Marseilleviridae are giant viruses that have the ability to infect amoebas. Such viruses were initially described in 2009. Since then, this family has grown, and diverse members have been found in different environments and geographic locations. Previous phylogenetic analyses suggested the existence of four marseillevirus lineages. A fourth lineage was described with the discovery of the Brazilian marseillevirus (BrMr), isolated from Pampulha Lake, Brazil. Here we describe the isolation and characterization of the Golden marseillevirus (GMar), a new marseillevirus isolated from golden mussels (Limnoperna fortunei) in South of Brazil. This new representative of Marseilleviridae has circular, double-stranded (dsDNA) that contains 360, 610 base pairs and encodes 483 open read frames (ORFs). The complete virus genome was sequenced and phylogenic analyses indicated clear differences between this virus and other marseilleviruses. In addition, this is the only marseillevirus so far that has been isolated from mussels, and this report expands the diversity of environments from which giant viruses could be recovered.Members of the family  Acanthamoeba polyphaga mimivirus (APMV) in 2003Marseillevirus was isolated in 2007 on biofilm from a cooling tower near ParisMegavirus chilensis was recovered from a seawater sample from the shores of ChilePandoravirus, was recovered from Chilean shores and from a garden pond in AustraliaPithovirus sibericumMollivirus sibericumPoxyviridae, Asfaviridae, Iridoviridae, and Phycodnaviridae. The list of NCLDVs increased with the discovery of mimiviruses2The discovery of protist-infecting giant viruses started with the isolation of Marseilleviridae was isolated from the water of a cooling tower in Paris in 2007Lausannevirus, was discovered in water samples collected from the river Seine in 2011Cannes8 virus was discovered in southeastern France in water from a cooling towerTunisvirus and Fontaine Saint-Charles virus were discovered in freshwater from decorative fountains in Tunisia and France, respectively14Insectomime virus was isolated from the internal organs and digestive tract of a dipteran larva known as the drone flyPort-Miou virus, isolated from a sample from a brackish submarine springBrazilian marseillevirus, isolated from Pampulha Lake in Minas GeraisThe first member of Limnoperna fortunei). These mollusks, which are native to the freshwater systems of China, Southeast Asia, are a mytilid invasive species of the Plata Basin, accidentally introduced in 1991 by ballast waterL. fortunei included Guaíba Lake at Rio Grande do Sul (southern Brazil), where it was first reported in 1998As demonstrated in earlier studies, giant viruses may be isolated from diverse environments. Recently, using molecular and virological methods, one study has demonstrated that oysters are excellent sources for isolating mimiviruses, possibly due to their structure, which allows for the bioaccumulation of microorganisms like viruses and, probably, amoebasGolden marseillevirus (GMar), a new marseillevirus isolated from golden mussels collected in southern Brazil.In this manuscript, we demonstrate that mussels are possible sources for the isolation of marseilleviruses. The isolation of marseillevirus from mussels is important in order to increase the number of strategies for isolation using filter-feeding organisms. Here, we report the isolation, identification, and genomic analysis of the L. fortunei) were collected from Guaíba Lake, Rio Grande do Sul, Brazil, in July 2014 . The samples were pooled, totaling eight pools. These pools were homogenized with 1 mL of PBS, centrifuged at 10,000 × g, filtered through a 0.45-μm membrane and 10 U/μL of Penicillin-GIBCOAcanthamoeba polyphaga (genotype T4) were cultivated in 10 mL of Peptona-Yeast Extract-Glucose (PYG) medium at 30 °C in 25-cm3 culture flasks, supplemented with 50 μg of gentamicin. After 48 h, the cells were harvested and centrifuged. The pellet was re-suspended in sterile PAS , and 104 amoebas per well were cultured in 24-well microplates. After 24 h, 100 μL of the inoculum, prepared as mentioned above, were used to infect the A. polyphaga monolayers, which were then incubated for three days at 30 °C. This step was repeated by sub-culturing the primary culture onto fresh amoeba monolayers. Up to five blind passages were performed. The amoeba cells were assessed daily for the presence of viruses and for cytopathic effects on the cell such as lysis and cellular rounding. To evaluate the replication of GMar in different amoeba, monolayers of Acanthamoeba castellani was infected too.Cells of A. polyphaga monolayers in the 24-well microplates with the virus isolate at an multiplicity of infection (MOI) of 10. The viruses were allowed to adsorb for 1 h, and then the cells were washed with PAS and incubated at 30 °C. At 1–14, 24, 25 and 26 h post-infection (pi), the cultures were frozen and thawed three times and then titrated. The virus titration was performed in 96-well microplates containing approximately 4 × 104A. polyphaga cells per well in 100 μL of the PYG medium. Virus titers were calculated according to the method of Spearmann and Kärber and expressed as the log10 culture infectious doses per 50 mL (TCID50/50 μL).After the virus isolation, infectivity assays were performed by inoculating the A. polyphaga monolayers were infected at an MOI of 10. Uninfected cell cultures were used as controls. After 7 h of infection, the monolayers were washed twice with 0.1 M PBS and fixed with 2.5% glutaraldehyde (grade I) in 0.1 M PBS for 1 h at room temperature. Following the visualization of the full cytopathic effect (CPE), the cell monolayers were scraped off of the plates and pelleted by centrifugation at 900 × g for 5 min. The amoebae were then fixed with 2% osmium tetroxide and embedded in EPON resin. Ultrathin sections were stained with 2% uranyl acetate and examined using a Tecnai G2-Spirit FEI 2006 transmission electron microscope operating at 80 kV at the Microscopy Center, UFMG, Brazil.After the titrations, the A. polyphaga-infected cells were collected, vigorously vortexed, and centrifuged at 5,000 × g for 5 min. The cell-free virus particles were pelleted on a 25% sucrose gradient by ultracentrifugation  at 33,000 × g for 2 h at 4 °C. The pellet was re-suspended in Tris-EDTA-NaCl buffer (TEN). In order to remove the nucleic acids not protected by the capsid, the preparation was treated with 100 U of DNAse I (Roche) and 100 U of RNAse (Invitrogen) at 37 °C for 1 h. Next, the virus DNA was extracted using phenol-chloroform according to Sambrook and Russel® and Qubit apparatus (Life Technologies).The supernatants of the de novo assembled using Geneious® software. The transfer RNA (tRNA) sequences were analyzed by applying the tRNAscan-SE tool. Gene predictions were performed using FgenesVin silico and by the amplification of specific genomic fragments (data not shown).Next-generation sequencing was performed in a MiSeq (Illumina) apparatus with pair-end applications (2 × 150 pb). The pair-end samples were prepared with a Nextera XT DNA sample prep kit. After sequencing, a total of 510,511 reads were Megavirales core genes-namely the family B DNA polymerase, the D6/D11 helicase, the VV A18 helicase, the D5 primase-helicase, and major capsid protein. The amino acid sequences were aligned with Muscle software, and the phylogenetic tree was constructed in MEGA 7. We built a hierarchical-clustering based on the gene presence/absence patter of 5443 NCVOGs, using a MeV tool with Pearson correlation as distance metric. The average nucleotide identity using both best hits (one-way ANI) and reciprocal best hits (two-way ANI) between two genomic datasets as calculated.Phylogenetic reconstruction based on individual and concatenated alignment of the five A. polyphaga monolayers , a concentrated preparation of virus-infected eviruses .4/50 μL) were detected. After 5 h pi, high virus titers were detected, as shown in 11/50 μL). At this time, we observed cell rounding and cell chains formation. After 10 h pi, it was possible to visualize extensive cell lysis, showed in The replicative cycle of GMar was measured at different periods after infection. After 1 h pi, we observed cell alterations in the amoeba cytoplasm, described previously like as cell rounding. At this time, high virus titers . The virus genome of one isolate (GenBank submission KT835053) is a circular, double-stranded DNA molecule composed of 360, 610 bp, which is comparable in size to the genomes, described previously, which range from 346, 754 bp to 386, 631 bp for Lausannevirus and Insectomime virus respectively. The mean guanine-cytosine is 43.1% for GMar, which is also similar to other members of this family.The complete genome of three isolates that induced CPE in Brazilian marseillevirus genome . The GMar virus gene content consists of 48.03% uncharacterized proteins (232 of the 483 predicted proteins). The predicted proteins contained between 35 and 1293 amino acids. Moreover, we found numerous paralogous families, consisting of genes that code endonucleases (seven sequences), serine/threonine protein kinases (sixteen sequences), F-box containing protein (three sequences), putative MutH/archeal (four sequences) and ankyrin repeat (ten sequences). In addition, we found two histone-like proteins: a histone H2B/H2A fusion protein and a histone H3 containing a N-terminal Histone-like transcription factor, an archeal histone domain and C-terminal H3-like domain . The histone-like proteins in GMar resemble those predicted in other marseilleviruses, such as LausannevirusBrazilian marseillevirusA total of 483 open reading frames (ORFs) were identified after merging all coding sequence predictions. These ORFs were evenly distributed on both negative (263 ORFs) and positive (220 ORFs) strands, similar to the gene distribution on the A total of 440/483 ORFs (91%) from the GMar had a significant BLASTp matches to marseilleviruses protein sequences available in the NCBI non redundant database. The 43 ORFs that displayed no identity with previously described sequences were tentatively classified as ORFans. As for the other marseilleviruses, no tRNA-related genes were detected in the GMar genome.Marseilleviridae. Marseillevirus (Lineage A), Lausannevirus (Lineage B), Tunisvirus (Lineage C), Brazilian marseillevirus (Lineage D) and Golden marseillevirus. It is interesting to note that there are fourteen (14) non-shared genes, among these there are seven (7) GMar genes.Furthermore, we noticed a high proportion of orthologous genes  in membeA hierarchical clustering tree was constructed using presence/absence matrix of 5443 clusters of orthologous genes shared by NCLDVs. It shows in Marseillevirus, Senegalvirus, Melbournevirus