The First Isolation of Insect-Specific Alphavirus (Agua Salud alphavirus) in Culex (Melanoconion) Mosquitoes in the Brazilian Amazon.

Advances in diagnostic techniques coupled with ongoing environmental changes have resulted in intensified surveillance and monitoring of arbovirus circulation in the Amazon. This increased effort has resulted in increased detection of insect-specific viruses among hematophagous arthropods collected in the field. This study aimed to document the first isolation of Agua Salud alphavirus in mosquitoes collected within the Brazilian Amazon. Arthropods belonging to the family Culicidae were collected within a forest fragment located in the Environmental Protection Area of the metropolitan region of Belem. Subsequently, these specimens were meticulously identified to the species level. Afterward, the collected batches were macerated, and the resulting supernatant was then inoculated into C6/36 and Vero cell cultures to facilitate viral isolation. The presence of arboviruses within the inoculated cell cultures was determined through indirect immunofluorescence analysis. Furthermore, positive supernatant samples underwent nucleotide sequencing to precisely identify the viral strains present. Notably, a batch containing Culex (Melanoconion) mosquitoes was identified to be positive for the genus Alphavirus via indirect immunofluorescence. This study is the first report on insect-specific alphavirus isolation in Brazil and the first-ever description of Agua Salud alphavirus isolation within Amazon Forest remnants.

Molecular detection of multiple arboviruses in the city of Goiânia-Goiás-Brazil.

BACKGROUND: Healthcare systems are currently ill-equipped to diagnose arboviruses rapidly and efficiently or to differentiate between various viruses. METHODS: Utilizing molecular techniques, this study examined arbovirus infections in 459 patients from a public health unit in Goiânia-Goiás, Brazil, a region where arbovirus infection poses a significant public health challenge. RESULTS: Nearly 60% of the analyzed samples tested positive for at least one arbovirus, and over 10% of the patients were co-infected with more than one virus. CONCLUSIONS: Fast and accurate diagnostic tools are essential for informing public health policy and enhancing epidemiological surveillance.

Sylvatic Mosquito Viromes in the Cerrado Biome of Minas Gerais, Brazil: Discovery of New Viruses and Implications for Arbovirus Transmission.

Studies on animal virome have mainly concentrated on chordates and medically significant invertebrates, often overlooking sylvatic mosquitoes, constituting a major part of mosquito species diversity. Despite their potential role in arbovirus transmission, the viromes of sylvatic mosquitoes remain largely unexplored. These mosquitoes may also harbor insect-specific viruses (ISVs), affecting arboviral transmission dynamics. The Cerrado biome, known for rapid deforestation and its status as a biodiversity hotspot, offers an ideal setting for investigating mosquito viromes due to potential zoonotic spillover risks from land use changes. This study aimed to characterize the viromes of sylvatic mosquitoes collected from various locations within Minas Gerais state, Brazil. The total RNA was extracted from mosquito pools of Psorophora albipes, Sabethes albiprivus, Sa. chloropterus, Psorophora ferox, and Coquillettidia venezuelensis species, followed by high-throughput sequencing (HTS). Bioinformatic analysis included quality control, contig assembly, and viral detection. Sequencing data analysis revealed 11 near-complete viral genomes (new viruses are indicated with asterisks) across seven viral families and one unassigned genus. These included: Xinmoviridae (Ferox mosquito mononega-like virus* and Albipes mosquito Gordis-like virus*), Phasmaviridae (Sabethes albiprivus phasmavirus*), Lispiviridae (Pedras lispivirus variant MG), Iflaviridae (Sabethes albiprivus iflavivirus*), Virgaviridae (Buriti virga-like virus variant MG and Sabethes albiprivus virgavirus 1*), Flaviviridae (Psorophora ferox flavivirus*), Mesoniviridae (Alphamesonivirus cavallyense variant MG), and the genus Negevirus (Biggie virus variant MG virus and Coquillettidia venezuelensis negevirus*). Moreover, the presence of ISVs and potential novel arboviruses underscores the need for ongoing surveillance and control strategies to mitigate the risk of emerging infectious diseases.

