary exams. The complex composition of hospital costs result in waste and inefficiency in the public serviceThe tests of heterogeneous responses and robustness were used to verify if the estimators were accurate. The main hypothesis of our study was that the program can reduce hospitalization costs, making public hospital management more efficient.Data on the dependent variable, namely hospitalization costs, were collected in the Department of Informatics of the Unified Health System (DATASUS), using the microdata set of the Hospital Admission system of the Brazilian Unified Health System (SUS) from 2010 to 2013, totaling 3,609,384 observations in the three years of analysis. The values of the variable of interest, hospitalization costs, were deflated; thus, the series suffers real impact without inflationary influence.In 2011, the average hospitalization cost was R$ 38.67 per patient/day in the municipalities covered by the program. In 2013, this value reached R$ 42.78 per patient/day, a 10.6% increase within three years. In municipalities not covered, the average was R$ 30.83 per patient/day in 2011 and R$ 33.51 in 2013, an 8.7% increase within three years. Based on these data, we can affirm this program is indicated to municipalities with higher hospitalization costs.Data were collected in Datasus and the National Register of Health Establishments (NRHOSP), both organs of the Ministry of Health, to analyze the effects of MemC. We used a dummy to identify the municipalities covered by the MemC. They were named “treated” and coded with the value 1, while the municipalities not covered were named “controls” and coded with the value 0.Based on the data, we observed the program included 23 of the 5,570 Brazilian municipalities in 2011, increasing to 90 municipalities in 2012, and to 184 in 2013. The coverage rate increased from 0.41% of the Brazilian territory in 2011 to 3.3% in 2013.10 , but do not interfere or generate any bias in the results.The covariates or control variables used were age and hospitalization costs of patients in the intensive care unit. These data were obtained from Datasus database, from 2010 to 2013. The control variables are used to greater precision to the estimated values11 due to the adherence criterion related to a cutoff point and exogenous probability, namely “cutoff.” The established cutoff were municipalities with more than 20,000 inhabitants. Thus, we estimate the following equation:Our article analyzed how home care  affects the hospitalization costs. As an empirical strategy, we used the regression discontinuity designYimp is the variable of interest of the model for the individual i , in the municipality m, and in the year p ; MemC is the Melhor em Casa program, which takes value equal to 1, if the individual’s municipality is covered by the program, and 0 if it is not covered, for the individual i , in the municipality m, and the year p ; The Tip indicates if the municipality is above or below the cutoff, cited previously, for the individual i , in the municipality m, and in the year p ; Finally, εip is an error term. Due to the sensitivity of considering an increase in probability – but not from zero to one, since the attribution to treatment may depend on additional factors –, the regression discontinuity model fuzzy was applied . This ensures a causal identification among the selected variables, enabling the confirmation of the effect of MemC on hospitalization costs with higher statistical security.In which 16 This variation of bandwidth is also a way to guarantee our empirical strategy, emphasizing that we test our model for a linear and quadratic specification.To perform local regressions, you need to enter a window (bandwidth) that will determine the size of the cutoff distance at the level of sample observations. Then, we used the methodology of Calonico et al., that is, we made our regressions with different cutoffs than the one stipulated by the program. Finally, we tested the covariates of our model. The tests should not generate statistically significant estimators, guaranteeing the parameters of our model.In addition to the aforementioned tests, we applied three more robustness tests to confirm our empirical strategy. First, we verified possible effects of other programs or actions of years prior to the beginning of the program. Another test was the cutoff alteration We also performed heterogeneous test responses to understand and confirm the effects of MemC on sub-sample situations, namely risk groups (pregnant and older adults), which require a longer hospitalization time. We analyzed if the program can affect municipalities with fewer working equipment than the national average, which would cause a longer hospitalization due to the delay in the results of the exams. Finally, we verified the effect of MemC on the group of people hospitalized without urgency, or elective, which would show the speed of care and the reduction of costs with this type of hospitalization.We initially verified if discontinuity occurs at the 20,000 inhabitants’ cutoff point to guarantee the statistical results. The Regarding the heterogeneous responses, based on the estimators shown in In addition to the heterogeneous responses, we applied robustness tests to statistically guarantee the assertion of the initial hypothesis. In We continue with the robustness tests in Concluding our tests, in Melhor em Casa program affected the hospitalization costs. The program proposes the provision of services by teams composed of several professionals in the health area to promote patient recovery in the comfort of their household (home care). Besides the recovery in a healthier environment, it also promotes hospital bed vacancy, reducing daily costs of hospitalization.Our study analyzed how the After ensuring that the results present causal characteristics, we verified that the MemC can effectively reduce hospitalization costs, with reductions in approximately 4.7% in 2011, 5.8% in 2012 and 10.2% in 2013. The results heterogeneous responses and the robustness test were the ones expected, confirming the results of our model and statistically supporting our estimators.We could observe that the risk groups (pregnant women and older adults), even with higher costs due to specific care, also had their costs reduced with the program. MemC can effectively reduce hospitalization costs of municipalities with fewer hospital equipment than the national average. The program can also be efficient when considered the elective hospitalizations, that is, those that are not urgent. Thus, we conclude that the program helps reduce hospitalization costs, especially in risk and more vulnerable groups, showing to be an efficient public policy. O objetivo principal deste trabalho é verificar se o programa Melhor em Casa  consegue reduzir os custos com internações hospitalares. Entende-se que o atendimento hospitalar é uma forma de política social na área da saúde de suma importância para a manutenção da vida e uma forma obrigatória de restabelecer os princípios constitucionais com a população brasileira. O atendimento hospitalar deve ser conduzido de forma suficientemente eficaz, para não ocasionar o colapso do sistema, principalmente em um momento de recursos orçamentários escassos.2 . O programa visa uma articulação com a atenção domiciliar (AD) – ou seja, o paciente atendido na unidade de saúde e que está apto para concluir sua recuperação no conforto de seu lar, junto aos seus familiares, poderá ser transferido. Assim, há uma redução dos custos com a internação e diminuição da mortalidade dentro dos hospitais.Diante disso, para conter problemas referentes às internações hospitalares e reduzir os custos desnecessários para a população, o governo federal criou em 2011 o MemC3 .No entanto, atualmente não verificamos empenho dos gestores de saúde. O que se observa, de fato, são casos de má administração e más condições, tanto técnicas como estruturais, que reduzem a eficiência dos serviços hospitalares. As consequências são custos irrecuperáveis, tanto econômicos quanto ligados à vida9 . Dessa forma, a contribuição deste trabalho, além de verificar o efeito do MemC, também está na aplicação da estratégia empírica do modelo de regressão descontínua (RD), que garante respostas sem viés endógeno dos estimadores, confirmando ou não a eficiência do programa.Um dos grandes problemas do atendimento hospitalar são os custos dos serviços prestados na unidade de saúde, com níveis elevados de desperdício, o qual chega à cifra de R$ 3,6 bilhões por ano gastos com consultas, internações e exames desnecessários. A composição complexa dos custos hospitalares propicia o desperdício e gera ineficiência no âmbito do serviço públicoOs testes de repostas heterogêneas e de robustez foram empregados para verificar se os estimadores foram precisos. A hipótese principal do estudo era a de que o programa consegue reduzir os custos com as internações hospitalares, tornando a gestão hospitalar pública mais eficiente.Os dados da variável dependente, despesas ou gastos com internações hospitalares, foram coletados no Departamento de Informática do Sistema Único de Saúde (Datasus), usando o conjunto de microdados do Sistema de Internações Hospitalares do Sistema Único de Saúde (SIH-SUS) referente ao período de 2010 a 2013, totalizando 3.609.384 observações nos três anos de análise. Os valores da variável de interesse, despesas por internação hospitalar, foram deflacionados; assim, a série sofre impacto real, sem influência inflacionária.No ano de 2011, nos municípios onde existe o programa, houve um gasto médio com internação de R$ 38,67 por paciente/dia. Em 2013, esse valor passou a ser de R$ 42,78 por paciente/dia, um aumento de 10,6% em três anos. Nos municípios que não recebem o programa, a média de gastos era de R$ 30,83 por paciente/dia em 2011 e de R$ 33,51 por paciente/dia em 2013, com um aumento de 8,7% em três anos. Esses dados apontam que existe uma motivação para que