and Cannes8 virus). Three other clades appear to delimit the phylogeny of the Marseilleviridae family, composing the lineages previously known as B, C and D and a fifth comprising of GMar. These trees show the GMar within the family Marseilleviridae, but far from the other members already described. The analysis of nucleotide identity between the BrMr, recently founder of a new lineage and geographically closer to GMar, displays 74.96% of similarity. In addition to the concatenated tree, five trees with separate genes were also generated to support this result, based on the sequences available in the database. In spite of some variations in the positions, the clusters formed were the same, reinforcing the proposition of a new lineage to the virus here described , for concatenated alignment  show fouescribed .The discovery of a new strain of marseillevirus was attained by analyzing golden mussels, invasive organisms that are found worldwide. The source of the GMar is the inner water collected from golden mussels found in a local lake in Porto Alegre, Brazil, and this is the first report on the use of this organism for the isolation of marseilleviruses. Although this is a small-scale study, our results suggest that the golden mussels seem appropriate for the recovery of GMar and, perhaps, other marseilleviruses. It is likely that the filtration process necessary for the ingestion of nutrients of these organisms may be responsible for the accumulation of such viruses in their tissues. Oysters have been previously shown to be excellent sources for the isolation of mimiviruses due to their highly efficient filtration capacity, filtering more than 400 liters of water per dayMarseilleviridae already described (Marseillevirus and Insectomime) can replicate in A. castellani, even if they have been isolated in A. polyphaga2Two members of the Marseilleviridae family, of which GMar would be the first member. This suggestion is validated by genome analyses emphasizing several divergences between GMar and other marseilleviruses. When we look at the shared orthologues genes, we observe that seven genes are unique to GMar. It is noteworthy that the core genome analysis clearly distinguishes GMar from other lineages, drawing attention to the similar conserved gene content of the presently known marseilleviruses. More remarkably, phylogeny based on gene presence/absence patterns NCVOG reflects the gene loss and gain history of the giant viruses, clustering GMar into a distinctive clade in Marseilleviridae family. Conclusively, phylogenetic analysis show the four lineages of Marseilleviridae, which are presently composed by A, B, C and D. Recently a marseillevirus isolated in a river in TokyoMarseilleviridae, phylogenetic analysis shows that it is distant to Mimiviridae and the other giant viruses already identified. The analysis of separate genes also reinforces and consistently maintains the GMar in a separate branch in tree, indicating the possibility of a new strain. Finally, phylogenetic analysis of both the concatenated genes how the individual genes (according to availability in the database), shows clearly the four strains already described and characterized the GMar in one branch distant.In this study, we propose the foundation of a new lineage (E) in the Marseilleviridae with the inclusion of new members. In brief, these data increase the knowledge about the biological and genomic characteristic of marseilleviruses, currently incomplete.As the knowledge about the giant viruses is something recent, and the database grows with each new study, it is obvious that very soon there will be a review of annotation of genomes available, and probably a reorganization of the family How to cite this article: dos Santos, R. N. et al. A new marseillevirus isolated in Southern Brazil from Limnoperna fortunei. Sci. Rep. 6, 35237; doi: 10.1038/srep35237 (2016).


Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental condition in childhood , with substantial overall financial burden to children/adolescents, their families, and society. The aims of this study were to describe the clinical characteristics of children and adolescents with ADHD in Spain, estimate the associated direct/indirect costs of the disorder, and assess whether the characteristics and financial costs differed between children/adolescents adequately responding to currently available pharmacotherapies compared with children/adolescents for whom pharmacotherapies failed.This was a multicenter, cross-sectional, descriptive analysis conducted in 15 health units representative of the overall Spanish population. Data on demographic characteristics, socio-occupational status, social relationships, clinical variables of the disease, and pharmacological and non-pharmacological treatments received were collected in 321 children and adolescents with ADHD. Direct and indirect costs were estimated over one year from both a health care system and a societal perspective.P = 0.024), the difference being mainly due to significantly higher direct costs, particularly with larger expenses for non-pharmacological treatment (P = 0.012).The estimated average cost of ADHD per year per child/adolescent was €5733 in 2012 prices; direct costs accounted for 60.2% of the total costs (€3450). Support from a psychologist/educational psychologist represented 45.2% of direct costs and 27.2% of total costs. Pharmacotherapy accounted for 25.8% of direct costs and 15.5% of total costs. Among indirect costs (€2283), 65.2% was due to caregiver expenses. The total annual costs were significantly higher for children/adolescents who responded poorly to pharmacological treatment  contains supplementary material, which is available to authorized users. Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental condition in childhood, with an estimated worldwide prevalence in children/adolescents ranging from 5.3% to 7.1%, with little variation across countries and regions . In addiADHD significantly impairs psychosocial functioning and adjustment, and children/adolescents usually have difficulties in interpersonal relationships with peers/family , 7. FurtHealth-economics literature has consistently shown that the overall financial burden of ADHD to children/adolescents, their families, and society is substantial , 16–18. Systematic reviews of the literature have shown that the pharmacological treatment of ADHD is associated with a substantial benefit in outcomes in the short term, and is cost-effective compared with placebo, no treatment, or behavioral therapy only , 21. AnaThe objective of this multicenter, cross-sectional study was to estimate the associated direct and indirect costs from both a health care system and a societal perspective over a 1-year time period in Spanish children/adolescents with ADHD. In addition, we assessed whether the characteristics and financial costs differed between children/adolescents responding well (good responders) versus those who responded poorly (poor responders) to currently available pharmacotherapies.This was a multicenter, cross-sectional, descriptive study in a sample of children/adolescents with ADHD in the context of the Spanish research project “Plan of Action in ADHD (PANDAH)”. A total of 321 children/adolescents were recruited between 1 December 2012 and 30 April 2013 from 15 health care centers (7 privately and 8 publicly funded) representative of the overall Spanish population.The study included children/adolescents with DSM-IV Text Revision (DSM-IV-TR) diagnostic criteria for ADHD, aged ≤18 years, and without intellectual disability (i.e. intelligence quotient ≥70), as estimated by the treating physician. Children/adolescents were excluded if they had one of the following conditions: a severe medical disease other than asthma , neurological disorders (e.g. epilepsy), severe psychiatric disorders , symptoms of major depression before the onset of ADHD, or ADHD symptoms during episodes of depression.To minimize selection bias, child/adolescent psychiatry or pediatric neurology units initially recruited the first child/adolescent who attended on a given day who fulfilled study inclusion criteria. When additional children/adolescents attended on the same day, only those consecutive to the first one were enrolled.To detect a mean cost difference of €2000 between good responders and poor responders, and assuming a standard deviation (SD) of €5000 and €7000, respectively, we calculated that the total sample size needed was 350 children/adolescents . As the study was conducted in 15 different health care centers, each one should recruit between 20 and 30 children/adolescents.The central Ethics Committee that approved the study was the Niño Jesús Hospital Committee (Madrid), and all children/adolescents or their legal representatives signed a written informed consent form prior to participation in the study.A case report form (CRF) to be completed by the investigators was specifically designed for the study. First, a pilot study was conducted at 3 of the participating centers and data entered for 2 children/adolescents; this served to detect possible misleading questions, or other problems encountered by the investigators when completing the questionnaires. Second, all participating investigators were trained in the usage of the final CRF through a conference call. The CRFs were collected each week from all participating centers to build a common database.At the time of recruitment, the following data were collected and recorded by the investigators during the interview: a) current demographic characteristics of children/adolescents; b) socio-occupational status; c) social relationships; d) date of diagnosis, ADHD subtype, and psychiatric comorbidities based on DSM-IV-TR criteria ; e) ADHDDescriptive analyses were summarized by means (SDs) or medians and interquartile ranges for continuous variables, and absolute numbers and percentages for categorical variables.The study was performed using a health care system and a societal perspective to estimate both direct and indirect costs, and adopted a 1-year time period.All costs are expressed in 2012 Euros. Direct medical costs were defined as the cost of resources consumed to detect and treat the disorder, including costs of diagnostic tests, blood tests, emergency/outpatient visits, hospital admissions, and medication use. Direct medical costs were estimated by multiplying the amount of consumed resources by unit costs. These were obtained from the individual official reimbursed process in each particular region because, in Spain, health care is a competence of each autonomous community. Transport to treatment center was classified as a non-medical direct cost, and estimated based on public transport or taxi tickets given to