Mosquito (Diptera: Culicidae) diversity and arbovirus detection across an urban and agricultural landscape.

Fragmented landscapes in Mexico, characterized by a mix of agricultural, urban, and native vegetation cover, presents unique ecological characteristics that shape the mosquito community composition and mosquito-borne diseases. The extent to which landscape influences mosquito populations and mosquito-borne diseases is still poorly understood. This work assessed the effect of landscape metrics -agriculture, urban, and native vegetation cover- on mosquito diversity and arbovirus presence in fragmented tropical deciduous forests in Central Mexico during 2021. Among the 21 mosquito species across six genera we identified, Culex quinquefasciatus was the most prevalent species, followed by Aedes aegypti, Ae. albopictus, and Ae. epactius. Notably, areas with denser native vegetation cover displayed higher mosquito species richness, which could have an impact on phenomena such as the dilution effect. Zika and dengue virus were detected in 85% of captured species, with first reports of DENV in several Aedes species and ZIKV in multiple Aedes and Culex species. These findings underscore the necessity of expanding arbovirus surveillance beyond Ae. aegypti and advocate for a deeper understanding of vector ecology in fragmented landscapes to adequately address public health strategies.

Hard ticks (Ixodida: Ixodidae) in the Colombian Caribbean harbor the Jingmen tick virus: an emerging arbovirus of public health concern.

BACKGROUND: Ticks are obligate hematophagous ectoparasites involved in transmitting viruses of public health importance. The objective of this work was to identify the Jingmen tick virus in hard ticks from the Colombian Caribbean, an arbovirus of importance for public health. METHODS: Ticks were collected in rural areas of Córdoba and Cesar, Colombia. Taxonomic identification of ticks was carried out, and pools of 13 individuals were formed. RNA extraction was performed. Library preparation was performed with the MGIEasy kit, and next-generation sequencing (NGS) with MGI equipment. Bioinformatic analyses and taxonomic assignments were performed using the Galaxy platform, and phylogenetic analyses were done using IQ-TREE2. RESULTS: A total of 766 ticks were collected, of which 87.33% (669/766) were Rhipicephalus microplus, 5.4% (42/766) Dermacentor nitens, 4.2% (32/766) Rhipicephalus linnaei, and 3.0% (23/766) Amblyomma dissimile. Complete and partial segments 1, 2, 3, and 4 of Jingmen tick virus (JMTV) were detected in the metatranscriptome of the species R. microplus, D. nitens, and A. dissimile. The JMTVs detected are phylogenetically related to JMTVs detected in Aedes albopictus in France, JMTVs detected in R. microplus in Trinidad and Tobago, JMTVs in R. microplus and A. variegatum in the French Antilles, and JMTVs detected in R. microplus in Colombia. Interestingly, our sequences clustered closely with JMTV detected in humans from Kosovo. CONCLUSIONS: JMTV was detected in R. microplus, D. nitens, and A. dissimile. JMTV could pose a risk to humans. Therefore, it is vital to establish epidemiological surveillance measures to better understand the possible role of JMTV in tropical diseases.

Advancing arbovirus diagnosis in Brazil: strengthening diagnostic strategies and public health data collection.