o programa seja implantado onde o gasto com internação é maior.dummy em que eles foram denominados “tratados” e receberam o valor 1, enquanto os municípios que não receberam o MemC foram denominados “controles” e recebem o valor 0.Para analisar os efeitos do MemC, foram coletados dados do Datasus e do Cadastro Nacional de Estabelecimentos de Saúde (CNES), ambos órgãos do Ministério da Saúde. Como forma de identificar os municípios que recebem o MemC, utilizou-se uma Pelo conjunto de dados obtidos, observamos que em 2011 o programa contemplou 23 dos 5.570 municípios brasileiros existentes. Já em 2012 esse número saltou para 90 municípios, e em 2013 a quantidade de municípios contemplados passou a ser de 184, evidenciando um salto de 0,41% de cobertura do programa em 2011 para 3,3% em 2013.10 , mas não devem interferir ou gerar qualquer viés nos resultados encontrados do modelo.As covariáveis ou variáveis de controle utilizadas foram a idade e os gastos com pacientes na unidade de terapia intensiva. Esses dados foram coletados no banco de dados Datasus, referentes ao período de 2010 a 2013. É importante frisar que as variáveis de controle são utilizadas para dar maior precisão aos valores estimados11 pois o programa possui um critério de adesão relacionado a um ponto de corte e de probabilidade exógeno, que chamamos de cutoff . O cutoff estabelecido foram municípios com mais de 20.000 habitantes. Assim, estimamos a seguinte equação:Neste trabalho, buscamos compreender o efeito da atenção domiciliar  sobre o valor das despesas com as internações hospitalares. Como estratégia empírica, utilizamos o desenho de regressão descontínuaYimp é a variável de interesse do modelo para o indivíduo i , no município m e no ano p ; MemC é o programa Melhor em Casa, que leva valor igual a 1, caso o município do indivíduo receba o programa, e 0 caso contrário, para o indivíduo i, no município m e no ano p ; o Tip indica se o município está acima ou abaixo do cutoff , citado anteriormente, para o indivíduo i, no município m e no ano; por fim, εip é um termo de erro. Devido à sensibilidade de considerar um aumento de probabilidade – mas não de zero para um, pois a atribuição ao tratamento pode depender de fatores adicionais –, aplica-se o modelo de regressão descontínua fuzzy . Isso garante uma identificação causal entre as variáveis selecionadas, possibilitando a confirmação do efeito do MemC sobre as despesas hospitalares com maior segurança estatística.onde bandwidth ) que determinará o tamanho da distância do ponto de corte no nível de observações da amostra. Utilizamos, então, a metodologia de Calonico et al.16 Essa variação de bandwidth também é uma forma de garantir nossa estratégia empírica, ressaltando que testamos o nosso modelo para uma especificação linear e quadrática.Para realizar as regressões locais, é preciso introduzir uma janela , que necessitam de um tempo maior de internação. Procuramos analisar se o programa consegue afetar os municípios com um número efetivo de equipamentos menor que a média nacional, o que provocaria um maior tempo de internação devido à demora dos resultados dos exames. Por último, verificamos o efeito do MemC para o grupo de pessoas internadas sem caráter de urgência, ditas eletivas, o que demonstraria a rapidez no cuidado e a redução dos custos com esse tipo de hospitalização.Para garantir os resultados estatísticos, inicialmente verificamos se ocorre descontinuidade no ponto de corte de 20.000 habitantes. O resultado desse teste inicial é apresentado na A existência da descontinuidade, fator básico para que possamos calcular nossos estimadores, é confirmada na Em relação às respostas heterogêneas, pelos estimadores apresentados no Painel A, da No Painel A da Para garantir estatisticamente a afirmação da hipótese inicial, além das respostas heterogêneas, aplicamos testes de robustez. Na cutoffs, de 10.000 habitantes e de 50.000 habitantes, os resultados estimados não têm significância estatística. Isso era esperado, pois certifica que os efeitos acontecem apenas no critério de corte determinado pelo programa, que é de 20.000 habitantes, demonstrando que não se trata de acaso estatístico.Na Finalizando nossos testes, no Painel C da home care ). Além da recuperação em um ambiente mais saudável para o paciente, também promove a desocupação de leitos, e com isso, consegue uma redução das despesas diárias com a internação.O presente estudo analisou os efeitos do Melhor em Casa sobre os gastos com internações hospitalares. O programa propõe a prestação de serviços, por parte de equipes compostas de diversos profissionais da área de saúde, para promover a recuperação do enfermo no conforto de seu lar , mesmo com gastos maiores devido aos cuidados específicos, também têm gastos reduzidos pelo programa. Quanto aos municípios com um número de equipamentos hospitalares menor do que a média nacional, verificou-se que o MemC consegue ser efetivo e também reduz as despesas com as internações. Nos casos relativos às internações eletivas, ou seja, aquelas que não são de urgência, o programa também consegue ser eficiente. Dessa forma, pode-se afirmar que o programa ajuda na redução das despesas com as internações, principalmente em grupos de risco e com maior vulnerabilidade, mostrando-se uma política pública eficiente.


Acute genital ulcers are painful and distressing to women. Lipchutz Ulcer is an uncommon disease that typically occurs in sexually inactive young women. The main differential diagnosis are sexually-transmitted or non-infectious diseases which cause genital or oro-genital ulcerations. This article aims to review the diagnosis of acute genital ulcers and, through a rare case of acute genital ulcerations, to discuss diagnostic procedures. A 13-year-old girl of Caucasian origin presented to the paediatric emergency room reporting acute vulvodynia, preceded by prodromal influenza-like symptoms  present for the past 48 hours.The patient had no relevant medical history, nor recurrent buccal or genital apotheosis. She didn’t receive any regular medication. She was sexually inactive, and hadn’t applied any cream or anything else on the vulva. There was no trauma nor history of sexual abuse.This is the first time she had experienced such symptoms.The patient had an antalgic gait. Gynecologic examination showed her labia minor with two centimetric, fibrinous, soft ulcerations on their inner aspect. These lesions had a symmetrical appearance, known as « kissing lesions » . FurtherIntense pain caused stress for both the adolescent and her mother. Nevertheless, the patient didn’t present other clinical, extra-genital signs .Blood count, viral , parasitic (toxoplasmosis) and treponemal (TPHA-VDRL) serologies as well as samples for further microbiological explorations  have been performed. While results were pending, hospitalization in a paediatric ward was required with up to step II analgesic (paracetamol and tramadol) were administered in association with local hygiene  and wound care. An empirical antiviral treatment (acyclovir per os 200 mg 5X/ day) was initiated and the bladder was catheterized.Performed explorations only showed an isolated inflammatory syndrome without hyperleukocytosis (CRP up to 20 mg/L). Viral and bacterial tests were negative and serologies didn’t bring any proof of recent infection. In light of these results, acyclovir was terminated. Ulcers eventually fully healed in a few weeks.Acute vulvar ulceration, also referred to as a Lipschutz Ulcer, is a clinical diagnosis based on both:— detailed history with acute painful genital ulcerations associated to fever in a sexually inactive, adolescent girl — physical examination showing centimetric, fibrinous, soft necrotic ulcerations with red-violaceous border and inguinal adenopathies. Pathogenesis is unclear but a hypersensitivity reaction secondary to viral or bacterial infection with deposition of immune complex in the dermal vessels causing micro-thrombosis, and eventually leading to deep, necrotizing, painful, aphtoid lesions is suspected.One hypothesis suggests a manifestation of EBV primary infection .The disease typically begins with prodromal influenza or mononucleosis-like symptoms followed by the onset of 1mm to 2,5cm, single or multiple , vulvar ulcerations .The ulcerations typically involve the labia minor but can extend to the labia major, perineum vestibule and lower vagina. There is no risk of transmission. Spontaneous healing is complete in two to six weeks (this feature being a retrospective diagnostic argument), usually without scarring.The clinical diagnosis is one of exclusion. As a matter of fact, sexually transmitted infections , auto-immune diseases , trauma and other causes of acute genital ulcerations need to be excluded. Biopsy is often not required.Rosman et al. have developed an algorithm for evaluation and treatment of acute genital ulcerations after HSV-infection exclusion  Rosman .Patient care consists of pain control including topical and systemic analgesia and wound care. Patient and parents should be reassured that the disease is benign and its lack of recurrence. In case of severe pain, hospitalization with bladder catheterization might be required to avoid any secondary urine retention .The main differential diagnosis of Lipschutz ulcerations are sexually transmitted diseases and non-infectious diseases which cause genital or oro-genital ulcerations.The most common diseases in this regard are :genital herpesHIV infectionsyphiliscomplex aphthosisBehcet’s syndromeCrohn’s diseasepyoderma gangrenosumchildhood vulval pemphigoidLipchutz Ulcer is an uncommon disease that typically occurs in young, sexually inactive women.The diagnosis is one of exclusion and other causes of genital ulcerations such as genital herpes, sexually transmissible infections  and auto-immune disease  must first be ruled out.Healing without scarring is spontaneous under proper analgesia and wound care, this feature being an important diagnostic argument.