the child/adolescent and parent/caregiver. Total direct costs were the sum of medical and non-medical costs. Indirect costs were those faced because of ADHD symptoms, and defined as the costs of the child/adolescent’s parents or caregivers, and the cost of lost productivity – as a consequence of hours spent in medical visits or missing workdays due to sick leave – applied to parents or caregivers. The cost of lost productivity was calculated by multiplying the estimated number of lost working hours by the average wage per working-hour . More inP-values were unadjusted for multiplicity.Good responders were defined as children/adolescents treated pharmacologically for at least 9 months who had an ADHD-RS-IV total score ≤ 18  for at least the 3 previous months , 31. ConA total of 321 children/adolescents were enrolled in the study; 262 were recruited by child/adolescent psychiatrists, 59 by neuropediatricians. Table Most children/adolescents were currently living with their parents and siblings (63.6%); their relationship with their family was good in 53.0% of cases. However, because of the ADHD symptoms, 17.8% of parents had to give up their job to care for their child/adolescent, and 11.5% of children/adolescents had a caregiver other than their parents, of whom 54% were relatives.Relationship with peers was good overall (59.5% of cases), but, in 43.0% of cases, parents reported that the child/adolescent had some type of social difficulty such as being led by others, provoking and annoying others, or being sore losers .All children/adolescents were attending school except one who was unemployed; 51% were enrolled in partially State-funded institutions, 35% in public schools, and 14% in private institutions. At the academic level, 19.1% of children/adolescents required an adapted school curriculum, and 51.9% received reinforcement/tutoring lessons about 4 times/week (mean [SD] = 4.2 [3.8]). A total of 13.7% of children/adolescents had been expelled from school at least once, with a mean (SD) age at first expulsion of 12 (2.5) years, and a mean (SD) of 3.3 (4) expulsions. Finally, 33.6% repeated a grade (mean [SD] repetitions = 1.1 [0.3]), at a mean (SD) age of 10.6 (2.8) years for the first time.The average estimated total cost of ADHD per year per child/adolescent was €5732.64, with direct costs accounting for 60.2% of total costs (€3450.04) , followed by visits to a primary care center . Support from an educational psychologist or a psychologist  was needed by 37.4% and 32.7% of children/adolescents, respectively, in the majority of cases held at private centers , and represented 45.2% of direct costs and 27.2% of total costs. Pharmacotherapy accounted for 25.8% of direct costs and 15.5% of total costs. Stimulants, specifically MPH OROS (65.4%) and MPH IR (25.5%) were the most commonly prescribed drugs. The mean duration of treatments during the previous year was 288 and 252 days for MPH OROS and MPH IR, respectively (data not shown). Costs due to hospital admissions, diagnostic tests, and transportation to treatment centers represented 2% of total costs each.Among the indirect costs estimated, 65.2% was due to caregiver expenses, which were paid by a relative in 35.1% of cases (data not shown), and represented 26% of the total costs. Moreover, a parent or caregiver had to leave their job to care for the child/adolescent in 17.8% of cases, and the average number of lost working days in the previous year was 8.5, of which 5 (SD 7.1) days were due to ADHD itself  of children/adolescents received pharmacological treatment, and treating physicians considered that 58.8% (n = 139) were good responders.In the overall sample, 73.5% (n = 139) had less severe ADHD measured at baseline, greater improvement from baseline, and were less frequently diagnosed with oppositional defiant disorder (Table n = 97), the disorder had a more negative impact on several aspects of their lives, including poorer global functioning, with worse relationships with friends and family.There were no differences between good and poor responders with respect to type of treatment or treatment duration, but good responders n = 19 had lesP = 0.024). This was mainly due to significantly higher direct costs for poor responders versus good responders , attributable to higher expenses in non-pharmacological treatment and transport   driven by significantly longer time spent at visits (P = 0.011).The difference in total annual costs of ADHD between groups was statistically significant, and higher for poor responders compared with good responders  have been estimated to be between €716 and €3888 , 32. TheThe main contributor to direct costs corresponded to non-pharmacological interventions , and accounted for 45.2% of total direct costs and 27.2% of total costs. Most cost-of-illness studies in ADHD include these services together with costs of other health care professionals, and therefore the specific weight of costs due to psychological interventions is difficult to compare. Nonetheless, these results are similar to a German study that considered the costs of behavioral therapy separately from other types of interventions, and reported that this expenditure represented 46.6% of costs for children/adolescents aged 6–17 years, and up to 74.4% in children ≤5 years of age . MoreoveOne striking finding of our study was the high cost associated with the employment of a caregiver, which represented 65.2% of indirect costs, and 26% of all costs. Mental health-related problems in caregivers and families of children/adolescents with ADHD include stress, anxiety, and depression , but theIn this study, poor responders were observed to have worse global functioning, and poorer relationships with friends and family. This is in line with the results of several systematic reviews, which reported that treatment has a beneficial effect on multiple short- and long-term outcomes, including self-esteem, social functioning, academic performance, and antisocial behavior, among others, when compared with children/adolescents without ADHD or those with untreated ADHD , 22, 23.Poor responders were found to incur higher total financial expenses than good responders, a difference mainly due to direct medical costs, and particularly because of significantly higher costs of non-pharmacological treatment. Although not directly comparable, these results are in line with previous systematic reviews showing that treatment is associated with less use of ancillary services, including school-, health-, and work-related services; financial assistance services; use of the justice system; and fewer emergency room visits , 23.The main advantage of the current study is that the studied population is representative of different regions in Spain and data were complete for almost all variables studied. However, the results must be interpreted in the light of considerable limitations. First, the cross-sectional nature of the study, with information obtained retrospectively through interview for some variables, means that results may be more sensitive to potential recall bias. Second, children/adolescents recruited in this study were treated by psychiatrists and neuropediatricians, and may therefore be representative of a subset of children/adolescents with more severe ADHD, thus incurring higher costs, as for adults . Third, ADHD is a highly prevalent disorder associated with a significant high personal, familial, and financial impact on the family, health system, and society. An adequate response to pharmacological treatment was associated with lower overall expenses in the management of the disorder.Additional file 1: Table S1.Non-significant differences in clinical and demographic baseline characteristics between children/adolescents with good response versus those with poor response to pharmacological treatment. The table presents analysis results and is provided as a supplement to Table


Cryptococcus spp. is typically a difficult undertaking. Collecting samples in the field is costly in terms of travel, personnel time and materials. Furthermore, the recovery rate of Cryptococcus spp. may be very low, thereby requiring a large number of samples to be taken without any guarantee of success. Ecological niche modeling is a tool that has traditionally been used to forecast the distribution of plant and animal of species for biodiversity and conservation purposes. Here, we use it in a public health application to produce risk area maps for cryptococcal disease in Colombia. The Genetic Algorithm for Ruleset Production (GARP) was used to create models for Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii), based on environmental sampling and clinical records data recorded since 1987. These maps could be used to focus public health messaging related to cryptococcal disease, and it enables us to characterize the ecological niche for Cryptococcus in Colombia. We found that the ecological niche for C. gattii in Colombia is quite diverse, establishing itself in sub-tropical and temperate ecoregions within the country. This suggests that C. gattii is highly adaptive to different ecological conditions in Colombia and different regions of the world.The environmental isolation of  Cryptococcus is a fungal organism (yeast cells or blastoconidia ~1–2 micrometers in size) that can cause illness in humans and animals through environmental exposure. Of particular medical interest are Cryptococcus neoformans  and Cryptococcus gattii (serotypes B and C). Cryptococcus neoformans (C. neoformans) has a worldwide distribution and is typically found in bird droppings in cosmopolitan settings; those infected by C. neoformans are predominantly immunocompromised [Cryptococcus gattii (C. gattii) was traditionally understood to be limited to tropic and sub-tropic regions in association with Eucalyptus spp. trees [C. gattii in the Pacific Northwest region of North America, a temperate rainforest region, has redefined the ecological niche of C. gattii [C. gattii infections occur in immunocompetent persons.. gattii ,4. The mC. neoformans and C. gattii for Colombia to assist clinicians in the diagnosis of cryptococcal disease, and to direct public health messaging related to cryptococcal disease. This study also describes the environmental characterization of the ecological niche for C. neoformans and C. gattii for Colombia based on the environmental predictor data used in the modeling.Risk maps are useful for delineating the geographical areas where potential exposure to environmental pathogens may occur. The purpose of this study was to create ecologically-based risk maps for n = 1074; 96.6%) belong to C. neoformans var. grubii (serotype A), five isolates (0.4%) to C. neoformans var. neoformans (serotype D), and 33 isolates (3.0%) to