BACKGROUND: The last five decades have seen a surge in viral outbreaks, particularly in tropical and subtropical regions like Brazil, where endemic arboviruses such as Dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) pose significant threats. However, current diagnostic strategies exhibit limitations, leading to gaps in infection screening, arbovirus differential diagnoses, DENV serotyping, and life-long infection tracking. This deficiency impedes critical information availability regarding an individual's current infection and past infection history, disease risk assessment, vaccination needs, and policy formulation. Additionally, the availability of point-of-care diagnostics and knowledge regarding immune profiles at the time of infection are crucial considerations. OBJECTIVES: This review underscores the urgent need to strengthen diagnostic methods for arboviruses in Brazil and emphasizes the importance of data collection to inform public health policies for improved diagnostics, surveillance, and policy formulation. METHODS: We evaluated the diagnostic landscape for arboviral infections in Brazil, focusing on tailored, validated methods. We assessed diagnostic methods available for sensitivity and specificity metrics in the context of Brazil. RESULTS: Our review identifies high-sensitivity, high-specificity diagnostic methods for arboviruses and co-infections. Grifols transcription-mediated amplification assays are recommended for DENV, CHIKV, and ZIKV screening, while IgG/IgM ELISA assays outperform Rapid Diagnostic Tests (RDTs). The Triplex real-time RT-PCR assay is recommended for molecular screening due to its sensitivity and specificity. CONCLUSION: Enhanced diagnostic methods, on-going screening, and tracking are urgently needed in Brazil to capture the complex landscape of arboviral infections in the country. Recommendations include nationwide arbovirus differential diagnosis for DENV, ZIKV, and CHIKV, along with increased DENV serotyping, and lifelong infection tracking to combat enduring viral threats and reduce severe presentations.

Temporal and Spatiotemporal Arboviruses Forecasting by Machine Learning: A Systematic Review.

Arboviruses are a group of diseases that are transmitted by an arthropod vector. Since they are part of the Neglected Tropical Diseases that pose several public health challenges for countries around the world. The arboviruses' dynamics are governed by a combination of climatic, environmental, and human mobility factors. Arboviruses prediction models can be a support tool for decision-making by public health agents. In this study, we propose a systematic literature review to identify arboviruses prediction models, as well as models for their transmitter vector dynamics. To carry out this review, we searched reputable scientific bases such as IEE Xplore, PubMed, Science Direct, Springer Link, and Scopus. We search for studies published between the years 2015 and 2020, using a search string. A total of 429 articles were returned, however, after filtering by exclusion and inclusion criteria, 139 were included. Through this systematic review, it was possible to identify the challenges present in the construction of arboviruses prediction models, as well as the existing gap in the construction of spatiotemporal models.

Alphavirus Identification in Neotropical Bats.

Alphaviruses (Togaviridae) are arthropod-borne viruses responsible for several emerging diseases, maintained in nature through transmission between hematophagous arthropod vectors and susceptible vertebrate hosts. Although bats harbor many species of viruses, their role as reservoir hosts in emergent zoonoses has been verified only in a few cases. With bats being the second most diverse order of mammals, their implication in arbovirus infections needs to be elucidated. Reports on arbovirus infections in bats are scarce, especially in South American indigenous species. In this work, we report the genomic detection and identification of two different alphaviruses in oral swabs from bats captured in Northern Uruguay. Phylogenetic analysis identified Río Negro virus (RNV) in two different species: Tadarida brasiliensis (n = 6) and Myotis spp. (n = 1) and eastern equine encephalitis virus (EEEV) in Myotis spp. (n = 2). Previous studies of our group identified RNV and EEEV in mosquitoes and horse serology, suggesting that they may be circulating in enzootic cycles in our country. Our findings reveal that bats can be infected by these arboviruses and that chiropterans could participate in the viral natural cycle as virus amplifiers or dead-end hosts. Further studies are warranted to elucidate the role of these mammals in the biological cycle of these alphaviruses in Uruguay.

Thrombotic microangiopathy associated with arboviral infection: Report of 3 cases.

