Aedes Mosquito Virome in Southwestern Cameroon: Lack of Core Virome, But a Very Rich and Diverse Virome in Ae. africanus Compared to Other Aedes Species.

In Cameroon, Aedes mosquitoes transmit various arboviruses, posing significant health risks. We aimed to characterize the Aedes virome in southwestern Cameroon and identify potential core viruses which might be associated with vector competence. A total of 398 Aedes mosquitoes were collected from four locations (Bafoussam, Buea, Edea, and Yaounde). Aedes albopictus dominated all sites except for Bafoussam, where Aedes africanus prevailed. Metagenomic analyses of the mosquitoes grouped per species into 54 pools revealed notable differences in the eukaryotic viromes between Ae. africanus and Ae. albopictus, with the former exhibiting greater richness and diversity. Thirty-seven eukaryotic virus species from 16 families were identified, including six novel viruses with near complete genome sequences. Seven viruses were further quantified in individual mosquitoes via qRT-PCR. Although none of them could be identified as core viruses, Guangzhou sobemo-like virus and Bafoussam mosquito solemovirus, were highly prevalent regionally in Ae. albopictus and Ae. africanus, respectively. This study highlights the diverse eukaryotic virome of Aedes species in southwestern Cameroon. Despite their shared genus, Aedes species exhibit limited viral sharing, with varying viral abundance and prevalence across locations. Ae. africanus, an understudied vector, harbors a rich and diverse virome, suggesting potential implications for arbovirus vector competence.

Simultaneous detection of arboviruses by a multiplex RT-qPCR assay in Tocantins, a northern state of Brazil.

In Brazil, Dengue, Zika and Chikungunya viruses constitute a major threat to the public health system. Simultaneous circulation of these arboviruses occurs in many regions of the world due to the expansion of transmission vectors. The infection by these arboviruses triggers similar symptoms during their acute phase. However, in some cases, severe symptoms may occur, leading to different types of disabilities and even death. In this context, considering the similarity of the symptoms, the problems caused by the infection of these arboviruses, and the increasing risk of coinfection in humans, the differential diagnosis of these infections is essential for clinical management and epidemiological investigation. Thus, this study aimed to identify, through diagnosis via Quantitative Polymerase Chain Reaction with Reverse Transcription, arbovirus coinfection in patients from the Tocantins state (Northern Brazil). A total of 495 samples were analyzed, three from which were determined to be a coinfection of Dengue and Chikungunya viruses. The data obtained here indicate the co-circulation and coinfection by Dengue and Chikungunya viruses in the Tocantins state. These results highlight the importance of monitoring the circulation of these arboviruses for the development of health actions that aim their prevention and combat, as well as their clinical and therapeutic management.

Identification and molecular characterization of Wanghe virus: A novel member of the Solinviviridae family from mosquitoes, China.

Arboviruses have always been a significant public health concern. Metagenomic surveillance has expanded the number of novel, often unclassified arboviruses, especially mosquito-borne and mosquito-specific viruses. This report presents the first description of a novel single-stranded RNA virus, Wanghe virus, identified from mosquitoes that were collected in Shandong Province in 2022. In this study, a total of 4,795 mosquitoes were collected and then divided into 105 pools according to location and species. QRT-PCR and nested PCR were performed to confirm the presence of Wanghe virus, and its genomic features and phylogenetic relationships were further analyzed. Our results revealed that Wanghe virus was detected in 9 out of the 105 mosquito pools, resulting in a minimum infection rate (MIR) of 0.19 % (9/4,795). One complete genome sequence and three viral partial sequences were obtained from the Wanghe virus-positive pools. Pairwise distance analysis indicated that these amplified sequences shared high nucleotide identity. Phylogenetic analysis demonstrated that Wanghe virus is most closely related to Guiyang Solinvi-like virus 3, which belongs to Solinviviridae. Further analyses indicated that Wanghe virus is a new, unclassified member of Solinviviridae.

Molecular Detection and Genetic Characterization of Two Dugbe Orthonairovirus Isolates Detected from Ticks in Southern Senegal.

Dugbe virus (DUGV) is a tick-borne arbovirus first isolated in Nigeria in 1964. It has been detected in many African countries using such diverse methods as serological tests, virus isolation, and molecular detection. In Senegal, reports of DUGV isolates mainly occurred in the 1970s and 1980s. Here, we report a contemporary detection of three novel DUGV isolates upon screening of a total of 2877 individual ticks regrouped into 844 pools. The three positive pools were identified as Amblyomma variegatum, the main known vector of DUGV, collected in the southern part of the country (Kolda region). Interestingly, phylogenetic analysis indicates that the newly sequenced isolates are globally related to the previously characterized isolates in West Africa, thus highlighting potentially endemic, unnoticed viral transmission. This study was also an opportunity to develop a rapid and affordable protocol for full-genome sequencing of DUGV using nanopore technology. The results suggest a relatively low mutation rate and relatively conservative evolution of DUGV isolates.