C. gattii, with Norte de Santander the department with the highest frequency of cryptococcosis cases (77%) caused by C. gattii in the immunocompetent population [Previous clinical and epidemiological research performed by the Colombian Group for the Study of Cryptococcosis identified an increased incidence related to the AIDS pandemic. The mean annual incidence of cryptococcal disease in the general population between 1997–2005 and 2006–2010 remained the same at 2.4 cases per million population; whereas, in AIDS patients the incidence rate rose from 3000 cases per million population during 1997–2005 up to 3300 cases per million population during 2006–2010. The vast majority of clinical isolates recovered  than in the dry season [C. gattii serotypes B and C and humidity, temperature, evaporation and solar radiation in Colombia was also described [Several environmental approaches have been undertaken in Colombia to describe the environmental importance of the fungus and its potential association with clinical isolates. These reports have documented the isolation of g others ,8,9. Anay season , but a described .Cryptococcus spp. is a difficult undertaking. Collecting samples in the field, and subsequent testing in the laboratory, is costly in terms of travel, personnel time and materials; C. neoformans and C. gattii cannot be identified with the naked eye, and these fungal organisms are not believed to adversely affect the health of its plant hosts. Furthermore, the recovery rate of Cryptococcus spp. may be very low, thereby requiring a large number of samples to be taken without any guarantee of success [Risk maps are typically the outcomes of models of disease transmission based on epidemiological, climatic and environmental data . These m success .i.e., aerial or satellite imagery) sources, and compatibility with GIS and species distribution modeling software [However, when the information is available, disease risk maps generated from ecological niche modeling can provide accurate forecasts of a pathogenic organism’s ecological niche based on the environmental characteristics of its observed locations . Supportsoftware . Environi.e., geographical space and environmental components); whereas, the realized niche of a species is comprised of the actual distribution of a species determined by a variety of factors such as dispersal, history, and physical barriers  [The concept of the ecological niche of a species is defined as the set of physical and biological conditions under which the species can maintain its population without immigration . For a ssponses) ,17,18. Ei.e., potential misclassification of the place of species occurrence). Ideally, the disease risk model would be based on environmental findings collected under a random location-sampling scheme with a high number of species presence observations from a wide geographical area. Environmental sampling results are heavily dependent on sampling effort though. Repeat sampling is often performed at historic presence locations or near the environs of reported cases. This produces reassuring results for the investigators, but from an ecological niche modeling/risk map creation perspective, it does not produce additional data points to inform the creation of the model. The likelihood of obtaining positive Cryptococcus spp. results from random sampling however are typically very low [Cryptococcus spp. or if replicate findings after a period of time is necessary. Additionally, it is difficult to determine how many negative environmental sampling results are sufficient to classify an area as having a definitive absence of an organism.The use of clinical records for disease risk mapping and assessment has proved useful in some applications ,19 but ivery low . FurtherGuizottia abyssinica agar plates supplemented with Byphenil and antibiotics. Plates were observed daily during one month, and suspected brown colonies were streaked onto Sabouraud glucose agar plates for further phenotypic characterization and species determination [Canis lupus familiaris. Clinical cases recorded in the National Epidemiology Survey (a passive disease surveillance system) were geocoded to their address of residence.Environmental and clinical data were managed in an Excel table  and imported into a GIS  for mapping and analysis. Approximate latitude and longitude coordinates from where the environmental samples were collected were derived from Google Maps . Environmental sampling data were gathered from the information collected by the Colombian Microbiology Group of the National Institute of Health for the period 1987–2011; additionally, extensive environmental sampling was performed in 13 geographical departments of Colombia during the years 2012–2014 for the purpose of increasing the number of input data points to support the ecological niche modeling . Environmination . AdditioNumerous ecological niche-modeling algorithms are available  . Howeveri.e., predicted presence—observed presence and predicted absence—observed absence) by the total number of predictions . A commission error occurs when the model predicts a species to occur where it does not, and an omission error occurs when the model fails to predict a species occurrence where it does in fact occur.Predictive accuracy of the model is calculated by dividing the number of correct predictions  and clinical cases  to verify the predictive accuracy of the model. Details of the isolations of C. neoformans and C. gattii biogeography in the ecological niche modeling: temperature, precipitation and elevation layers from WorldClim [Cryptococcus spp. using Desktop GARP. The C. neoformans and C. gattii input datasets were split into 50% training and 50% testing subsets to build and test the models. The environmental data layers were jack-knifed individually through 30 model runs for C. neoformans and C. gattii. Each model run comprised of up to 1000 iterations or until convergence was reached.We used 38 environmental predictor layers  believedorldClim , land coorldClim  and the orldClim , soil tyorldClim  and maxiorldClim . The spaC. neoformans and C. gattii. For these final model runs, the input C. neoformans and C. gattii data were again divided into training (50%) and testing (50%) subsets, and 50 model runs were performed. Each model run produced a binary value output data layer with 1 indicating areas with suitable ecological conditions to support C. neoformans and C. gattii, respectively, and 0 indicating unsuitable areas. The 50 output data layers where overlaid and summed to produce a final, composite ecological niche map for C. neoformans and C. gattii.Environmental data layers with training and testing accuracies ≥80% were used to create the final ecological niche models for C. neoformans and C. gattii. We reclassified geographical areas above 3000 m elevation as “not suitable” for C. neoformans and C. gattii because the Páramo climate altitudinal zone (3000–4000 m above sea level) is characterized by temperature below 10 °C with icy winds and frequent snowfall, and the glacial climate altitudinal zone (>4000 m) is characterized by glaciers [C. neoformans and C. gattii based on previous environmental sampling findings in Colombia, and observations from Vancouver Island, Canada [Lastly, we used our expert opinion to critically assess the predicted ecological niche model output for glaciers . These c, Canada ,14,32.C. neoformans environmental isolations  along the Pacific coast and Amazon River basin, and the Orinoquia region in western Colombia  was performed in 13 departments where cases of cryptococcal disease have been reported, where interest . EnvironC. gattii clinical case data to augment the environmental observations for the predictive modeling. This introduces uncertainty as to whether the case’s place of residence was in fact the place of exposure to C. gattii in the environment, and it has the potential to result in modeling error. However, Mak et al. [C. gattii disease surveillance data. Furthermore, validation of the C. neoformans model based on field observations was accomplished by overlaying the clinical reports of cryptococcal disease due to C. neoformans . Of the 51 clinical records, 50 were located within the forecasted ecological niche of C. neoformans, thereby further validating the predictive accuracy of the model.Another limitation of this study is the use of k et al.  have demC. neoformans into C. neoformans var. grubii and C. neoformans var. neoformans as separate species, and the five species within C. gattii—in recognition of the new taxonomy of the C. neoformans/C. gattii species complex recently proposed by Hagen and colleagues [Further refinement of the prediction models may include dividing lleagues . HoweverC. neoformans and C. gattii describe the ecological niche areas for these fungal species at the regional scale. The actual habitat suitability and ultimate presence of C. neoformans and C. gattii at a specific location at the site level is highly variable because there will most likely be considerable ecological heterogeneity within each 1 km by 1 km spatial unit in the ecological niche model. Instead, the maps can be used by clinicians to consider the diagnosis of cryptococcal disease for patients residing in or having recent travel to these geographical areas.Lastly, it is important to note that the prediction models for C. neoformans and C. gattii for Colombia. We found that the ecological niche for C. gattii in Colombia is quite diverse, establishing itself in sub-tropical and temperate ecoregions within the country. This suggests that C. gattii is highly adaptive to different ecological conditions in Colombia and different regions of the world. Furthermore, the modeling software (Desktop GARP) and environmental predictor layers used in this study are available at no cost, and the methodology is applicable to other jurisdictions and fungal organisms.In this study we describe the utility of ecological niche modeling to produce risk maps for