Dengue fever and chikungunya are viral diseases that have spread rapidly throughout the world in recent decades. The occurrence of complications is well known, including prerenal acute kidney injury (AKI), which is usually thought to be caused by dehydration and fluid loss. Thrombotic microangiopathy (TMA) is an uncommon aggravation of dengue fever and chikungunya, with only a few cases described in the medical literature. The aim of this study is to present 3 cases of TMA associated with arboviral infection. Three patients with clinical history, laboratory test, and kidney biopsy results compatible with TMA were selected for the study, 2 of whom had a serological diagnosis of dengue fever and 1 of chikungunya. The 3 patients were followed up at the Federal University of Maranhão Hospital's Nephrology Service in 2018. A targeted gene panel sequencing (TGPS) plus multiple to atypical hemolytic uremic syndrome (aHUS) multiplex ligation-dependent probe amplification (MLPA) was performed in 2 of the patients and revealed in the patient 1 a heterozygous pathogenic variant in the gene THBD, as well as heterozygous deletions in CFH, CFHR1, and CFHR3. In the patient 2, there were heterozygous pathogenic variant in the genes CFI and CFB, in addition to heterozygous deletions in the genes CFHR1 and CFHR3. Both received treatment with eculizumab and undergone recovery of renal function. The third patient had TMA not classified as either aHUS or thrombotic thrombocytopenic purpura (TTP); he abandoned the treatment and returned to the service after 2 years for a dialysis emergency. Patients with arboviral infectious disease and changes that suggest TMA should have appropriate support to establish early diagnosis and useful treatment.

Microclimate and the vertical stratification of potential bridge vectors of mosquito­borne viruses captured by nets and ovitraps in a central Amazonian forest bordering Manaus, Brazil.

In the Americas, some mosquito-borne viruses such as Zika, chikungunya, and dengue circulate among humans in urban transmission cycles, while others, including yellow fever and Mayaro, circulate among monkeys in sylvatic cycles. The intersection of humans and wildlife at forest edges creates risk for zoonotic virus exchange. We built a scaffold tower at the edge of a treefall gap in rainforest bordering Manaus, Brazil, to identify vectors that may bridge transmission between humans and monkeys. We vertically sampled diurnally active, anthropophilic mosquitoes using handheld nets at 0, 5, and 9 m and container-breeding mosquitoes in ovitraps at 0, 5, 10, and 15 m. Haemagogus janthinomys and Psorophora amazonica were present in high relative abundance in nets at each height sampled, while anthropophilic species were uncommon in ovitraps. Hg. janthinomys was more abundant at elevated heights than at ground level, while Ps. amazonica abundance was not significantly stratified across heights. The presence of each species increased with increasing 7-day rainfall lagged at 1 week, and at 1 and 4 weeks prior to collection, respectively. In addition, Hg. janthinomys was most frequently collected at 29.9 °C, irrespective of height. These data provide insight into the potential role of each species as bridge vectors.

Prevalence of arbovirus antibodies in young healthy adult population in Brazil.

BACKGROUND: The emergence and re-emergence of infectious diseases are a cause for worldwide concern. The introduction of Zika and Chikungunya diseases in the Americas has exposed unforeseen medical and logistical challenges for public health systems. Moreover, the lack of preventive measures and vaccination against known and emerging mosquito-transmitted pathogens, and the occurrence of unanticipated clinical complications, has had an enormous social and economic impact on the affected populations. In this study, we aimed to measure the seroprevalence of endemic and emerging viral pathogens in military personnel stationed in Manaus, Amazonas state. METHODS: We measured the seropositivity of antibodies against 19 endemic and emerging viruses in a healthy military personnel group using a hemagglutination inhibition assay (HIA). RESULTS: Overall, DENV positivity was 60.4%, and 30.9% of the individuals reacted against ZIKV. Also, 46.6%, 54.7%, 51.3% and 48.7% individuals reacted against West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilheus virus (ILHV) and Rocio virus (ROCV), respectively. Individuals with high DENV HIA titer reacted more frequently with ZIKV or WNV compared to those with low HIA titers. Observed cross-reactivity between Flaviviruses varied depending on the virus serogroup. Additionally, 0.6% and 0.3% individuals were seropositive for Oropouche virus (OROV) and Catu virus (CATUV) from the family Peribunyaviridae, respectively. All samples were negative for Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalomyelitis virus (WEEV), Mayaro virus (MAYV), Mucambo virus (MUCV) and CHIKV from the family Togaviridae. CONCLUSIONS: A high proportion of individuals in our high-risk population (~ 60%) lacked antibodies against major endemic and emerging viruses, which makes them susceptible for further infections. Military personnel serving in the Amazon region could serve as sentinels to strengthen global infectious disease surveillance, particularly in remote areas.