The dependent variables were rupture strength , size , and sphericity . The heating temperature (X1) was the independent variable that had a significant effect on the rupture strength (Y1) and size (Y2). Rupture strength (Y1) increased as the heating temperature (X1) increased; at the same time, the CAG beads size (Y2) decreased. With all conditions, the values of sphericity (Y3) were over 94%. SEM images revealed that increase in the rupture strength of the CAG beads by heat treatment resulted from their porous structures. Loss of moisture by syneresis, occurring with heat treatment, was judged to create a dense porous structure of CAG beads. Our findings offer useful information for cooking or sterilizing food products utilizing CAG beads. In addition, thermal treatment could be applied to produce hard CAG beads with a high rupture strength.Calcium alginate gel (CAG) has been widely investigated for the development of artificial foods; however, there are few studies on its thermal stability. This study aimed to monitor changes in the physical properties of CAG beads during heat treatment using response surface methodology. Heating temperature (X Alginate is a linear anionic polysaccharide derived from brown seaweeds and is composed of 1-4)-linked β-D-mannuronic (M) and α-L-guluronic acid (G) residues -4-linked. It can In recent years, there has been an increasing interest in CAG as a biomaterial for making artificial or imitative foods. Some researchers have studied the optimization of the processing of fish roe ,8 and efThis study aimed to understand the effects of heat treatment on the physical properties of CAG beads for its application to fish roe analogs. The central composite design (CCD) of response surface methodology (RSM) was adopted to monitor the effects of heat treatment. RSM is a statistical procedure frequently used for optimization and monitoring of food processes. The basic principle of RSM is to describe model equations defining the effect of test variables on responses and determine interrelationships among test variables in any response . The CCDSodium alginate  and calcium lactate  were used as the functional materials for CAG beads. All chemicals and reagents used in this study were of an analytical grade.w/v) was dropped through a single nozzle 20G  using a peristaltic pump  into a calcium lactate solution . The stirring speed of calcium lactate solution (250 mL) in the reactor (500 mL) was 300 rpm. The distance between the nozzle tip and the surface of the calcium lactate solution was 8 cm. The allowed gelation time was 20 min. The CAG beads were rinsed with distilled water to remove any remaining calcium lactate. A schematic diagram for preparation of CAG beads is shown in A sodium alginate solution  and were represented as diameter (mm). Sphericity (%) was determined by the percent ratio of the minor diameter to major diameter, obtained from the size measurements; five beads were randomly selected from each experimental condition for measurement.2; and load cell force, 0.1 kN. Five samples were measured for each experiment.The rupture strength was measured using a rheometer  with the following conditions: plunger diameter, 25 mm; penetration speed, 80 mm/min; adaptor area, 4.91 cm2 factorial points, 4 axial points (α = 1.414), with 3 replicates of the central points. Heating temperature  and time  were chosen as independent variables. The range and center point values of the two independent variables were based on the results of preliminary experiments , size , and sphericity , indicating the physical characteristics of the CAG beads. The experiments were randomized to minimize the effects of unexpected variability in the observed responses.To monitor the effects of heat treatment on the physical properties of the CAG beads, a central composite design (CCD) was adopted in the optimization of the CAG beads. CCD in this design comprises 2eriments . The depUsing the response surface methodology of MINTAB statistical software , Equation (1) was used to fit results .(1)Y=β0Y is a dependent variable , 0β is a constant, iβ, ii, βijβ are regression coefficients, iX and jX are levels of the independent variables. The target value of Y1 maximum and response optimization were calculated by the response optimizer in the MINITAB software. Three-dimensional response surface plots were developed using Maple software  and represented a function of two independent variables.Here, To investigate the influence of heat treatment on CAG bead microstructure, the CAG beads were immersed in liquid nitrogen and cut with a knife to obtain the cross section. The CAG beads then underwent lyophilization in a freeze-dryer  for 24 h. Moreover, the CAG beads were fixed to a sample with a gold layer using an ion sputter device , and viewed by SEM  at an accelerating voltage of 15 kV.Water content (%) was measured with a moisture analyzer . The CAG beads were weighed and placed on the analyzer and heated at 100 °C until no weight change was observed. The difference between the original and final weight was considered as the water content. Weighing was performed on a digital balance . Water content and weight are expressed as the mean of three replications.D is the density of the beads; M is the weight of the beads; V is the volume of the beads; r is the radius of the beads.The mean weights and diameters of the beads were measured and used to calculate densities of beads using the following Equation (2):R2) value indicates that the model equations described the experimental designs adequately [R-squared values , the better the fit [p-value smaller than 0.05 implies that the corresponding model term is significant [R2 values of Y1 (rupture strength), Y2 (size), and Y3 (sphericity) were 0.904, 0.888 (p < 0.05), and 0.935 (p < 0.01), respectively. The statistical significance of the quadratic polynomial model equation was evaluated by an analysis of variance (ANOVA), shown in F-values of Y1, Y2, and Y3 were 9.57, 8.63, and 1.57, respectively; the related p-values were not significant  (p > 0.05). These results indicate that the models were suitable for accurately predicting variation [equately . In gene the fit . A p-valnificant . The R2 ariation .1, X2), quadratic , and interaction (X1X2) terms for the three dependent variables . The effects of heating temperature (X1) and heating time (X2) on rupture strength (Y1), size (Y2), and sphericity (Y3) are expressed as a three-dimensional plot  increased when the heating temperature (X1) increased from 40 °C (−1.414) to 100 °C (+1.414); Y1 decreased when heating time (X2) increased from 5 min (−1.414) to 60 min (+1.414) (1 was significant (p < 0.01), while their square terms and interaction terms were not significant (p > 0.05) (1 term for Y1 was significant (p < 0.01), while the X2 term for Y1 was not significant (p > 0.05) (1) is a significant factor affecting the rupture strength (Y1); however, there was no statistically significance between rupture strength (Y1) and heating time (X2). This result supports previous study [The rupture strength (Y(+1.414) a. The li > 0.05) . The X1  > 0.05) . We founus study , which sus study . The autus study .2) sharply decreased with an increase in the heating temperature (X1) from 40 °C (−1.414) to 100 °C (+1.414); however, there was no significant relationship between size (Y2) and heating time (X2) from 5 min (−1.414) to 60 min (+1.414) (p > 0.05) (2 were significant (p < 0.01), while their square terms and interaction terms were not significant (p > 0.05) (1 term for Y2 was significant (p < 0.01), while X2 term for Y2 was not significant (p > 0.05) (1) is a significant factor affecting the size (Y2); however, there was no significant relationship between the size (Y2) and heating time (X2). This finding is consistent with previous study results [The size (Y > 0.05) b. The li > 0.05) . The X1  > 0.05) . Here, i results , where t results  found th results ,27,28. A3) increased as the coded values of independent variables approached zero (3 were not significant (p > 0.05), while their square terms were significant (p < 0.01) (3 were not significant a (p > 0.05), while their square terms were significant (p < 0.01) (The sphericity (Yhed zero c. The li < 0.01) . The lin < 0.01) . Previou1) and size (Y2) affected dependent variables, with heating temperature (X1) being the most important factor. In addition, as the CAG beads’ heating temperature (X1) increased, the rupture strength (Y1) increased and the size (Y2) decreased.In conclusion, the rupture strength (YX1) increased from 70 °C to 100 °C, the CAG beads gradually formed a more compact gel network with a homogeneous distribution of small pores, while the CAG beads, after heat treatment at 40 °C exhibited void pores. The change of microstructure in the CAG beads could explain the change in physical properties. Our findings reveal that at higher temperatures, the rupture strength increased  and weight were correlated . Here, data show that the higher the heated temperature of the CAG beads, the more significant the size (Y2) and weight reduction. Moreover, size (Y2) and water content were correlated . However, the water content of the CAG beads decreased as the heat treatment increased. The weight of the non-heated CAG beads was 11.5 mg and the weight of the heated CAG beads at 100 °C was 5.8 mg. The water content of the non-heated CAG beads was 95.9%, while that of the CAG beads heated at 100 °C was 93.3%. Our findings reveal that the absolute water content 11.03 mg was reduced to 5.39 mg and there was a 51.2% water loss.Heating at 70 and 100 °C significantly reduced the CAG bead size believed to result from water loss ,27,28. TIn conclusion, the SEM images, with the water content, weight, and size correlations show that the denser the structure of the CAG beads after exposure to higher heating temperatures, the less available space there is for water, resulting in an increased rupture strength and reduced size.1), size (Y2), and sphericity (Y3) and were verified (1), size (Y2), and sphericity (Y3) of the unheated CAG beads were 3450 ± 112.47 kPa, 2.60 ± 0.05 mm, and 96.5 ± 2.14%, respectively. According to the results of CCD, the optimal conditions of X1 (heating temperature) and X2 (heating time) were −0.6649 (56.0371 °C) and −1.414 (5 min), respectively. To facilitate the operation, the optimal process conditions of heating time and temperature were 56 °C and 5 min, respectively. The predicted values of Y1, Y2, and Y3 