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.

In vivo rescue of arboviruses directly from subgenomic DNA fragments.

Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the "Infectious Subgenomic Amplicons" (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo. Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo. It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

Maintenance of persistent transmission of a plant arbovirus in its insect vector mediated by the Toll-Dorsal immune pathway.

Throughout evolution, arboviruses have developed various strategies to counteract the host's innate immune defenses to maintain persistent transmission. Recent studies have shown that, in addition to bacteria and fungi, the innate Toll-Dorsal immune system also plays an essential role in preventing viral infections in invertebrates. However, whether the classical Toll immune pathway is involved in maintaining the homeostatic process to ensure the persistent and propagative transmission of arboviruses in insect vectors remain unclear. In this study, we revealed that the transcription factor Dorsal is actively involved in the antiviral defense of an insect vector (Laodelphax striatellus) by regulating the target gene, zinc finger protein 708 (LsZN708), which mediates downstream immune-related effectors against infection with the plant virus (Rice stripe virus, RSV). In contrast, an antidefense strategy involving the use of the nonstructural-protein (NS4) to antagonize host antiviral defense through competitive binding to Dorsal from the MSK2 kinase was employed by RSV; this competitive binding inhibited Dorsal phosphorylation and reduced the antiviral response of the host insect. Our study revealed the molecular mechanism through which Toll-Dorsal-ZN708 mediates the maintenance of an arbovirus homeostasis in insect vectors. Specifically, ZN708 is a newly documented zinc finger protein targeted by Dorsal that mediates the downstream antiviral response. This study will contribute to our understanding of the successful transmission and spread of arboviruses in plant or invertebrate hosts.

Monitoring Aedes populations for arboviruses, Wolbachia, insecticide resistance and its mechanisms in various agroecosystems in Benin.

Aedes mosquitoes are the main vectors of arboviruses in Benin. Cases of dengue have been reported in Benin with all four serotypes of the virus actively circulating in this region. Some agricultural settings are known to harbor Aedes vectors responsible for the transmission of arboviruses. The massive use of certain insecticides in agricultural settings has probably contributed to insecticide resistance in these vectors. In Benin, the susceptibility of arbovirus vectors to insecticides is poorly studied. In addition, the distribution of Wolbachia spp., which is used against some arboviruses is unknown. Moreover, there is limited information regarding the vectors responsible for the transmission of arboviruses in Benin. This present study monitored the species composition, arboviruses, and Wolbachia symbiont status, as well as the phenotypic and molecular insecticide resistance profile of Aedes populations from three agroecosystems in Benin. Aedes species identification was performed morphologically and confirmed using qPCR. (RT)-qPCR assay was applied for monitoring the presence of DENV, CHIKV, ZIKV, and WNV pathogens as well as for naturally occurring Wolbachia symbionts. Insecticide resistance was assessed phenotypically, by permethrin (0.75%) exposure of Adults (F0) using World Health Organization (WHO) bioassay protocols, and at the molecular level, using TaqMan (RT)-qPCR assays for assessing knock-down resistance (kdr) mutations (F1534C, V1016G/I, and S989P) and the expression levels of eight detoxification genes (P450s from the CYP9 and CYP6 families, carboxylesterases and glutathione-S-transferases). Aedes aegypti (Ae. aegypti) mosquitoes were the most abundant (93.9%) in the three agroecosystems studied, followed by Aedes albopictus (Ae. albopictus) mosquitoes (6.1%). No arboviruses were detected in the study's mosquito populations. Naturally occurring Wolbachia symbionts were present in 7 pools out of 15 pools tested. This could influence the effectiveness of vector control strategies based on exogenously introduced Wolbachia, all present in the three agroecosystems. Full susceptibility to permethrin was observed in all tested populations of Ae. albopictus. On the contrary, Ae. aegypti were found to be resistant in all three agroecosystem sites except for banana plantation sites, where full susceptibility was observed. Molecular analysis revealed that individual target site resistance kdr mutations F1534C and V1016G/I were detected in most Ae. aegypti populations. Additionally, double mutant (F1534C + V1016G/I) mosquitoes were found in some populations, and in one case, triple mutant (F1534C + V1016G/I + S989P) mosquitoes were detected. Metabolic resistance, as reflected by overexpression of three P450 genes (CYP6BB2, CYP9J26, and CYP9J32), was also detected in Ae. aegypti mosquitoes. Our study provides information that could be used to strategize future vector control strategies and highlights the importance of continuing vector surveillance. Future studies should assess the effect of piperonyl butoxide (PBO) on metabolic resistance and identify the different strains of Wolbachia spp., to choose the best vector control strategies in Benin.