Wolbachia and its involvement in reducing flavivirus titer in insects. Finally, we highlight the similarities of the known immune pathways and identify potential targets for future studies aimed at improving antiviral therapeutic and vaccination design.Vector-borne diseases, including arboviruses, pose a serious threat to public health worldwide. Arboviruses of the flavivirus genus, such as Zika virus (ZIKV), dengue virus, yellow fever virus (YFV), and West Nile virus (WNV), are transmitted to humans from insect vectors and can cause serious disease. In 2017, over 2,000 reported cases of WNV virus infection occurred in the United States, with two-thirds of cases classified as neuroinvasive. WNV transmission cycles through two different animal populations: birds and mosquitoes. Mammals, particularly humans and horses, can become infected through mosquito bites and represent dead-end hosts of WNV infection. Because WNV can infect diverse species, research on this arbovirus has investigated the host response in mosquitoes, birds, humans, and horses. With the growing geographical range of the WNV mosquito vector and increased human exposure, improved surveillance and treatment of the infection will enhance public health in areas where WNV is endemic. In this review, we survey the bionomics of mosquito species involved in Nearctic WNV transmission. Subsequently, we describe the known immune response pathways that counter WNV infection in insects, birds, and mammals, as well as the mechanisms known to curb viral infection. Moreover, we discuss the bacterium  Culex genus. Mosquitoes can then reinfect the bird population, further perpetuating enzootic infection  belongs to the flavivirus genus, which also includes dengue virus (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). WNV is endemic to the United States (U.S.) and Canada, Africa, Europe, the Middle East, and West Asia WHO, . WNV hasn Figure , or can n Figure . It is an Figure . In thisCulex quinquefasciatus is located between the latitudes 36°N and 36°S  challenge via the RNAi pathway  that interacts with Dicer and Argonaute 2   pathway is conserved across diverse phyla and provides host protection against virus infection, including arboviruses  in Ae. aegypti mosquitoes is correlated with a reduction in DENV titer  can increase WNV titer in Cx. tarsalis . The study concluded that both somatic infection and stable transinfection of Wolbachia lead to inhibition of DENV and WNV replication and transmission  has been shown to manipulate this pathway for its own survival     during the initial spread of WNV in the U.S. The study determined that the vaccine is safe for California condors, stimulates protective antibodies, and protects against naturally circulating WNV  first and then WNV have an antibody response to WNV. Interestingly, finches first challenged with WNV and then SLEV have an elevation in WNV antibody titers, but no increase in SLEV antibody titers during the second infection  response. In one study that used WNV (Kunjin subtype) authors found increased levels of type I and type II interferon in blood leukocytes, lymph nodes, and spleen. They also noted increases in IFN-α, CXCL10, TLR3, ISG15, and IRF-7 in the brain, but no neuroinvasion of the virus  to induce apoptosis and reduce WNV titer  mice and B cell activating factor receptor (BAFFR)-deficient mice are susceptible to infection, but, if treated with immune sera from a wild-type mouse with antibodies to WNV, can be protected from infection  and envelope (E) proteins from WNV into the yellow fever 17D clone (Arroyo et al., Wolbachia into the mosquito population. As discussed in an earlier section, Wolbachia reduces flavivirus titer in mosquitoes, and models predict that WNV eradication is possible with the introduction of Wolbachia (Farkas et al., Wolbachia into the Culex population would have any detrimental effects on the greater ecosystem.Despite these successful early clinical trials, no WNV vaccine has moved into phase III trials. Some challenges for a phase III trial for a WNV vaccine are discussed in Ishikawa et al. . One notLA wrote the manuscript in consultation with AG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We detected Zika virus in breast milk of a woman in Brazil infected with the virus during the 36th week of pregnancy. Virus was detected 33 days after onset of signs and symptoms and 9 days after delivery. No abnormalities were found during fetal assessment or after birth of the infant. Flaviviridae and was first described in 1947. The first outbreak of infection with this virus was on Yap Island, Micronesia, in 2007 , rash , rash , myalgiaGeneral examinations were requested, and a PCR for Zika virus was repeated for blood and urine samples. Virus was detected only in urine. Serologic analysis detected dengue virus IgM and IgG; no antibodies against nonstructural protein 1 of this virus were detected. Results of reverse transcription PCR (RT-PCR) were negative for chikungunya virus. These findings were compatible with acute or recent Zika virus infection.4 copies/mL)  was observed. The mother and the medical team supported a decision to avoid breast-feeding once RT-PCR confirmed presence of the virus.No studies have confirmed Zika virus transmission by breast-feeding or provided knowledge about the pathophysiology of infection. Our report describes a case of Zika virus infection in a patient at 36 weeks of pregnancy. The patient and baby remained well after delivery, with no evidence of transmission of Zika virus to the newborn. However, we detected persistence of virus by RT-PCR in breast milk from samples during the pregnancy (in colostrum) 33 days after onset of signs and symptoms (in breast milk).4 copies/mL by RT-PCR but no replicative virus in culture  and breast milk 9 days after birth  by PCR. We also observed cytopathic effect in virus culture, which showed infectivity of the virus. Our data provide evidence that Zika virus can persist in some tissues for a long period. Moreover, viral culture showed potential infectivity of the virus.We detected Zika virus in colostrum (2.44 × 10The World Health Organization does not recommend that mothers avoid breast-feeding in cases such as the one mentioned in this report. However, with indications that virus might be present and persistent in breast milk, further studies should be performed to elucidate the potential transmission of Zika virus to the newborn.


In the article titled “A Rare Cause of Childhood Cerebellitis-Influenza Infection: A Case Report and Systematic Review of Literature” , Dr. Can“CSF cultures were bacteriologically sterile. Polymerase chain reaction [PCR] assays of CSF for influenza virus, herpes simplex virus 1 and 2, adenovirus, enterovirus, cytomegalovirus, human herpesvirus- 6, epstein-barr virus, and varicella zoster virus were all negative” should be corrected to “Multiplex polymerase chain reaction [PCR] assays of CSF for herpes simplex virus 1 and 2, adenovirus, enterovirus, cytomegalovirus, human herpesvirus-6 and -7, Epstein-Barr virus, varicella zoster virus, parechovirus, parvovirus B19  and influenza virus type A and B, parainfluenza virus, adenovirus, respiratory syncytial virus, human metapneumovirus, human bocavirus, human coronavirus, enterovirus, and rhinovirus  were all negative.”“Serologic tests of his blood showed negative results for epstein-barr virus, herpes simplex virus, varicella-zoster virus, cytomegalovirus, measles, mumps, rubella, and mycoplasma pneumoniae. Respiratory viruses such as adenovirus, rhinovirus, respiratory syncytial virus, parainfluenza virus, human bocavirus, human metapneumovirus, and coronavirus were not detected in the nasopharyngeal swab specimen by multiplex PCR. However, we identified influenza A H1N1 virus on the third day of the onset of the symptoms, which was when we started treatment with oseltamivir as 4  mg/kg orally twice a day. The patient was diagnosed with influenza-associated cerebellitis based on the clinical findings” should be corrected to “Serologic tests of his blood showed negative results for Epstein-Barr virus, herpes simplex virus, varicella zoster virus, cytomegalovirus , measles, mumps, rubella, and mycoplasma pneumoniae . Respiratory viruses including adenovirus, rhinovirus, respiratory syncytial virus, parainfluenza virus, human bocavirus, human metapneumovirus, and coronavirus were not detected in the nasopharyngeal swab specimen by multiplex PCR .”Additionally, there were errors in the Case Representation section which should be corrected as follows:


Arboviruses are emerging infectious diseases with the ability to expand geographically and rapidly affect large populations.The recent epidemic caused by the Zika virus in the Americas and congenital Zika syndrome associated with maternal infection has called out attention to the importance of studying arboviruses during pregnancy.This is a review on selected arboviruses infections during gestation, including Zika, Chikungunya, Dengue and Yellow Fever viruses. Issues such as historical overview, pathogenesis, transmission, clinical conditions, diagnosis, treatment and prevention are addressed. Arboviruses are emerging infectious diseases which have gained importance in the last 50 years due to their ability to expand geographically and rapidly affect large populations. The spread of these diseases in the human population occurs through the introduction of the pathogen into the environment, associated with other factors such as ecological changes and human behavior. As the mosquito vectors breed in standing water, events like heavy rain with flooding and construction of dams are associated with the emergence of infections by arboviruses. In addition, urbanization, migration and international travel are also contributing factors to the transmission of these disease , 2.Recently, the epidemic caused by the Zika virus in the Americas has led the World Health Organization to declare a state of international emergency, providing global visibility to the issue, which has become a public health priority.The present study consists in a review on arboviruses during gestation, addressing infections caused by the Zika, Chikungunya, Dengue and Yellow Fever viruses.Arboviruses are thus designated since they are viruses essentially transmitted by arthropods, predominantly mosquitoes. At least 135 types of arboviruses are capable of causing human infection, with the vast majority being RNA viruses [Aedes aegypti mosquito [The Zika virus was initially isolated in 1947 from Rhesus monkey blood during epidemiological studies on Yellow Fever in the Zika Forest, in Uganda  and, in mosquito . ThroughThe first recognized major outbreak occurred on Yap Island, Micronesia, in 2007 , 9.In 2015, the Zika virus reached the Americas, and was initially identified in Brazil, where a phylogenetic analysis indicated it was the Asian strain of the virus . AccordiThe Chikungunya virus was reported for the first time in Tanzania in 1952. After the first epidemic, the virus became endemic in Africa. It reached Asia in 1958 and was responsible for several outbreaks in subsequent years .The virus remained restricted to the African and Asian continents until 2013, when it was introduced in the Latin Caribbean and established the first human cycle in the Americas .In September 2014, the first autochthonous case was confirmed in Brazil, in the northern region. After 7 days, new cases were identified in the northeastern region and, in just 1 month, the Ministry of Health of Brazil