for each optimal condition were 2993 kPa, 2.60 mm, and 96.8%, respectively. To verify the accuracy of the predicted Y1, Y2, and Y3 values, the CAG beads were prepared under each of the optimal conditions and tested. Percentage error evaluation is helpful in establishing the validity of generated model equations and also to describe the domain of applicability of the optimization model [1, Y2, and Y3 were 2844 ± 66.64 kPa, 2.55 ± 0.02 mm, and 96.0 ± 2.25%, respectively, similarly to the predicated values, with error values of 4.98, 1.92, and 0.83, respectively  was the factor that had the greatest effect on the rupture strength (Y1) and size (Y2). The CAG beads increased in rupture strength and decreased in size as the heating temperature increased because of water loss. RSM was successfully employed to optimize the non-heated CAG beads. Under each optimal condition, Y1, Y2, and Y3 were 2844 ± 66.64 kPa, 2.55 ± 0.02 mm, and 96.0 ± 2.25%, respectively. Our results indicate that the heat treatment of CAG can be used not only for sterilization and cooking but also as a processing technique by controlling the physical properties.Calcium alginate gel (CAG) is used to make artificial or imitative foods because of its unique gelling abilities and is a promising biomaterial in the food industry. In the food industry, it is important to verify stability and process suitability for artificial or imitative foods using CAG; the effects of heat treatment on the physical properties of CAG beads can be utilized in several ways. Our findings reveal that the heating temperature (X


Arthropod-borne viruses (arboviruses) can be maternally transmitted by female insects to their offspring, however, it is unknown whether male sperm can directly interact with the arbovirus and mediate its paternal transmission. Here we report that an important rice arbovirus is paternally transmitted by the male leafhoppers by hitchhiking with the sperm. The virus-sperm binding is mediated by the interaction of viral capsid protein and heparan sulfate proteoglycan on the sperm head surfaces. Mating experiments reveal that paternal virus transmission is more efficient than maternal transmission. Such paternal virus transmission scarcely affects the fitness of adult males or their offspring, and plays a pivotal role in maintenance of viral population during seasons unfavorable for rice hosts in the field. Our findings reveal that a preferred mode of vertical arbovirus transmission has been evolved by hitchhiking with insect sperm without disturbing sperm functioning, facilitating the long-term viral epidemic and persistence in nature. Arbovirus vertical transmission is commonly mediated by transovarial passage of female insect vectors. Here, the authors show that Rice gall dwarf virus can be transmitted by male leafhoppers via interactions of the viral capsid and heparan sulfate proteoglycan on the surface of sperm heads. For example, Rice stripe virus (RSV) transmitted by planthoppers has caused a serious agricultural threat in Asian rice-growing countries3, and Zika virus transmitted by mosquitoes has caused a recent public health threat in Americas4. Frequently, arthropod-borne viruses (arboviruses) can be vertically transmitted to the vector progeny population to ensure survival during adverse conditions for horizontal transmission7. Thus, vertical transmission is an important endemic maintenance mechanism for arboviruses in nature. Vertical virus transmission by insect vectors in nature may include maternal or paternal transmission10. Maternal transmission of arboviruses through transovarial passage has been extensively investigated14, however, whether sperm-mediated paternal virus transmission occurs remain undetermined. Oocytes accumulate a large quantity of cytoplasm that provide a room for viral infection, whereas sperms discard their cytoplasm during spermatogenesis and transform into a streamlined shape with the small head consisting of condensed nucleus and the slender tail made of microtubule bundles for motility15. Therefore, if arboviruses can be paternally transmitted via insect sperm, a possible target may be the outer membrane of the sperm. Considering the extremely streamlined sperm structure, viral infection to the sperm head is expected to impair normal functioning of the sperm18. For example, the presence of human immunodeficiency virus in the human sperm and Zika virus in mice sperm would damage sperm normal functioning18, and, thus, sperm-mediated paternal virus transmission may seem unlikely to occur. In this study, however, we demonstrate that a preferred mode of parental virus transmission has been evolved by hitchhiking with the sperm of male insect vectors without disturbing sperm functioning in a leafhopper-borne plant reovirus.Many devastating plant, animal, and human pathogens are vectored by arthropod insects19. The mechanisms for vertical transmission of persistent plant viruses between an infected female and its offspring through transovarial passage have been demonstrated14. For example, we have determined that RSV, a tenuivirus, and Tomato yellow leaf curl virus, a begomovirus, exploit the existing oocyte entry paths of vitellogenin to overcome transovarial transmission barriers in planthopper or whitefly vectors12. We also have shown recently that transovarial transmission of Rice dwarf virus (RDV), a plant reovirus, is mediated by the specific interaction of the viral capsid protein with the outer membrane protein of an obligate symbiotic bacterium of the vector green rice leafhoppers20. Rice gall dwarf virus (RGDV), also a plant reovirus, causes epidemic outbreaks and extensive rice yield losses in Asian rice-growing countries, and has long been thought to be transmitted by a transovarial mechanism in green rice leafhoppers24. However, we observed recently that the percentage of transovarial transmission (~ 20%) in the main vector of RGDV, the green rice leafhopper Recilia dorsalis, is much lower than the overall percentage (~ 80%) of vertical transmission24. Unlike RDV, RGDV virions encounter strong barriers to enter the oocytes in female vectors for maternal transmission24. The significant disparity between the percentages for the vertical and maternal transmission suggested that RGDV may have evolved to be paternally transmitted by male insects to the offspring. Here, we report that a high efficiency of sperm-mediated paternal transmission route of RGDV by male R. dorsalis occurs without affecting the fitness of male insects or their offspring, which may play a vital role in long-term maintenance and spread of RGDV in the field.More than 75% of plant viruses can be transmitted by aphids, leafhoppers, planthoppers, whiteflies, and other vectors in a persistent, semi-, or non-persistent manner, thereby providing fertile ground for mechanistic studies on vector transmission+) male (♂) or female (♀) R. dorsalis reared under controlled greenhouse conditions  ♂ leafhoppers, 22% were positive for RGDV  in Guangdong, Southern China when rice plants are rarely present, the weed Alopecurus aequalis becomes the primary habitat of rice leafhoppers for up to two generations  was generated as bait to screen the sperm proteins extracted from R. dorsalis   Fig. . The 12 73) Fig. . Yeast t73) Fig. . The spe73) Fig. . RT-qPCRons Fig.  and the ons Fig. . It is cons Fig. .Nephotettix cincticeps. We first observed that the viruliferous rates of natural R. dorsalis and N. cincticeps population in Guangdong were ~ 58% and 5% during planting seasons, respectively  is evidently higher than the maternal rate (~ 20%) in field-collected R. dorsalis populations. Thus, during the cold seasons unfavorable for virus-infected rice hosts in the field, the maintenance efficiency of RGDV through up to two insect generations tends to decrease. We deduce that such a sperm-mediated paternal transmission is a more powerful type of vertical virus transmission than maternal transmission by insect vectors in many cases, and  plays a vital role in the efficient maintenance of RGDV during the cold seasons in the field. Taken together, a preferred mode of vertical arbovirus transmission has been evolved by hitchhiking with insect sperm, thereby explaining a natural long-term endemic pattern of RGDV throughout Southern China for > 30 years.A handful of studies have described the mechanistic basis of vertical maternal transmission of arboviruses and its role in epidemic persistence of viruses30. It seems that La Crosse virus and Zika virus in mosquitoes can be venereally transmitted by male accessory sex gland fluid rather than by sperm, which agrees with the findings for the symbiotic rhabdoviruses in Drosophila31. We cannot rule out the possibility that paternal transmission of RGDV occurs not through sperm but through seminal fluid. However, even though RGDV can enter into female oocytes from the infected male seminal fluid, it would encounter strong ovarian barriers24, and, thus it appears to be of a low level of occurrence and constitute a relatively minor component of the vertical transmission rate. Given the widespread occurrence of long-term epidemic for RGDV in nature, the virus has gained an evolutionary advantage by hitchhiking with the insect sperm for vertical propagation. We anticipate other arboviruses may have also evolved a similar strategy to ensure viral survival during adverse conditions for horizontal transmission.At present, whether other vector-borne viral pathogens can be carried by insect sperms and then be paternally transmitted with high efficiency in nature is as yet unknown. Interestingly, arboviruses such as La Crosse virus and Zika virus can be paternally transmitted by male mosquitoes with low efficiency, but viral antigens were not observed within spermsOur study reveals a mode of vertical transmission of an arbovirus. Lack of virus-infected host plants in cold seasons presents a bottleneck for arbovirus transmission so that sperm-mediated paternal transmission in vector populations may be critical to viral persistence in the field. Our data lay a foundation for further investigation of the mechanisms underlying paternal virus transmission and ecological significance, and provide insights into the development of efficient approaches to attenuate viral epidemic by targeting sperm-mediated paternal transmission mechanism.R. dorsalis adults from a laboratory-reared colony and a field-caught colony, respectively. In each treatment, one newly emerged adult female was