Metagenomic analysis of individual mosquito viromes reveals the geographical patterns and drivers of viral diversity.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.

Haemovigilance survey and screening strategy for arthropod-borne viruses in blood donors from Argentina.

Arthropod-borne viruses (arboviruses) count among emerging infections, which represent a major challenge for transfusion safety worldwide. To assess the risk of arboviruses-transmission by transfusion (ATT), we performed a survey to evaluate the potential threat for transfusion safety. Samples were retrospectively and randomly collected from donors who donated during the peak of dengue incidence in Cordoba (years: 2016 and 2019-2022). A cost-efficient strategy for molecular screening was implemented with a nucleic acid test (NAT) configured with Flavivirus and Alphavirus-universal degenerated primers targeting conserved gene regions. Besides, we evaluated the neutralizing antibody (NAb) prevalence by plaque reduction neutralization test (PRNT). A total of 1438 samples were collected. Among the NAT-screened samples, one resulted positive for Flavivirus detection. Subsequent sequencing of the PCR product revealed Saint Louis Encephalitis Virus (SLEV) infection (GeneBank accession number OR236721). NAb prevalence was 2.95% for anti-Dengue, 9.94% anti-SLEV, 1.09% anti-West Nile Virus, and 0% anti-Chikungunya. One of the NAb-positive samples also resulted positive for IgM against SLEV but negative by ARN detection. This is the first haemovigilance study developed in Argentina that evaluates the potential risk of ATT and the first research to determine the prevalence of NAb against Flavivirus through PNRT to avoid possible cross-reactions between Ab against Flavivirus. Herein, the finding of one SLEV-viremic donor and the detection of anti-SLEV IgM in a different donor demonstrated a potential threat for transfusion safety and emphasized the need for increased vigilance and proactive measures to ensure the safety of blood supplies.

Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes.

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.

Arboviruses: Transmission and Host Resistance.

In this review, there is a complete description of the classes of arboviruses, their evolutionary process, virus characterization, disease transmission methods; it also describes about the vectors involved in transmission and their mood of transmission, both biologically as well as non-biologically and, about host, the resistance mechanism in host, and artificial methods of preventing those viral transmissions. Arboviruses transmitted to hosts by some vectors such as mosquitoes, ticks, etc. The virus replicates in the host can be prevented by some host resistance mechanisms like RNA interference (RNAi), which degrade virus RNA by its antiviral activity, insect repellents, IGRs, and PI technology.

Arbovirus researchers unite: expanding genomic surveillance for an urgent global need

Arthropod-borne viruses (arboviruses) are transmitted by arthropod vectors to humans, leading to disease outbreaks mainly in tropical regions of the world.1 Although many arboviruses are known, three Aedes-spp-borne viruses are particularly concerning for humans at a global scale due to recurring large and expanding epidemic outbreaks of dengue virus, Zika virus, and chikungunya virus. These viruses cause a heavy disease burden with mild to potentially life-threatening symptoms, resulting in substantial short-term and long-term morbidity and mortality.2 Epidemiological estimates underscore the impact of these viruses, with half of the world population at risk of dengue virus infection and around 100­400 million cases and 20 000 deaths reported each year.3 In addition to worsening outbreaks in tropical areas, there has been an increase in disease in subtropical and temperate regions, driven by the expansion of the main vectors Aedes aegypti and Aedes albopictus to new areas associated with urbanisation, globalisation, human mobility, and climate change.4 An additional 100 million people are estimated to be at risk of infection owing to wider transmission seasons in high-altitude areas and temperate regions of the world in the next 50 years.5 To strengthen the mitigation of epidemics of these arboviruses, WHO launched the Global Arbovirus Initiative in March, 2022, to build a coalition of key stakeholders to improve surveillance and prevention.

A Multiplex Nanopore Sequencing Approach for the Detection of Multiple Arboviral Species.