had already confirmed 682 cases of this virus infection .Although numerous epidemics with Dengue-like characteristics have been described for centuries, the Dengue virus was first isolated in 1943 in Japan (DENV1) and in 1945 in Hawaii (DENV2) .Aedes aegypti mosquito, created an environment conducive to the virus transmission, which reached a pandemic status after World War II [The spread of its vector, the d War II .In the Americas, Dengue was controlled through a campaign conducted by the Pan American Health Organization in 1947 to eliminate mosquitoes. However, in the 1970s, the region was reinfested and, since then, the incidence of the disease has increased in the Americas, as well as in other regions .Aedes aegypti mosquito and, in the 1930s, two live attenuated virus vaccines were developed. The combination of extensive vaccination campaigns and vector control significantly reduced the number of cases of this disease [Yellow Fever originated in Africa and was brought to the Americas in the seventeenth century as a consequence of the slave trade, and was responsible for the deaths of thousands of people in the 18th and 19th centuries. In 1900, transmission was confirmed through the Over the years, outbreaks in Africa and South and Central America continued to occur . In DeceA. aegypti and A. alpobictus.The Zika, Dengue and Yellow Fever viruses belong to the Flaviviridae family and Flavivirus genus, while the Chikungunya virus belongs to the Togaviridae family and Alphavirus genus. All are transmitted in the urban cycle, mainly by mosquito species Arboviruses display a transmission cycle between the mosquito and the human host, with periods of both extrinsic and intrinsic incubation. Extrinsic incubation refers to the period that elapses between the vector infection until the virus spreads to the mosquito’s saliva, which then infects the new host. The intrinsic incubation period refers to the time interval between inoculation in the human host until the virus reaches the bloodstream.The pathogenesis of arboviruses still remains unknown in several respects, but it is recognized that, upon primary inoculation, viral particles infect target cells such as fibroblasts, monocyte, dendritic cells and host endothelial cells. The viruses then replicate, reaching the regional lymph nodes, and, subsequently, the bloodstream .Sexual transmission of the Dengue, Chikungunya or Yellow Fever viruses has not been described. However, transmission of the Zika virus through infected semen has already been well established . In addiA review of a study of sexually active women of Rio de Janeiro highlights the contribution of sexual transmission of the Zika virus. Women between the ages of 15 and 65 presented 90% more cases recorded in 100,000 women than men of the same age group. This was not repeated in groups <15 years and > 65 years .Arboviruses can also be transmitted by blood transfusion or transplantation of organs or tissues from infected and asymptomatic donors at the time of donation. In the case of Yellow Fever, transmission is also possible during the viremia period after donor vaccination –30.The occurrence of arboviruses during pregnancy is of additional concern, due to the possibility of vertical transmission and fetal involvement. In 2015, a significant increase in the number of newborns diagnosed with microcephaly in several regions of Brazil presenting a high incidence of Zika virus was observed. Maternal-fetal transmission of the virus was verified during all gestation stages –34.Torres et al. found a vertical transmission rate for the Chikungunya virus ranging from 27.7 to 48.29%, occurring more frequently when maternal viremia coincided with the time of birth. However, the birth route showed no relation to the incidence of congenital disease .Infection of pregnant women with the Dengue virus is associated with the risk of miscarriage, fetal death, prematurity and vertical transmission. Although gestation is considered a risk factor for the clinical course of the disease, no association was found between the severity of the maternal infection and neonatal disease , 37.Few studies on yellow fever are available, but perinatal transmission of the disease has been described .The clinical presentation of these arboviruses during pregnancy is supposed to be similar to the general population. However, there is a lack of studies specifically addressing pregnant women.The incubation period of this disease is of 4 to 5 days. The most frequently reported symptoms are a rash, fever, arthritis or arthralgia, and conjunctivitis. Other symptoms include myalgia, headache, retro-orbital pain, edema and vomiting. It is estimated that only 20% of the people infected with the Zika virus manifest symptoms .In a cohort of pregnant women in Rio de Janeiro, where exanthema was used as the inclusion criterion, in addition to macular or maculopapular exanthema in 100% of the cases, the following symptoms were also observed: pruritus (90%), arthralgia (62%), conjunctivitis (58%), fatigue and malaise (52%), myalgia (41%) and a short mild fever in less than 1/3 of women with acute infection . AnotherSince its discovery, four different genotypes of the Chikungunya virus have been identified: East-Central-South-African (ECSA), West African, Asian and Indian Ocean lineage (IOL). The incubation period is usually from 3 to 7 days (ranging from 1 to 12 days) and 80% of the cases are symptomatic .Fever is typically elevated, with a sudden onset, associated with arthralgia in almost all cases. Joint pain is usually symmetrical and can be disabling, more intense in the morning, improving with moderate exercise. The joints most often involved are the ankle, wrists and fingers. About 50% of the patients present a maculopapular rash and, occasionally, vesicular-bullous lesions and ulcers. Other symptoms include headache, myalgia, conjunctivitis, nausea and vomiting .Symptoms typically resolve within 7 to 10 days, but some patients may still present rheumatologic symptoms for long periods after the initial infection.Pregnant women are considered a risk group and are more likely to progress to more severe forms of the illness and death , 44.Four serotypes of the Dengue virus exist: DENV-1, DENV-2, DENV-3 and DENV-4. Primary infection by one serotype confers immunity against reinfection by the homologous virus, but when infection by another serotype occurs, which is common in endemic areas, the risk of severe disease is increased due to a phenomenon known as antibody-dependent enhancement .The incubation period varies from 4 to 10 days. About 50% of the infections are symptomatic and may present a broad clinical spectrum. The classification for dengue severity is divided into Dengue without warning signs, Dengue with warning signs, and severe Dengue. For the symptomatic patients three phases may occur:1st phase - febrile, lasting from 2 to 7 days. The first manifestation is fever, usually high, associated with headaches, myalgia, arthralgia and retro-orbital pain. The rash, predominantly maculopapular, is present in 50% of the cases. Nausea and vomiting may be present. Most patients present improvements in their general state, but 5% progress to the critical phase.Abdominal pain or tenderness;Persistent vomiting;Clinical fluid accumulation ;Mucosal bleeding;Lethargy or restlessness;Liver enlargement (≥2 cm);Increases in hematocrit concurrent with rapid decrease in platelet count.2nd phase - critical, beginning with the defervescence of the fever, between the third and seventh day of the onset of the disease, accompanied by the appearance of one or more warning signs, including:3rd phase - recovery, lasting from 3 to 5 days, after which there is progressive clinical improvement of patients who have suffered the critical phase.Severe plasma leakage, leading to shock or fluid accumulation with respiratory distress;Severe bleeding;Severe organ involvement .Severe Dengue is defined in the presence of one of the following criteria:The process that determines the infection progression to severe forms appears to be multifactorial, involving individual immunological status and virus-related factors.Like other infectious diseases, Yellow Fever has a wide spectrum of severity, ranging from asymptomatic patients to fatal cases.The incubation period of the disease ranges from 3 to 6 days. Most symptomatic patients present with fever, headache, chills, muscle aches, and nausea, usually lasting a week. About 10 to 25% develop hemorrhagic manifestations and hepatic and renal impairment, with mortality occurring in 20 to 50% of the symptomatic cases .The disease manifests itself in three stages: infection, remission and intoxication. In milder cases, patients recover without sequelae within a few days. In severe cases, patients develop a more severe form of the disease after the remission period, lasting from 24 to 48 h, called the intoxication period, and may present coagulopathy, haemorrhagic manifestations, jaundice, urinary albuminuria and acute renal failure .A cohort of pregnant women with suspected Zika virus infections was carried out in Rio de Janeiro, between September 2015 and May 2016. Among the 125 pregnant women with confirmed diagnoses, 46.4% presented adverse pregnancy outcomes. Acute infection occurred between 6 and 39 weeks of gestation and complications were observed in 55% of cases when the infection occurred in the first trimester, 52% when infection occurred in the second trimester and 29% when the pregnant women were infected in the last trimester. Abortion cases accounted for 25% of first trimester outcomes and 3% of second trimester outcomes, with 2 fetal deaths among 34 pregnant women with a positive third trimester examination .Of the 117 neonates, 42% displayed abnormal findings in the first month of life, with almost all cases presenting central nervous system abnormalities. Microcephaly was diagnosed in 3.4% of cases and restriction of fetal growth in 9%. The neurological abnormalities, similar to other studies , 48, incThe neurological findings, observed even in late infections, result from central nervous system tropism of the virus, leading to the occurrence of fetal microcephaly and Guillain-Barré syndrome in adults , 50.A previous case–control study, had already established the association of the Zika virus with the occurrence of fetal and neonatal neurological abnormalities. The analysis included 32 neonates with microcephaly, considered cases, and 62 neonates without microcephaly as controls, born in eight public maternity units in the metropolitan north-eastern region of Brazil. Eleven of 32 cases showed severe microcephaly and significantly higher proportions of cases compared to controls were born with low birth weight and were small for