crossed with one newly emerged adult male as follows: (i) V− female (V−♀) × V− male (V−♂); (ii) V+ female (V+♀) × V− male (V−♂); (iii) V− female (V−♀) × V+ male (V+♂); and (iv) V+ female (V+♀) × V+ male (V+♂). The V− and V+R. dorsalis populations have been established in our laboratory  assay, and the females were left in each of the tubes to lay eggs for another 5 days and the seedling in each tube was renewed daily. The potential V+ females were then collected for virus detection by RT-PCR assay. At 7 days after the removal of females, the seedlings were dissected to collect eggs for RGDV detection by RT-PCR assay. For each of the four mating types in the three independent experiments with the laboratory-reared colony, the number of eggs detected in each independent experiment was as follows: V−♀ × V−♂, n1 = 51, n2 = 65, n3 = 69; V+♀ × V−♂, n1 = 72, n2 = 72, n3 = 76; V−♀ × V+♂, n1 = 78, n2 = 77, n3 = 81; V+♀ × V+♂, n1 = 51, n2 = 62, n3 = 72. For each of the four mating types in the three independent experiments with the field-caught colony, the number of eggs detected was as follows: V−♀ × V−♂, n1 = 66, n2 = 66, n3 = 50; V+♀ × V−♂, n1 = 65, n2 = 66, n3 = 55; V−♀ × V+♂, n1 = 78, n2 = 77, n3 = 81; V+♀ × V+♂, n1 = 55, n2 = 62, n3 = 84.Four treatments of crossing were conducted for boratory . Our preR. dorsalis were fed on diseased rice plants for 2 days and then transferred to non-infected rice seedlings. At different days after eclosion, the reproductive system from 30 males was excised, fixed, immunolabeled with virus-specific IgG conjugated to rhodamine (virus-rhodamine) (0.5 μg/μl) and actin dye phalloidin-fluorescein isothiocyanate (FITC) , and then processed for immunofluorescence microscopy. For visualizing viral association with sperm, mature sperms were excised from the seminal vesicles of V+ males and then smeared on poly-lysine-treated glass slides. The sperms were fixed and immunolabeled with virus-rhodamine (0.5 μg/μl), and then stained with 4′,6-diamidino-2-phenylindole (DAPI) . For visualizing the virus in females after transfer from males, virgin V− female adults were mated one on one with V+ male adults in individual glass tubes for 3 days. Males were then collected and confirmed for RGDV-positive by RT-PCR assay. The reproductive system was excised from each of the 30 female adults at 3, 6, and 10 days after mating with the V+ males, and then immunolabled with virus-rhodamine (0.5 μg/μl) and phalloidin-FITC  to visualize the route of viral transfer.For visualizing viral infection to the male reproductive system, second instar nymphs of + male adult R. dorsalis or the spermatheca of female adult R. dorsalis at 3 days after crossing with V+ males were excised, fixed with 2% v/v glutaradehyde and 2% v/v paraformaldehyde in posphate-buffered saline (PBS) for 2 h at room temperature, and then postfixed with 1% w/v osmium tetroxide in PBS for 1 h at room temperature. The fixed samples were dehydrated in grade series of ethanol up to 100% and embedded in Spurr’s resin (SPI Ltd). Sections were observed with a transmission electron microscope .The seminal vesicles of VEscherichia coli strain Rosetta, and the proteins were purified using nickel-nitrilotriacetic acid resin (Qiagen). Sperm smears were also incubated with the purified proteins (0.5 μg/μl) for 60 min and then stained with P2- or P8-specific IgG conjugated to rhodamine, P2-rhodamine (0.5μg/μl) or P8-rhodamine (0.5 μg/μl), respectively, and then processed for confocal microscopy. Alternatively, purified RGDV virions were pre-incubated with P2 or P8 antibodies (0.5 μg/μl) for 10 min, and then incubated with live sperms. The samples were stained with virus-rhodamine and DAPI, and then processed for confocal microscopy.Mature, live sperms from the seminal vesicles were smeared on poly-lysine-treated glass slides, and then incubated with purified RGDV virions (0.01 μg/μl) for 5, 30, or 90 min For detecting the binding of P2 or P8 of RGDV with sperm in vitro, His-tag-fused P2 or P8 was expressed in R. dorsalis using a GST pull-down assay. In brief, the GST-fused P8 or GST was bound to GST-Sepharose 4B beads (GE) for 3 h at 4 °C. Sperms were collected from the seminal vesicles of the males, suspended in PBS and lysed by ultrasounds. Sperm lysates were then centrifuged at 15,000×g for 15 min, and the supernatants were incubated with GST or GST-P8 conjugated Sepharose beads for 4 h at 4 °C. After extensive wash of the column, bound proteins were eluted, and then the eluted proteins were treated with dithiotreitol (1 mm) for 30 min followed by alkylation with iodoacetamide (50 mm) for 30 min The mixture was incubated with trypsin (1 μg/50 μg protein) (Promega) overnight, centrifuged at 14,000 × g for 2 min and the pellets were collected. Peptides were eluted into autosampler vials with 50 μl buffer (84% ACN in 0.5% acetic acid). Organic solvent was removed in a SpeedVac concentrator and the final sample volume was adjusted with buffer (2% ACN in 0.1% TFA) to 12 μl. A nanoflow high performance liquid chromatography instrument (Thermo Fisher Scientific) was coupled on-line to a Q Exactive (Thermo Fisher Scientific) with a nanoelectrospray ion source (Proxeon). The peptide mixture (5 μg) was loaded onto a C18-reversed phase column  and separated with a linear gradient of 4–100% buffer B (80% ACN and 0.5% acetic acid). Data processing was performed using MaxQuant v1.6.0.1 software. The experiment was replicated three times. Peptides identified specifically from GST-P8 bound proteins were selected for analysis. Around 12 peptides targeted HSPG were identified in each replicate. We then used PCR, 5′ and 3′ RACE approaches to clone the full-length HSPG cDNA based on the transcriptome database of R. dorsalis. It was a unique gene in the transcriptome database of R. dorsalis. The full-length cDNA  of HSPG was obtained, and it contained five domains (I–V)  using a DUALmembrane starter kit (DUALsystems Biotech) according to the manufacturer’s manual. The P8 gene of RGDV was cloned into the bait vector pBT3-STE (PBT-STE-RGDV P8), and the five domain sequences of HSPG of R. dorsalis as well as HSPG domain III of N. cincticeps and P. alienus were cloned into the prey vector pPR3-N  (Supplementary Table Interaction between HSPG of E. coli strain BL21. GST-HSPG-DoIII was incubated with glutathione-Sepharose beads (Amersham) for 4 h at 4 °C, then the mixture was centrifuged for 5 min at 500 g, and the supernatant was discarded. His-P8, His-P2, or His-GFP was added to the beads, and the mixture was incubated for 4 h at 4 °C. The beads were collected and washed with washing buffer . Immunoprecipitated proteins were detected by western blot with His-tagged antibodies  and GST-tagged antibodies  separately.A GST pull-down assay was used to confirm the interaction between the protein encoded by the domain III fragment of the HSPG gene and the P8 gene of RGDV. In brief, the domain III sequence of the HSPG gene was cloned into PGEX-3 for fusion with GST. The P8 gene of RGDV, P2 gene of RGDV and the GFP gene were cloned into pBT30a for His-tag fusion  at the 5′ end. PCR products were used for the synthesis of dsRNAs targeting HSPG domain III (dsHSPG) according to the protocol for the T7 RiboMAX Express RNAi System kit (Promega). The 630-bp segment of the GFP gene was used for in vitro dsRNA syntheses (dsGFP) as the control  with a Nanoject II Auto-Nanoliter Injector (Spring), and then placed on non-infected rice seedlings. Different concentrations of the dsHSPGs were microinjected into newly emerged male adult leafhoppers, and the mortality of insects was assessed every day. For each concentration, 30 insects were microinjected, and three replicates were performed. To measure the effects of dsRNA on the transcript levels of the P8 gene of RGDV and HSPG gene of R. dorsalis, the male reproductive systems were excised from dsRNA-treated male adults at 5 days after microinjection, and then total RNAs were extracted using Trizol reagent (Invitrogen). The transcript levels of the P8 gene of RGDV and HSPG gene were quantified by relative RT-qPCR assay with the SYBR Green PCR MasterMix kit (Promega) in a Mastercycler Realplex4 real-time PCR system (Eppendorf) in accordance with the manufacturer’s instructions. Relative levels of gene expression were normalized to a housekeeping gene elongation factor 1alpha gene  and estimated by the 2−△△Ct (cycle threshold) method. A pool of 20 insects was used for each replicate, and the experiment was replicated three times. Furthermore, the total proteins were extracted from dissected male reproductive systems of 40 dsRNA-treated leafhoppers. P8 and HSPG protein levels were analyzed by western blot assay using P8- and HSPG-specific IgGs, respectively. To determine whether dsHSPG treatment inhibited paternal transmission, at 3 days after microinjection, one dsHSPG- or dsGFP-treated V+ virgin male was placed with one V− virgin female in a glass tube containing a rice seedling for 3 days. Ten pairs were performed for each treatment. The male leafhoppers were then assayed for RGDV, and female leafhoppers were left in the tubes for oviposition. The offspring of each cross  were tested for RGDV by RT-PCR assay.A DNA fragment spanning a 774-bp segment targeting HSPG domain III was amplified by PCR using forward primer 5′-P<0.05 significance level. Comparisons between two means were conducted using Student’s t test. The data were back-transformed after analysis in the text, figures, and tables.All data were analyzed with SPSS . Percentage data were arcsine square root-transformed before analysis. Multiple comparisons of the means were conducted using a one-way analysis of variance followed by Tukey’s honestly significant difference test at the Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting Summary