Spatial Distribution and Burden of Emerging Arboviruses in French Guiana.

Despite the health, social and economic impact of arboviruses in French Guiana, very little is known about the extent to which infection burden is shared between individuals. We conducted a large multiplexed serological survey among 2697 individuals from June to October 2017. All serum samples were tested for IgG antibodies against DENV, CHIKV, ZIKV and MAYV using a recombinant antigen-based microsphere immunoassay with a subset further evaluated through anti-ZIKV microneutralization tests. The overall DENV seroprevalence was estimated at 73.1% (70.6-75.4) in the whole territory with estimations by serotype at 68.9% for DENV-1, 38.8% for DENV-2, 42.3% for DENV-3, and 56.1% for DENV-4. The overall seroprevalence of CHIKV, ZIKV and MAYV antibodies was 20.3% (17.7-23.1), 23.3% (20.9-25.9) and 3.3% (2.7-4.1), respectively. We provide a consistent overview of the burden of emerging arboviruses in French Guiana, with useful findings for risk mapping, future prevention and control programs. The majority of the population remains susceptible to CHIKV and ZIKV, which could potentially facilitate the risk of further re-emergences. Our results underscore the need to strengthen MAYV surveillance in order to rapidly detect any substantial changes in MAYV circulation patterns.

The virome of vector mosquitoes.

Mosquitoes are the major vectors for arthropod-borne viruses (arboviruses) of medical importance. Aedes aegypti and A. albopictus are the most prolific and widespread mosquito vectors being responsible for global transmission of dengue, Zika and Chikungunya viruses. Characterizing the collection of viruses circulating in mosquitoes, the virome, has long been of special interest. In addition to arboviruses, mosquitoes carry insect-specific viruses (ISVs) that do not directly infect vertebrates. Mounting evidence indicates that ISVs interact with arboviruses and may affect mosquito vector competence. Here, we review our current knowledge about the virome of vector mosquitoes and discuss the challenges for the field that may lead to novel strategies to prevent outbreaks of arboviruses.

Assessing the reporting of Dengue, Chikungunya and Zika to the National Surveillance System in Colombia from 2014-2017: A Capture-recapture analysis accounting for misclassification of arboviral diagnostics.

BACKGROUND: Chikungunya, dengue, and Zika are three different arboviruses which have similar symptoms and are a major public health issue in Colombia. Despite the mandatory reporting of these arboviruses to the National Surveillance System in Colombia (SIVIGILA), it has been reported that the system captures less than 10% of diagnosed cases in some cities. METHODOLOGY/PRINCIPAL FINDINGS: To assess the scope and degree of arboviruses reporting in Colombia between 2014-2017, we conducted an observational study of surveillance data using the capture-recapture approach in three Colombian cities. Using healthcare facility registries (capture data) and surveillance-notified cases (recapture data), we estimated the degree of reporting by clinical diagnosis. We fit robust Poisson regressions to identify predictors of reporting and estimated the predicted probability of reporting by disease and year. To account for the potential misclassification of the clinical diagnosis, we used the simulation extrapolation for misclassification (MC-SIMEX) method. A total of 266,549 registries were examined. Overall arboviruses' reporting ranged from 5.3% to 14.7% and varied in magnitude according to age and year of diagnosis. Dengue was the most notified disease (21-70%) followed by Zika (6-45%). The highest reporting rate was seen in 2016, an epidemic year. The MC-SIMEX corrected rates indicated underestimation of the reporting due to the potential misclassification bias. CONCLUSIONS: These findings reflect challenges on arboviruses' reporting, and therefore, potential challenges on the estimation of arboviral burden in Colombia and other endemic settings with similar surveillance systems.

Phylogenetic Characterization of Arboviruses in Patients Suffering from Acute Fever in Rondônia, Brazil.