their gestational age. Of the 27 cases investigated by brain imaging, 11 presented one or more abnormalities .Another study analyzed the data extracted from the epidemiologic reports published by the Secretary of Health Surveillence of the Brazilian Ministry of Health, that monitors cases of microcephaly associated with congenital infection in Brazil. Considering the confirmed cases of microcephaly and related deaths associated with Zika virus in Brazil, from November 2015 to October 2016, the estimated case fatality rate is 8.3% .Maternal-fetal transmission occurs mainly when there is maternal viremia at the time of delivery, with onset of neonatal symptoms 3 to 9 days after birth.The main clinical manifestations in symptomatic newborns are fever, irritability, exanthema, lower or generalized lower limb edema, hyperalgesia, vesicular-bullous dermatosis, meningoencephalitis and hemodynamic instability . Some caA 2-year follow-up of a cohort of children exposed to the virus during the perinatal period showed a delay in global neurodevelopment, with low developmental rates of those exposed compared to negative controls for infections .Two possible mechanisms may be responsible for fetal and neonatal morbidity due to dengue virus infection during pregnancy: the presence of maternal hemodynamic changes, which may compromise the placenta and cause fetal hypoxia, and the direct effect of infection on the fetus.Paixão et al. conducted a systematic review and meta-analysis of adverse fetal outcomes resulting from Dengue infection during gestation. Sixteen studies were included in the systematic review and eight were eligible for the meta-analysis, totaling 292 women exposed to the Dengue virus during gestation. The review indicated an association of Dengue in pregnancy with abortion (OR 3.51), fetal death (RR 6.7), preterm birth (OR 1.71) and low birth weight (OR 1.41) . Other sRibeiro et al. studied the fetal outcome of 24 pregnant women with laboratory-confirmed Dengue infections and observed an increase in the incidence of abortions , fetal death (8.3%) and prematurity (12.5%). After birth, 29% of the newborns presented symptoms of the disease .The Dengue virus is endemic in many localities and the prevalence of infection during pregnancy, with consequent fetal impairment, may vary according to the local scenario and the cumulative period of exposure to the virus , 60. TheMost studies regarding Yellow Fever analyzed fetal and neonatal effects after inadvertent vaccinations during pregnancy.In 2009, a case of perinatal transmission of Yellow Fever was described during an epidemic in the state of São Paulo, Brazil. The 30-year-old woman presented with fever, headache and jaundice 3 days before vaginal delivery. The female newborn was discharged after 2 days and on the third day was readmitted with fever and cyanosis, progressing with hemorrhage, liver enzyme alterations, oliguria and death with 12 days of life. The neonate underwent antibiotic therapy and intensive support during hospitalization. Confirmation of newborn infection was obtained by RT-PCR harvested after 5 days of the onset of symptoms. The maternal infection was confirmed by IgM positive for Yellow Fever and negative for Dengue .Due to the similarity between these arboviruses symptoms and the possibility of rapid evolution to severe forms, it is recommended that infection by the Dengue virus be considered as the main differential diagnosis in patients who have been in endemic areas. Infections with the Zika and Chikungunya virus should also be considered, since they present the same vector and many regions are endemic for different arboviruses.Table Some endemic areas display multiple arboviruses which present the same symptoms, such as fever, rash, myalgia, and arthralgia. Therefore, laboratory evaluation of the Dengue, Zika and Chikungunya viruses, as well as other TORCH group diseases  is recommended to establish the diagnosis.In the acute phase, which occurs within the first 7 days after the onset of the symptoms, virus RNA can be identified in the patient’s serum, with RT-PCR as the test of choice for the diagnosis of Dengue, Zika and Chikungunya. In the case of Dengue infection, the NS1 antigen can also be detected in the acute phase by the ELISA test. However, this test is not widely available. A negative result, however, does not exclude the infection, and a serological test for IgM antibodies is necessary.In the convalescence phase, the ELISA test for IgM antibodies, which are usually detectable between 2 and 12 weeks after infection, should be performed for Dengue, Zika and Chikungunya. However, due to cross-reactions, a positive serology (IgM) for Dengue or Zika only indicates a recent flavivirus infection and it is not possible to distinguish the type of virus in question. In case of positive IgM antibodies, the Plaque Reduction Neutralization Test (PRNT), which characterizes and quantifies circulating levels of anti-Dengue virus neutralizing antibody, is necessary to confirm the diagnosis. Patients who have received a Yellow Fever vaccine in the past may also present serology cross-reactivity , 62.The Trioplex RT-PCR is a laboratory test designed to detect Zika, Dengue and Chikungunya virus RNA, authorized by the FDA to be used under an Emergency Use Authorization (USA) .The diagnosis of a Yellow Fever infection follows the same protocol with RT-PCR, and the detection of the NS1 antigen with high sensitivity and specificity, possible during the acute phase. The identification of Yellow Fever antibodies by IgM ELISA can also be performed. Serology assays, however, present limitations due to the possibility of cross-reactivity with other flaviviruses .In addition to serum, RT-PCR can also be performed on urine and saliva samples. Musso et al. analyzed saliva samples from symptomatic patients during the French Polynesia epidemic from October 2013 to March 2014, and, although there was no increase in the virus detection window, the saliva sample increased the molecular detection rate of viral RNA .There is no specific medication for infections caused by Dengue, Zika, Chikungunya or Yellow Fever viruses. The treatment consists in rest, drinking fluids to prevent dehydration and the use of analgesics, such as acetaminophen. In the presence of warning signs (section The use of aspirin or other non-steroidal anti-inflammatory drugs must be avoided. The use of these medicines may aggravate the hemorrhagic condition in case of Dengue virus infection .The Dengue vaccine CYD-TDV has recently been licensed but still remains largely unused. It is a tetravalent live attenuated viral vaccine, licensed for the prevention of Dengue illness caused by Dengue virus serotypes 1, 2, 3 and 4 in individuals ranging from 9 to 45 years old or 9 to 60 years old, depending on the license, living in Dengue endemic areas. The vaccination schedule consists of 3 0,5 mL injections administered at 6-month intervals, with an average efficiency of approximately 60%.There is no recommendation concerning CYD-TDV during pregnancy and breastfeeding, due to lack of sufficient data in this population. However, the limited data collected during the clinical trials on unintentional immunization in pregnancy have yielded no evidence of harm to the fetus or pregnant women. To date, no studies evaluating the effects of inadvertent use in pregnant women are available .Yellow Fever 17D vaccine is widely used for the prevention of Yellow Fever in travelers, for routine immunization in endemic areas and for emergency response during outbreaks. It is given in a volume of 0,5 ml by the subcutaneous route in a dose of no less than 1000 International Units. Approximately 90% of vaccines seroconvert by 10 days and 99% seroconvert by 30 days post immunization.The Yellow Fever vaccine is not recommended during gestation or breastfeeding, since it is composed of a live attenuated virus, except in situations of a severe epidemic or unavoidable trip to high-risk locations . HoweverIf vaccination is required during pregnancy, it is preferable to be administered during the first trimester, when the immune response appears to be more satisfactory. The vaccine may be administered during breastfeeding, which should be discontinued for 10 days during the post-vaccine viremia phase. Regarding breastfeeding, a case of vaccine-induced encephalitis in one infant was reported 8 days after the mother’s primary vaccination .At the time of publication of this article, no vaccines against Zika and Chikungunya were available. Future vaccine programs should consider the serological cross-reaction between the Zika virus and Dengue virus and the implications for disease pathogenesis .The preventive strategy considered most efficient is vector control, through the use of repellents and long-sleeved clothes, placement of screens in houses, sleeping in rooms with air conditioning and avoiding the accumulation of standing water, which serves as a breeding ground for the mosquito vectors. The WHO recommends the use of repellents containing DEET, IR3535, aminopropionic or icaridine .The CDC recommends sexual abstinence or condom use for men suspected of contracting a Zika virus infection over a period of 6 months. For those residing or returning from a region with active transmission of the virus but who do not show symptoms, such measures should be adopted for at least 8 weeks after the person’s return.Women who are interested in childbearing and pregnant women should not travel to areas with the risk of Zika. If travel is unavoidable, or with regard to pregnant couples living in Zika areas, condoms from start to finish every time sex is performed is recommended, during the entire pregnancy. Populations in endemic areas or who have been exposed should conduct early and serial ultrasounds during pregnancy .Arboviruses are emerging infectious diseases endemic in several localities, but with unpredictable and devastating potential, as seen recently with the viral epidemics in the Americas.Despite the recent publications on infections by the Zika virus and the repercussions of congenital infection, research in this area is still modest and many arboviruses aspects need to be clarified, especially regarding gestational infection.Some issues that should be focused on are the improvement of diagnostic tests, including specific and sensitive molecular tests, associated to ultrasound screening; more accurate estimates of maternal, fetal and neonatal risks and the development of preventive vaccines, among other therapeutic options.Only after the advancement of these issues will it be possible to develop effective intervention strategies aimed at the prevention or eradication of arbovirus infections and their deleterious maternal-fetal outcomes.