Rapid transmission and high mortality of Ebola virus disease (EVD) highlight a urgent need of large scale, convenient and effective measure for Ebola virus screening. Application of monoclonal antibodies (mAbs) are crucial for establishment of an enzyme-linked immunosorbent assay (ELISA) with high sensitivity and specificity.The traditional cell fusion technique was used to generate a panel of hybridomas. Two mAbs were characterized by SDS-PAGE, Western blot, Indirect immunofluorescence assay (IFA). A sandwich ELISA was established using the two mAbs. The detection capability of the ELISA was evaluated.In the current study, we produced two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), the major viral transmembrane spike protein associated with viral attachment. It was shown that 6E3 and 3F21 recognized GP1 and GP2 subunits of the GP respectively. Furthermore, 6E3 and 3F21 bound to corresponding epitopes on GP without reciprocal topographical interpretation. Subsequently, a sandwich ELISA based on the two mAbs were established and evaluated. The detection limit was 3.6 ng/ml, with a linear range of 3.6–100 ng/ml. More importantly, Ebola virus like particles (eVLPs) were able to be detected by this established virus detection measure.We produced and characterized two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), and established a sandwich ELISA based on the mAbs. It was suggested that the sandwich ELISA provided an alternative method for specific and sensitive detection of Ebola virus in the field setting. Ebola virus infection results in severe hemorrhagic fever in humans with high case-fatality rates of up to 90% . Over 11There were five distinct species of EBOV: Zaire ebolavirus (ZEBOV), Sudan Ebola virus (SEBOV), Ivory Coast Ebola virus (ICEBOV), Reston Ebola virus (REBOV), and Bundibugyo Ebola virus . Ebola vAccumulating studies revealed that two virus-specific mAbs were used to set up an effective ELISA kit for pathogen diagnosis , 12. In 2 humidified atmosphere. Recombinant Zaire Ebola virus GPdTM  was mature, recombinant, His-tagged Zaire Ebola virus Glycoprotein minus the transmembrane domain (rZEBOV GPdTM). Plasmid pEAK13-GP (Zaire) expressing Zaire Ebola virus GP was a gift from Dr. C Jiang, Tsinghua University, China [HEK 293 T, Vero 1008 and RD cells were cultured in Dulbecco’s modified Eagle’s medium , supplemented with 10% heat-inactivated fetal bovine serum (FBS) , and penicillin/streptomycin at 37 °C in a 5% COThe preparation of Ebola virus GP-specific mAbs were generated as previously described . In brieWestern blot analysis was performed as described previously with minor modification . HEK 293Dot-blot analysis was performed as described previously . DiffereBinding assays were performed in 96-well microplates by Octet Red system (Ferbio) . FirstlyThe additive test analysis was performed as described previously . AI = {[IFA was performed as described previously . HEK 293Generation of eVLPs was performed as described previously . Briefly6 293 T cells were co-transfected with 10 μg of a pNL4–3.Luc.R-E- [4 293 T cell each well. At 48 h post infection (hpi), cells were subject to determine the luciferase activity by by a Luciferase Assay System according to the manufacturer’s instructions (Promega) using Tuner Biosystems Modulus II.To generate EBOV pseudotypes, 4 × 10Luc.R-E-  and 10 μLuc.R-E- . The effLuc.R-E- . Sera we2CO3, 60 mmol/L NaHCO3, pH 9.6) were adsorbed to the surface of 96-well flexible microplates  at 4 °C overnight. After discarding coating buffer, samples were incubated in the microplates for 1 h at 37 °C. After washing 5 times with PBST, the plates were incubated for 45 min at room temperature with HRP-conjugated 3F21 (1:5000). After washing with PBST seven times, immunoreactivity was visualized by means of a TMB substrate system  and the optical density values (OD630nm) were measured using an ELISA plate reader .Sandwich ELISA was performed as described previously with minor modification . BrieflyIn order to achieve mAbs towards Ebola virus GP, we used commercially available recombinant Zaire Ebola virus GPdTM (Glycoprotein minus the transmembrane domain) to immunize BALB/c mice. Subsequently murine-derived hybridoma cells were generated by traditional cell fusion technology , 22. ConThe subunits recognized by 6E3 and 3F21 were determined by Western blot. HEK 293 T cells were transfected with plasmids expressing GP1 or GP2 for 48 h. Cell lysates from transfected cell were analyzed by Western blot using mAb 6E3 or 3F21. As shown in Fig. The antibody affinity towards GP was primarily assessed by Dot-blot. rGPdTM, as well as HIV-1 GP120, were dropped onto the NC membrane for 2 h, mAbs 3F21, 6E3 and irrelative 5G10 were used as detector antibody. The results showed that 3F21 and 6E3 were able to specifically bind to Ebola virus GP  or irrelevant antibody 5G10. The layer thickness (in nm) on APS sensor reflected the interaction between molecules. It was observed that a rapid and direct interaction occurred between mAbs (6E3 or 3F21) and GP. Moreover, the affinity of 3F21 towards GP was stronger than that of 6E3 at the same antibody concentration   test was performed to identify whether there was reciprocal spatial interpretation between the two antibodies 3F21 and 6E3 when binding to GP at the same time. ELISA plate was coated with GPdTM at concentration of 0.5 μg/ml (lower concentration) at 4 °C overnight. The 6E3 and 3F21 were used as detector antibody. Additive index value (97) between 6E3 and 3F21 was close to 100 .The Ebola virus glycoprotein (GP), which interacts with host Niemann-Pick C1 (NPC1) receptor, mediates viral attachment and entry into host cells and is the major inducer for the host immune response . During Aim to establish a mAb-based antigen detection method with sandwich ELISA, the selected mAbs should be topographically unrelated. Thus the ELISA additive index test was used to determine whether or not the epitopes recognized by the different mAbs were overlapping . The FigDual mAs were helpful to enhance the specificity of the virus detection and monitoring. ReEBOV Antigen Rapid Detection Test (RDT) was the first RDT listed by the WHO. Following laboratory analytical validation as per the Food and Drug Administration guidelines, the specificity of this VP40-detecting test was evaluated at 95% on serum specimens and at 97% on whole blood specimens . Its LDTDue to biosafety concerns, we were not allow to manipulate infectious Ebola virus. We hence had to evaluate the detection capacity of the ELISA by testing eVLPs, instead of active Ebola virus. Despite successful observation Fig.  and deteIn summary, two murine-derived mAbs towards distinct antigenic sites of Zaire Ebola virus GP was developed. Based on the two mAbs with unique properties a sandwich ELISA was established for Ebola virus detection. We would take this research further by developing a more sophisticated system using these mAbs, aiming for a more accurate and convenient detection.


Foot-and-mouth disease (FMD) is a contagious viral disease of animals. Multiple serotypes and antigenic variation in the viral genome are probably the factors that reduce control of the disease. Currently, the vaccines employed against FMD use killed virus. The inactivation or killing of the virus makes it less immunogenic and reduces its immunoprophylactic potential. To cope with this situation, the present study was designed, anti-idiotype FMD virus antigen was prepared, and the immunogenic potential of the antigen was compared to that of commercial killed-virus vaccines. The overall results showed that a persistent and strong immune response occurred with anti-idiotype FMD virus antigen. Thus, anti-idiotype FMD virus antigen may serve as a potential surrogate of FMD virus vaccines. The immunoprophylactic potential of anti-idiotype (anti-id) foot-and-mouth disease (FMD) antigen (Ag) was evaluated in the calves. The idiotype antibodies (Ab1) were produced in experimental goats by injecting inactivated FMD virus. The Fab (fragment antigen binding) of Ab1 was injected into the layer birds to raise anti-id antibodies (Ab2). The Ab2 was purified from egg yolks. The Fab component of Ab2 was emulsified in Montanide (1:1) and used as a surrogate of FMD virus. The immune response to Montanide adjuvanted monovalent and trivalent anti-id FMD virus antigen was determined in mice. The comparative immune potentiation potentials of Montanide adjuvanted trivalent anti-id FMD virus antigen and trivalent FMD vaccine were determined in mice and calves. Montanide adjuvanted monovalent anti-id FMD virus antigens produced mean Ab titers of 78.80%, 81.30%, and 81.20% for serotypes A, Asia 1, and O, respectively, at 45 days postimmunization (p.i.) in mice. Montanide adjuvanted trivalent anti-id FMD Ag in mice produced the highest Ab titer, 81.60%, at day 45 compared to the 77.50% titer measured for Montanide adjuvanted FMD vaccine at day 45 p.i. A slow decrease of 1% to 2% was recorded for the Ab titer of Montanide adjuvanted trivalent anti-id FMD virus antigen in mice at day 60. In calves, the titer corresponding to the immune response seen with Montanide adjuvanted trivalent anti-id FMD virus antigen (80%) was persistent whereas the titer of Montanide adjuvanted FMD vaccine decreased to 74% at day 60 p.i. Anti-id FMD virus antigen induced a strong and persistent immunogenic response in terms of Ab titer compared to the inactivated virus vaccine. Anti-id FMD virus antigen may serve as a surrogate of FMD virus vaccine.IMPORTANCE Foot-and-mouth disease (FMD) is a contagious viral disease of animals. Multiple serotypes and antigenic variation in the viral genome are probably the factors that reduce control of the disease. Currently, the vaccines employed against FMD use killed virus. The inactivation or killing of the virus makes it less immunogenic and reduces its immunoprophylactic potential. To cope with this situation, the present study was designed, anti-idiotype FMD virus antigen was prepared, and the immunogenic potential of the antigen was compared to that of commercial killed-virus vaccines. The overall results showed that a persistent and strong immune response occurred with anti-idiotype FMD virus antigen. Thus, anti-idiotype FMD virus antigen may serve as a potential surrogate of FMD virus vaccines. Aphthovirus of family Picornaviridae. Foot-and-mouth disease (FMD) virus contains a genome of 8.5 kb; the open reading frame (ORF) of the virus is translated into a polyprotein that, on posttranscriptional modification, produces four structural and eight nonstructural proteins  titers (Another approach to evoke an effective immune response is through the use of anti-idiotype (anti-id/Ab2) vaccines. Anti-id vaccines represent alternatives to protein- and peptide-based vaccines. Anti-id vaccines are generated against the idiotype of the primary antibodies (Ab1). The use of anti-id vaccines has been successfully practiced against various types of the human and animal antigens . Ab2 gen) titers . Anti-id) titers . The curAnti-id FMD antibodies were prepared in the layer birds. The development of Ab2 in the egg yolks of layer birds at day 14 postimmunization (p.i.) was confirmed through the use of the agar gel immunoprecipitation test (AGPT).The primary treatment of diluted egg yolk with ammonium sulfate (AS) resulted in yellow pellet formation upon centrifugation. More lipid contents were observed in primary treatment. The secondary treatment with 2 M AS resulted in precipitation of IgY proteins and in the formation of a clear white pellet. The IgY protein concentrations obtained from the egg yolks were more than 10 mg/ml in most of the samples processed.The purified IgY antibodies, after fragmentation with pepsin was performed, proceeded to SDS-PAGE for the assessment of the Fab (fragment antigen binding) component. The sizes of the fragmented IgY proteins ranged between 55 to 70 kDa as represented in The presence of maximum Fab protein contents was recorded in group LIIA (serotype A), while group LIIA1 (serotype Asia 1) showed the second highest Fab protein concentrations as represented in The culture media inoculated with a suspension of monovalent Montanide adjuvanted anti-id FMD virus antigen (MATM) and trivalent Montanide adjuvanted anti-id FMD virus antigen (MATC) for all three serotypes  indicated sterile preparations at up to 7 days postincubation, and no turbidity was recorded in the culture broth. Furthermore, the inoculations on the blood agar plates were also negative for any bacterial growth.The change in body temperature of the inoculated rabbits was normal (101 ± 0.5°F to 103 ± 0.5°F), and no mortality was recorded. The other parameters were in normal range, with signs of mild swelling at the site of injections. The swelling from the inoculations was resolved after 24 h.The Montanide adjuvanted monovalent anti-id FMD virus antigen (MATM) performed similarly for all three FMD serotypes. The mean Ab titer of Montanide adjuvanted anti-id FMD virus antigen was initially low, but, over the course of the study, the Ab titer increased until day 45 p.i. The titer started decreasing at day 45 p.i.; the decline was slow as presented in Group MMA of serotype Asia 1 (MMA1) yielded a variable range of mean Ab titers: initially, a titer of 73.40% was measured at day 15 p.i. followed by a stable rise in the mean Ab titer to a maximum of 81.30% at day 45 p.i. A slow decrease in the mean Ab titer to 79.50% at day 60 p.i. was observed as depicted in A steady increase in the mean Ab titer for mice immunized against Montanide adjuvanted trivalent anti-id FMD virus antigen was recorded. The mean Ab titer was 72.50% at day 15 p.i. followed by a stable rise to 81% at day 45 p.i. The mean Ab titer decreased to 80% at day 60 p.i.; the decline in the mean Ab titer was slow, as presented in The immune status of Montanide adjuvanted trivalent anti-id FMD virus antigen (MATM) was compared to the commercially available Montanide adjuvanted trivalent FMD vaccine (CTVM). The mean Ab titer of MATM ranged from 74.30% to 79.70% compared to the mean Ab titer range of 73.10% to 75.80% for CTVM as described for The mean Ab titer of MATM was 74.30% at day 15 p.i., which represented a nonsignificant difference from the 73.10% Ab titer of CTVM. A factorial increase in the Ab titer (79.70%) of MATM was observed that was significantly different from the 77.40% Ab titer of CTVM at day 30 p.i. The Ab titer of MATM persistently increased and reached its maximum value of 81.60% at day 45 p.i., whereas that of CTVM remained constant at 77.50%. The differences in the mean Ab titers of MATM and CTVM were significant at day 45 p.i.A decline in the mean Ab titers of MATM and CTVM was recorded at day 60 p.i.; the mean Ab titer of MATM decreased to 79.70%, which was significantly different from the 75.80% mean Ab titer of CTVM at day 60 p.i. as depicted in The immune response to Montanide adjuvanted trivalent anti-id FMD virus antigen (MATC) was observed in calves.The Ab titer of Montanide adjuvanted trivalent FMD vaccine (CTVC)  remained in the range of 71% to 79% from day 15 to day 60 p.i. The Ab titer increased to its maximum value up to day 30 p.i. followed by a continuous decrease as presented in The mean Ab titer of MATC steadily increased to 81.33% at day 45 p.i. followed by a slow decline to 80.00% at day 60 p.i. The mean Ab titer of CTVC was significantly different from that of MATC at day 45 and day 60 p.i. The mean Ab titer of CTVC continuously decreased from 76.00% to 74.66% from day 45 to day 60 p.i., respectively, as depicted in The present study was performed to explore the potential of Ab2 as a surrogate viral antigen against FMD virus. The anti-id antigen was developed against three serotypes of FMD virus: serotypes A, Asia 1, and O. Anti-id antigen were generated from egg yolk antibodies. A study comparing responses to commercially available Montanide adjuvanted trivalent FMD vaccine and Montanide adjuvanted trivalent anti-id FMD virus antigen was performed in laboratory animals.Ab2 was separated from the egg yolk of layer birds (injected with idiotype FMD virus antigen) with AS precipitation. The AS precipitation yielded up to 17 mg/ml of anti-id antibodies. The results of anti-id separation were similar to the results reported by Ko and Ahn, who demonstrated that the AS precipitation method is superior to cation exchange chromatography in terms of yield and purity for separation of antibodies . The useThe recent development of Ab1 and Ab2 as vaccines against bacterial and viral diseases is a great achievement . Anti-id19–Picornaviridae family was reported by Sharpe et al. . Ab2 was raised against potent human DENV serotype 1-specific antibodies named HM14c10; structural mimicry by Ab2 was achieved after several interactions with the heavy chain of antibodies to neutralize the DENV . A neutre et al. . The mana et al. . The Ab2a et al. . In the a et al. . The ovea et al. . The meaa et al. . The resa et al. , where Ahe virus . In the he virus .The idiotype antibodies (Ab1) against FMD virus were developed in experimental goats. Briefly, all three of serotypes A, Asia 1, and O of FMD virus were inactivated and injected into the goats through the intramuscular (i.m.) route for the preparation of polyclonal Ab1. Fab (fragment antigen binding) from the Ab1 was separated and reinjected into layer birds through the i.m. route for the development of anti-id antibodies (Ab2). The development of anti-id FMD antibodies in the egg yolks was confirmed through the use of the agar gel immunoprecipitation test (AGPT). The eggs collected from layer birds previously injected with idiotype FMD virus antigen were processed separately for the purification of Ab2 against all three FMD serotypes . IgY was separated through the use of the ammonium sulfate (AS) precipitation method as described by Ferella et al. . The purThe total protein contents of Fab were adjusted to 10 mg/ml. The prepared Fab from Ab2 was emulsified in Montanide adjuvant (1:1) for the preparation of monovalent anti-id antigens separately for three serotypes  of FMD, while a volume containing 10 mg/ml anti-id protein concentrations for each of the three serotypes  was mixed at 1:1:1 to prepare a trivalent anti-id FMD virus antigen. The suspension was emulsified in Montanide adjuvant (1:1) and stored at <10°C until further use.The prepared anti-id FMD virus antigens  for all three serotypes  were studied to analyze their sterility and safety. Thioglycolate broth and blood agar media were inoculated with 1 ml of each suspension and observed at 37°C for 7 days. The rabbits were immunized subcutaneously (s.c.) in the neck region and observed for changes in body temperature and morbidity for up to 7 days postimmunization (p.i.) for safety studies.The immunogenicity of Montanide adjuvanted monovalent and trivalent anti-id FMD virus antigens was analyzed in albino white mice (25 to 30 g body weight).Fifty mice were divided into five groups (10 mice in each); the groups were named MMA1  and MMA and MMO . Similarly, additional groups were named MAT  and UIC (for “uninoculated control group”). A 0.2-ml volume of monovalent anti-id FMD virus antigen was injected through the subcutaneous (s.c.) route in the members of groups MMA1, MMA, and MMO for serotype Asia 1, A, and O, respectively. A 0.2-ml volume of Montanide adjuvanted trivalent anti-id FMD virus antigen was injected into group MAT, while group UIC remained an uninoculated control group. A booster dose of 0.2 ml was injected after 2 weeks followed by the collection of serum samples at days 0, 15, 30, 45, and 60. The Ab titer was determined through the use of a competitive ELISA kit  according to the protocols of the manufacturer. A titer above 70% at a 1/10 dilution was considered to represent a positive result.The immunogenicity of Montanide adjuvanted trivalent anti-id FMD virus antigen and commercial FMD vaccine was evaluated in albino white mice (25 to 30 g body weight).Thirty mice were divided into three groups, namely, the MATM group , the CTVM group , and the UICM group (for “the uninoculated control group”) (10 mice in each group). A 0.2-ml volume of commercial FMD vaccine  was injected s.c. in the members of the CTVM group, and 0.2 ml of Montanide adjuvanted trivalent anti-id FMD virus antigen was injected into members of the MATM group, while the members of the UICM group were kept as uninoculated controls. A booster dose of 0.2 ml was injected after 2 weeks followed by the collection of serum samples at days 0, 15, 30, 45, and 60. The Ab titer was determined through the use of a competitive ELISA kit.For the experimental trials in calves, animals from the two volunteer farmers were managed at their own farm under conditions of good management. A total of nine animals were segregated into the following three groups: a MATC group , a CTVC group , and a UICC group (uninoculated controls). The members of group CTVC were injected s.c. with a single dose of 5 ml commercially available FMD killed-virus vaccine. A single dose of 5 ml Montanide adjuvanted trivalent anti-id FMD virus antigen was injected through the s.c. route in the members of the MATC group. Group UICC, with three animals, was treated as the uninoculated control group. A booster dose was given after 1 week, and blood samples were collected for serum separation. The titers of the antibodies were checked through the use of a competitive ELISA kit.The results of the Ab titer determination experiments were evaluated statistically using factorial analysis. The comparative mean Ab titers were measured through the use of Fisher’s least-significant-difference test.