The purpose of the study was to classify, through phylogenetic analyses, the main arboviruses that have been isolated in the metropolitan region of Porto Velho, Rondônia, Brazil. Serum samples from patients with symptoms suggesting arboviruses were collected and tested by One Step RT-qPCR for Zika, Dengue (serotypes 1-4), Chikungunya, Mayaro and Oropouche viruses. Positive samples were amplified by conventional PCR and sequenced utilizing the Sanger method. The obtained sequences were aligned, and an evolutionary analysis was carried out using Bayesian inference. A total of 308 samples were tested. Of this total, 20 had a detectable viral load for Dengue, being detected DENV1 (18/20), co-infection DENV1 and DENV2 (1/20) and DENV4 (1/20). For Dengue serotype 3 and for the CHIKV, ZIKV, MAYV and OROV viruses, no individuals with a detectable viral load were found. A total of 9 of these samples were magnified by conventional PCR for sequencing. Of these, 6 were successfully sequenced and, according to the evolutionary profile, 5 corresponded to serotype DENV-1 genotype V, and 1 to serotype DENV-4 genotype II. In the study, we demonstrate co-circulation of the DENV-1 genotype V and the DENV-4 genotype II. Co-circulation of several DENV serotypes in the same city poses a risk to the population and is correlated with the increase of the most severe forms of the disease. Similarly, co-circulation of genetically distinct DENV and the occurrence of simultaneous infections can affect recombination events and lead to the emergence of more virulent isolates.

Optimization of convolutional neural network hyperparameters for automatic classification of adult mosquitoes.

The economic and social impacts due to diseases transmitted by mosquitoes in the latest years have been significant. Currently, no specific treatment or commercial vaccine exists for the control and prevention of arboviruses, thereby making entomological characterization fundamental in combating diseases such as dengue, chikungunya, and Zika. The morphological identification of mosquitos includes a visual exam of the samples. It is time consuming and requires adequately trained professionals. Accordingly, the development of a new automated method for realizing mosquito-perception and -classification is becoming increasingly essential. Therefore, in this study, a computational model based on a convolutional neural network (CNN) was developed to extract features from the images of mosquitoes and then classify the species Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. In addition, the model was trained to detect the mosquitoes of the genus Aedes. To train CNNs to perform the automatic morphological classification of mosquitoes, a dataset, which included 7,561 images of the target mosquitoes and 1,187 images of other insects, was acquired. Various neural networks, such as Xception and DenseNet, were used for developing the automatic-classification model based on images. A structured optimization process of random search and grid search was developed to select the hyperparameters set and increase the accuracy of the model. In addition, strategies to eliminate overfitting were implemented to increase the generalization of the model. The optimized model, during the test phase, obtained the balanced accuracy (BA) of 93.5% in classifying the target mosquitoes and other insects and the BA of 97.3% in detecting the mosquitoes of the genus Aedes in comparison to Culex. The results provide fundamental information for performing the automatic morphological classification of mosquito species. Using a CNN-embedded entomological tool is a valuable and accessible resource for health workers and non-taxonomists for identifying insects that can transmit infectious diseases.

High prevalence of Phasi Charoen-like virus from wild-caught Aedes aegypti in Grenada, W.I. as revealed by metagenomic analysis.

Arboviruses cause diseases of significant global health concerns. Interactions between mosquitoes and their microbiota as well as the important role of this interaction in the mosquito's capacity to harbor and transmit pathogens have emerged as important fields of research. Aedes aegypti is one of the most abundant mosquitoes in many geographic locations, a vector capable of transmitting a number of arboviruses such as dengue and Zika. Currently, there are few studies on the metavirome of this mosquito particularly in the Americas. This study analyzes the metavirome of A. aegypti from Grenada, a Caribbean nation with tropical weather, abundant A. aegypti, and both endemic and arboviral pathogens transmitted by this mosquito. Between January and December 2018, 1152 mosquitoes were collected from six semi-rural locations near houses in St. George Parish, Grenada, by weekly trapping using BG-Sentinel traps. From these, 300 A. aegypti were selected for analysis. The metavirome was analyzed using the Illumina HiSeq 1500 for deep sequencing. The generation sequencing library construction protocol used was NuGEN Universal RNA with an average read length of 125 bp. Reads were mapped to the A. aegypti assembly. Non-mosquito reads were analyzed using the tools FastViromeExplorer. The NCBI total virus, RNA virus, and eukaryotic virus databases were used as references. The metagenomic comparison analysis showed that the most abundant virus-related reads among all databases and assemblies was Phasi Charoen-like virus. The Phasi Charoen-like virus results are in agreement to other studies in America, Asia and Australia. Further studies using wild-caught mosquitoes is needed to assess the impact of this insect-specific virus on the A. aegypti lifecycle and vector capacity.