Small interference RNA has been widely used to suppress gene expression. Three different short hairpin RNAs (shRNAs) against dopamine D1 receptor (Drd1), driven by mouse U6 promoter in self-complementary AAV8 vector (scAAV8), were used to silence mouse striatal Drd1 expression. Transduction of mouse striatum with all three scAAV8-D1shRNA viruses, but not the control scAAV8 virus, causes extensive neuroinflammation, demyelination, and axon degeneration. RNA interference is known to be coupled to the innate immune system as a host cell defense against virus infection. Activation of the innate immune system may play a causal role in the development of neuroinflammation and white matter degeneration, providing a novel animal model for multiple sclerosis (MS) and other neuroinflammatory diseases. Among these vectors, self-complementary adeno-associated virus (scAAV) with a double-stranded DNA bypasses the rate-limiting step of second DNA strand synthesis in traditional AAV virus (single-stranded DNA) and thereby transduces target tissue much more efficiently . Among dStriatal dopamine D1 receptor (Drd1) plays an important role in a number of neurological diseases including Parkinson disease, Huntington disease, addiction, depression, and schizophrenia . Dopaminin vitro studies . Analysis of potential “off-target” effects for each D1shRNA was conducted at the MIT web site (http://jura.wi.mit.edu/bioc/siRNAext/). The second D1shRNA2 (shRNA2) target region was chosen from 1,024 to 1,046 of Drd1 mRNA, 5′ GGCCCTTGGATGGCAATTTTACT 3′. The third D1shRNA3 (shRNA3) target region was chosen from 2,778 to 2,800 of Drd1 mRNA, 5′ AAGAGCATATGCCACTTTGTATT 3′. These shRNAs were selected due to their relatively low potential off-target effects in mouse genome, particularly at position 2 to 13 of the guide strand. The potential off-target effects for each shRNA were also analyzed in silico, and no common unintended genes were found.The target sequence of the first Drd1 shRNA (shRNA1) was chosen according to  studies . The othRenilla green fluorescence protein (hrGFP). The CMV-hrGFP cassette was released from pAAV-CMV-hrGFP (kindly provided by Dr. Beverly Davidson) by double digestion with EcoR I and Xba I enzymes, and was used to replace CMV-EGFP cassette in scAAV vector pVm-CMV-EGFP digested with EcoR I and Xho I enzymes. The resultant scAAV-hrGFP vector (control) was used to clone all shRNAs at the EcoR I site. Short-hairpin RNA cassette was driven by mouse U6 promoter with a loop sequence (5′ CTTCCTGTCA 3′). Three different D1shRNAs  targeting different Drd1 regions were cloned under the control of mouse U6 promoter. AAV8 help plasmid (kindly provided by University of Pennsylvania) was used for the generation of recombinant scAAV8 virus. The titers of the four scAAV8 virus were about 2 × 1012.To minimize potential toxicity of fluorescence marker protein, EGFP was replaced with humanized ad libitum. After a week of acclimation, 10 to 20 mice were grouped for injection of each scAAV8 viruses . The virus was injected at both dorsal  and ventral striatum  of 3-month old C57BL/6J male mice. 1 µl volume of scAAV8 virus was injected slowly (2 min) using stereotaxic delivery at each site. The surgery and other procedures were approved by both the UCSD Animal Care and Use Committee  and local Veteran’s Administration Hospital ) prior to the onset of the experiments. Mice were maintained in American Association for Accreditation of Laboratory Animal Care approved animal facilities. This facility meets all Federal and State requirements for animal care.Male C57BL/6J adult mice were purchased from Jackson Laboratory. Mice were housed in a climate-controlled animal colony with a reversed day/night cycle. Food  and water were available Mice were euthanized with carbon dioxide, and the brains were quickly removed on ice. Both anterior and posterior striatum were dissected from coronal brain sections on ice. Tissues were homogenized using Dounce Homogenizer in 1X Passive Lysis Buffer  supplemented with Protease Inhibitor Cocktail . Protein concentration was measured with Bradford Assay Kit . 20 µg of total proteins were loaded on SDS-PAGE gel, and blotted to PVDF membrane  after separation with electrophoresis. Mouse monoclonal antibody anti-Drd1  and mouse monoclonal antibody anti-NMDAR1  were used as primary antibodies. HRP conjugated anti-mouse IgG and HRP conjugated anti-rabbit IgG, were obtained from Amersham and Sigma as the secondary antibodies. The concentration of primary antibodies for Western Blot was generally used as recommended by the manufacturers. Image J was used for quantification. Immunohistochemical staining on coronal brain sections was performed as previously described . Mouse mDrd1 gene, and the D1shRNA3 was selected to targeting the 3′ UTR of the Drd1 gene (Renilla green fluorescent protein (hrGFP). Recombinant scAAV8-hrGFP virus was generated as a control. Using stereotaxic delivery, each scAAV8 virus was injected only into mouse left striatum, whereas uninjected mouse right striatum served as a control within the same coronal brain section  than the control striatum transduced with scAAV8-hrGFP. Many mice injected with either scAAV8-shRNA1 or scAAV8-shRNA2 virus also displayed excessive mouse IgG antibodies in their striatum. Consistent with the Western blot, we found that almost all mice injected with scAAV8-D1shRNAs displayed IgG background in immunohistochemical staining with anti-mouse IgG antibodies  that have lengths ranging from 21 to 23 bp. Both the D1shRNA1 and the D1shRNA2 were designed to targeting the coding sequences of mouse rd1 gene . All thr section . Four we section . To eval section . Surpris section . Adjacen section . However section , suggest section . As expeout mice . Using rout mice –S1E. Conout mice . Westernout mice . We weree points . Mouse Ie points . Striatutibodies . We did n = 6) transduced with scAAV8-hrGFP virus. Almost all mouse brains transduced with scAAV8-D1shRNA virus displayed microglial activation  controls. However, It is likely that D1shRNA immunogenicity plays an important role in microglial activation and white matter degeneration, as indicted by AAV8 virus transduces both neuronal cells and glial cells . TransduCNS demyelination can be naturally induced by a variety of virus in animals including visna virus, caprine arthritis-encephalitis virus, Theiler’s murine encephalomyelitis virus, Semliki Forest virus, etc . All the10.7717/peerj.3905/supp-1Figure S1(A) Three different shRNAs were designed to target different sites of mouse Drd1 gene. Immunohistochemical staining of Drd1 was performed using rabbit anti-Drd1 antibody in mouse striatum transduced with either scAAV8-hrGFP (B) or scAAV8-D1shRNA1 (C) virus. The virus was injected into the left striatum only. Reduced Drd1 protein was observed in the left striatum injected with scAAV8-D1shRNA1 virus, but not with scAAV8-hrGFP virus. High magnification of Drd1 reduction was shown in the left striatum transduced with scAAV8-D1sdhRNA1 virus (D) in comparison with Drd1 expression in the right control striatum (E). Suppression of Drd1protein expression was also observed using scAAV8-D1shRNA2 (F) and scAAV8-D1shRNA3 (H) in comparison with Drd1 expression in their right control striatum (G) and (I). Scale bar: 50 µm.Click here for additional data file.10.7717/peerj.3905/supp-2Figure S2(A) Immunohistochemical staining of Iba-1 in mice injected with scAAV8-D1shRNA2 virus. Microglial cells were activated to surrounding blood capillaries in the left striatum transduced with the scAAV8-D1shRNA2 virus. (B) and (C) are the higher magnifications of mild neuroinflammation around the blood capillaries. Scale bar: 50 µm.Click here for additional data file.10.7717/peerj.3905/supp-3Figure S3There is no difference in MBP staining in the left corpus callosum transduced with scAAV8-hrGFP virus (A) in comparison with the control right corpus callosum without virus injection (B). Scale bar: 100 µm. MBP was reduced (black arrow) on the left corpus callosum (C) of mice injected with scAAV8-D1shRNA3 in comparison with the normal right corpus callosum (D). Scale bar: 200 µm. Under a higher magnification, MBP staining was also decreased in striatal white matter tracts that were swollen and blebbed (black arrowheads) in the left striatum transduced with scAAV8-D1shRNA3 (E) in comparison with normal white matter tracts in the control right striatum (F). Scale bar: 60 µm.Click here for additional data file.10.7717/peerj.3905/supp-4Figure S4Two consecutive paraffin sections from mice injected with scAAV8-hrGFP virus were immunostained 7 weeks post-injection with either anti-Iba-1 (A) or anti-NF-L (B) antibody. There was no activation of microglial cells in the left striatum transduced with scAAV8-hrGFP virus in comparison with the uninjected control right striatum. There was no reduction of NF-L staining in the corpus callosum on the left side (C) transduced with the virus in comparison with the control right side without virus infection (D). Scale bar: 150 µm.Click here for additional data file.10.7717/peerj.3905/supp-5Figure S5Two consecutive brain paraffin sections from mice injected with scAAV8-D1shRNA3 virus were immunostained with either anti-Iba-1 (A) or anti-NF-L (B) antibody. Extensive microglial activation as shown by anti-Iba-1 immunostaining was observed in the left striatum transduced with the scAAV8-D1shRNA3 virus in comparison with the control right striatum. Decreased NF-L staining was observed in the left corpus callosum (C) compared with the control right corpus callosum (D). Scale bar: 200 µm. Neurofilament staining was reduced in blebbed striatal white matter tracts (black arrow) in the left striatum transduced with the virus (E) in contrast to the control right striatum (F). Scale bar: 60 µm.Click here for additional data file.10.7717/peerj.3905/supp-6Data S1Click here for additional data file.10.7717/peerj.3905/supp-7Data S2Click here for additional data file.10.7717/peerj.3905/supp-8Data S3Click here for additional data file.10.7717/peerj.3905/supp-9Data S4Week 7, 8, 9, 15.Click here for additional data file.10.7717/peerj.3905/supp-10Data S5Normalization controls.Click here for additional data file.10.7717/peerj.3905/supp-11Data S6Normalization controls for week 7, 8, 9, and 15.Click here for additional data file.10.7717/peerj.3905/supp-12Table S1Click here for additional data file.