Arthritogenic alphaviruses are responsible for a dengue-like syndrome associated with severe debilitating polyarthralgia that can persist for months or years and impact life quality. Chikungunya virus is the most well-known member of this family since it was responsible for two worldwide epidemics with millions of cases in the last 15 years. However, other arthritogenic alphaviruses that are as of yet restrained to specific territories are the cause of neglected tropical diseases: O’nyong’nyong virus in Sub-Saharan Africa, Mayaro virus in Latin America, and Ross River virus in Australia and the Pacific island countries and territories. This review evaluates their emerging potential in light of the current knowledge for each of them and in comparison to chikungunya virus. Togaviridae family. As arboviruses, they circulate between an invertebrate vector (mosquitoes) and a vertebrate host. They are distributed worldwide and are separated into two groups: the New World alphaviruses responsible for disease with neurological involvement and the Old World alphaviruses (or Semliki forest virus group) that have a rheumatic tropism. The latter, also called arthritogenic alphaviruses, notably include chikungunya virus (CHIKV), now widely distributed, O’nyong’nyong virus (ONNV) located in Sub-Saharan Africa, Mayaro virus (MAYV) in Latin America, and Ross River virus (RRV) in Australia and the Pacific island countries and territories [Alphaviruses are a genus of enveloped positive-stranded RNA viruses belonging to the ritories   circulating in three distinct continents, to try to understand their emergence potential in the near future.Robinson first described chikungunya fever in 1955, after an epidemic of dengue-like fever with extremely severe joint pain in Southern Tanganyika (now Tanzania) in 1952–1953 . Its namThree main chikungunya virus genotypes, named after their original geographical distribution, are described: the West African, the East-Central-Southern African , and the Asian genotypes (that include the Caribbean strains) ,9. BeforThe first epidemic, caused by the Indian Ocean lineage, started in coastal Kenya in august 2004 with an attack rate of over 50% , and froIn December 2013, the Asian genotype was responsible for the second major outbreak that started in Saint Martin Island in the Caribbean Sea ,31. FromAedes albopictus, has recently established, enabling local epidemics, such as in Italy in 2007 [A particular and recent concern about CHIKV is its potential to cause outbreaks in temperate regions where the Asian tiger mosquito,  in 2007  and 2017 in 2007 ,34,35 an in 2007  and 2017 in 2007 .Although genetically distinct from CHIKV, ONNV is its most closely related relative . SimilarMAYV was first described in Trinidad where it caused mild to moderate short febrile illness in five humans in 1954 . In 1959Epidemic polyarthritis has been described since 1886 in rural Australia ,63. The Aedes mosquitoes  [Aedes aegypti is usually the main vector of CHIKV in African urban cycles, Aedes albopictus (widely present on La Reunion Island in the Indian Ocean) was the vector responsible for the transmission during the epidemic that bore its name. Indeed, a single mutation, A226V in the E1 glycoprotein, favored the infection and dissemination of the virus in the new Aedes albopictus vector [Aedes albopictus was suspected to be the vector of the disease. This convergent evolution illustrates the ease with which the virus evolves to improve its transmissibility [Between outbreaks, CHIKV circulates in a sylvatic cycle where non-human primates are the main host ,78,79,80Dalzieli , etc.) aDalzieli  and anthopictus) ,82. Intes vector ,84,85. IAnopheles funestus and more rarely Anopheles gambiae are the main vectors [Aedes aegypti and disseminate to their salivary glands [Aedes mosquitoes have not yet been demonstrated to be carriers in the wild. More likely, as for CHIKV, it switches to an urban cycle where humans are the amplifying host during bigger outbreaks.Mainly circulating in an enzootic cycle, the vertebrate host of ONNV is not clearly defined. Nevertheless, forest buffalos, mandrills, and duikers were positive for ONNV in a serosurvey in the Congo basin . Anophel vectors ,87,88,89y glands , but AedHaemagogus sp. [Mansonia sp. [Psophora sp. [Aedes aegypti positive for MAYV [Aedes albopictus [Anopheles species  were able to get infected and to transmit MAYV, adding other potent MAYV vectors distributed worldwide to the list [MAYV is circulating in an enzootic cycle between non-human primates ,92,93,94ogus sp. ,91, Mansonia sp. , and Psohora sp. ,6. Nonetfor MAYV , and expbopictus ,101,102.Aedes vigilax are the most common ones in coastal and subcoastal areas and Culex annulirostris in both rural and urban settings, but Aedes notoscriptus, Aedes funereus, Aedes camptorhynchus, and Ochlerotatus camptorhynchus are also incriminated [Aedes aegypti and albopictus are efficient vectors [While twenty-one vertebrate hosts have been described for RRV , marsupiiminated ,70,110.  vectors ,113,114,Contrary to what has been reported for most flaviviruses , moFollowing a 2.5-day incubation period (range of 2–12 days) ,162, theThe ONNV fever acute phase follows an eight-day or more incubation period . It is cAfter less than a week of incubation , MAYV inFrom traveler data, the incubation time of RRV infection is estimated to be 7–9 days   is very well described and represents the major burden of CHIKV infection because it can be responsible for very heavy and long term incapacity . A recenAt the opposite end of the spectrum, accounts of ONNV chronic manifestations are scarce. While Shore et al. mentioned joint pain associated with residual weakness and depression in some patients in 1965 , other sFor MAYV, recurrent arthralgia has been reported in one patient , and somAs for CHIKV, there is great heterogeneity between studies concerning chronic joint pain after RRV infection. Two studies reported them in 52.5% of patients at one year and 57% of patients at three years ,147. TwoFor CHIKV, standard biological tests usually show lymphopenia, more rarely associated with a moderate thrombocytopenia. Hypocalcemia is detected in more than half of the cases, and a mild rhabdomyolysis with moderately elevated creatinine kinase and transaminases (ASAT > ALAT) might be observed ,133,134.Viremia usually rises a few days before onset of symptoms. RT-PCR or virus isolation permit direct diagnosis; however, most of the time, RT-PCR can no longer detect virus seven days after onset of symptoms for CHIKV ,184  remains a gold standard for diagnostics ,194.Because an efficient curative treatment does not exist, prevention measures are crucial to avoiding or limiting epidemics caused by these arboviruses. These include general measures to avoid mosquito bites and vaccination.Public health response measures are essential to prevent and control any alphavirus outbreak. During disease-free periods, ongoing mosquito surveillance  has shown its effectiveness for predicting RRV epidemics in Australia . Early dBecause CHIKV infection produces a life-lasting protection against a second infection and due to the limited antigenic variability of the virus, one might think that a CHIKV vaccine should be relatively easy to produce, or at least easier so than a dengue virus vaccine . NeverthTwo candidates are currently in Phase II clinical trials. The first one is a recombinant measles virus encoding the CHIKV structural proteins. The first results of the Phase II trial are very encouraging with all vaccines fully immunized after two injections  and excellent safety and tolerability . The secWhile it still needs to be unequivocally demonstrated, a number of studies suggest that protection against one strain of CHIKV protects against all others ,225,226.Three vaccine candidates have been tested for MAYV. The first one was a formalin-inactivated vaccine with moderate efficiency in mice . More reSince RRV has been a public health issue for some time now in Australia, vaccine research there has led to several candidates in preclinical studies ,233,234,Symptomatic treatment with antalgics, such as paracetamol, and steroidal or non-steroidal anti-inflammatory drugs remains the main driver of action available ,238,239.Several studies have searched for efficient antiviral molecules that have a broad-spectrum activity inhibiting all arthritogenic alphaviruses ,245,246;Another approach is to use hyper-immune intravenous anti-CHIKV immunoglobulins to prevent CHIKV infection in neonates, who often develop very severe forms of the disease. Indeed, after a conclusive study in neonate mice , a Phasest Century, chikungunya virus is undoubtedly a re-emerged disease threatening both tropical and temperate regions [With two worldwide epidemics causing close to 10 million cases all together during the 21 regions ,4. While regions ,256,257, regions .Anopheles sp. for ONNV in Africa and the Middle East, Haemagogus sp., Mansonia sp., and Psophora sp. for MAYV in America. and Aedes vigilax and Culex annulirostris for RRV in Oceania and South-East Asia [One of the prerequisites to imagining a worldwide outbreak of any of these alphaviruses is a wide distribution of their mosquito vector. Indeed, no circulation of the disease would occur in a region free from competent mosquitoes. Interestingly, none of these four alphaviruses shares the same vector , and theast Asia . Howeverast Asia ,259 and ast Asia ,261,262;ast Asia .Aedes aegypti and/or albopictus have been proven to be competent vectors in experimental settings for ONNV, MAYV, and RRV [Aedes aegypti and/or albopictus compared to the original vector and trigger a worldwide epidemic, as has happened for CHIKV . BecausOn the other hand, the real risk and the severity of an outbreak are difficult to anticipate because little is known about ONNV, MAYV, and RRV. While a surveillance system has been well established in Australia for RRV , under-surveillance of ONNV and MAYV is an issue, as well as the low number of clinical studies. Thus, many open questions remain concerning possible under-the-radar outbreaks, the true incidence of the diseases, or their clinical manifestations: What is the real risk of chronicity? Can severe cases occur and which types? Can these infections be directly or indirectly lethal? Since CHIKV is more of a global concern, fundamental knowledge on alphaviruses in general is expanding, and many of these publications also tested their hypothesis on the related ONNV, MAYV, or RRV. As an example, recent findings on the still obscure entry pathway of alphaviruses identified Mxra8 as an entry mediator, not only for CHIKV, but also for ONNV, MAYV, and RRV .Another potential pitfall, if an epidemic were to happen, is the speed of identifying which virus is responsible. Indeed, despite some virus-specific trends such as lymphadenopathy for ONNV, arthritogenic alphaviruses share very similar clinical manifestations that are also common to other arboviruses or other infectious diseases. Because they co-circulate in many geographical areas, an etiological diagnosis without biological testing seems impossible. Moreover, many cases of arbovirus co-infections have been reported, adding a layer of complexity to this problem ,268,269.Despite the problems stemming from cross-reactivity, it could be a major help in vaccine design, allowing broad-spectrum protection across all arthritogenic alphaviruses. Indeed, broad neutralizing antibodies against CHIKV can neutralize MAYV and ONNV by inhibiting entry and egress ,279. WhiIn conclusion, enriched by lessons learned from recent CHIKV outbreaks, it is undeniable that ONN, MAYV, or RRV represent emerging threats favored by climatic changes, globalization, travel, migration, and trade. Surveillance is key for detecting any unusual activity of these diseases, so that preventive measures can be taken as soon as possible to tackle a starting epidemic. Hopefully, the availability of a CHIKV vaccine might enable a broad-spectrum activity on all arthritogenic viruses that would be key for future prevention.