Sloths host Anhanga virus-related phleboviruses across large distances in time and space.

Sloths are genetically and physiologically divergent mammals. Phleboviruses are major arthropod-borne viruses (arboviruses) causing disease in humans and other animals globally. Sloths host arboviruses, but virus detections are scarce. A phlebovirus termed Anhanga virus (ANHV) was isolated from a Brazilian Linnaeus's two-toed sloth (Choloepus didactylus) in 1962. Here, we investigated the presence of phleboviruses in sera sampled in 2014 from 74 Hoffmann's two-toed (Choloepus hoffmanni, n = 65) and three-toed (Bradypus variegatus, n = 9) sloths in Costa Rica by broadly reactive RT-PCR. A clinically healthy adult Hoffmann's two-toed sloth was infected with a phlebovirus. Viral load in this animal was high at 8.5 × 107  RNA copies/ml. The full coding sequence of the virus was determined by deep sequencing. Phylogenetic analyses and sequence distance comparisons revealed that the new sloth virus, likely representing a new phlebovirus species, provisionally named Penshurt virus (PEHV), was most closely related to ANHV, with amino acid identities of 93.1%, 84.6%, 94.7% and 89.0% in the translated L, M, N and NSs genes, respectively. Significantly more non-synonymous mutations relative to ANHV occurred in the M gene encoding the viral glycoproteins and in the NSs gene encoding a putative interferon antagonist compared to L and N genes. This was compatible with viral adaptation to different sloth species and with micro-evolutionary processes associated with immune evasion during the genealogy of sloth-associated phleboviruses. However, gene-wide mean dN/dS ratios were low at 0.02-0.15 and no sites showed significant evidence for positive selection, pointing to comparable selection pressures within sloth-associated viruses and genetically related phleboviruses infecting hosts other than sloths. The detection of a new phlebovirus closely-related to ANHV, in sloths from Costa Rica fifty years after and more than 3,000 km away from the isolation of ANHV confirmed the host associations of ANHV-related phleboviruses with the two extant species of two-toed sloths.

Sylvatic cycles of arboviruses in non-human primates.

Arboviruses infecting people primarily exist in urban transmission cycles involving urban mosquitoes in densely populated tropical regions. For dengue, chikungunya, Zika and yellow fever viruses, sylvatic (forest) transmission cycles also exist in some regions and involve non-human primates and forest-dwelling mosquitoes. Here we review the investigation methods and available data on sylvatic cycles involving non-human primates and dengue, chikungunya, Zika and yellow fever viruses in Africa, dengue viruses in Asia and yellow fever virus in the Americas. We also present current putative data that Mayaro, o'nyong'nyong, Oropouche, Spondweni and Lumbo viruses exist in sylvatic cycles.

Is the brazilian diverse environment is a crib for the emergence and maintenance of exotic arboviruses?

We review the potential of Amazon forest as a source for circulation and maintenance of native arboviruses as well its capacity to host exotic arboviruses introduced in Brazil during their process of adapting to the Amazon environment. After a brief introduction about arboviruses isolated in Amazon region and description of the main arboviruses pathogenic to humans, we highlight the history of the last two exotic viruses introduced in Brazil - Chikungunya virus (CHIKV) and Zika virus (ZIKV) - and their consequences to the public health. Finally, we discuss and hypothesize what will happen with them after the outbreak. We look to the past to predict the future.