The emergence and continued geographic expansion of arboviruses and the growing number of infected people have highlighted the need to develop and improve multiplex methods for rapid and specific detection of pathogens. Sequencing technologies are promising tools that can help in the laboratory diagnosis of conditions that share common symptoms, such as pathologies caused by emerging arboviruses. In this study, we integrated nanopore sequencing and the advantages of reverse transcription polymerase chain reaction (RT-PCR) to develop a multiplex RT-PCR protocol for the detection of Chikungunya virus (CHIKV) and several orthoflaviviruses (such as dengue (Orthoflavivirus dengue), Zika (Orthoflavivirus zikaense), yellow fever (Orthoflavivirus flavi), and West Nile (Orthoflavivirus nilense) viruses) in a single reaction, which provides data for sequence-based differentiation of arbovirus lineages.

Seroepidemiological Studies of Arboviruses in Africa

The literature on sero-epidemiological studies of flaviviral infections in the African continent is quite scarce. Much of the viral epidemiology studies have been focussing on diseases such as HIV/AIDS because of their sheer magnitude and impact on the lives of people in the various affected countries. Increasingly disease outbreaks caused by arboviruses such as the recent cases of chikungunya virus, dengue virus and yellow fever virus have prompted renewed interest in studying these viruses. International agencies from the US, several EU nations and China are starting to build collaborations to build capacity in many African countries together with established institutions to conduct these studies. The Tofo Advanced Study Week (TASW) was established to bring the best scientists from the world to the tiny seaside town of Praia do Tofo to rub shoulders with African virologists and discuss cutting-edge science and listen to the work of researchers in the field. In 2015 the 1st TASW focussed on Ebola virus. The collections of abstracts from participants at the 2nd TASW which focused on Dengue and Zika virus as well as presentations on other arboviruses are collated in this chapter.

Evaluation of arboviruses of public health interest in free-living non-human primates (Alouatta spp., Callithrix spp., Sapajus spp.) in Brazil

INTRODUCTION: The aim of the present study was to evaluate the presence of arboviruses from the Flavivirus genus in asymptomatic free-living non-human primates (NHPs) living in close contact with humans and vectors in the States of Paraná and Mato Grosso do Sul, Brazil. METHODS: NHP sera samples (total n = 80, Alouatta spp. n = 07, Callithrix spp. n = 29 and Sapajus spp. n = 44) were screened for the presence of viral genomes using reverse transcription polymerase chain reaction and 10% polyacrylamide gel electrophoresis techniques. RESULTS: All of the samples were negative for the Flavivirus genome following the 10% polyacrylamide gel electrophoresis analysis. CONCLUSIONS: These negative results indicate that the analyzed animals were not infected with arboviruses from the Flavivirus genus and did not represent a risk for viral transmission through vectors during the period in which the samples were collected. .

Caracterización molecular de cepas aisladas de dengue 2 en Perú; 2000-2010 / Molecular characterization of isolates strains of dengue 2 in Peru; 2000-2010

El virus Dengue (VDEN) es el responsable de más de 50-100 millones de casos anualmente en el mundo. La infección del dengue es causada por cuatro serotipos (VDEN-1, VDEN-2, VDEN-3 y VDEN-4) y el espectro de la enfermedad varía desde una fiebre indiferenciada, fiebre hemorrágica por dengue (FHD), síndrome de shock por dengue, y muerte. Información epidemiológica liga el desarrollo de FHD con una infección secundaria y además sugiere que ciertas cepas son más virulentas que otras. En 2001, aparecieron los primeros casos de FHD asociados con VDEN-2 en la costa del Perú. En el 2010 ocurrió un brote en Iquitos con casos severos convirtiéndose en el más largo en la historia de la región. Sin embargo estudios orientados en determinar el origen distribución y diversidad genética de cepas peruanas de VDEN-2 durante los diez últimos años no han sido realizados. Para atender este vacío de conocimiento en la epidemiología del VDEN-2 en Perú, extractos de ARN de 30 aislamientos virales en células C6/36 (Aedes albopictus) fueron procesados por RT-PCR. Secuencias del gen de la envoltura (E) fueron determinadas y comparadas con muestras globales de VDEN-2. El análisis filogenético reveló la circulación de dos genotipos en Perú: Americano (hasta el 2000) y Americano/Asiático (2000-2010). Adicionalmente se identificaron cepas variantes del genotipo Americano/Asiático distribuidos en dos clados principales (1 y 2) que ingresaron al Perú por la costa norte (Ecuador) y por la selva (Brasil o Bolivia). Con el aparente incremento de la virulencia relacionada a cepas Americano/Asiático del clado 2, nuestros resultados soportan la necesidad de un continuo monitoreo de cepas emergentes de nuevos variantes o genotipos de dengue que podrían estar asociados a casos severos de la enfermedad.

Dengue virus (DENV) is responsible for more than 50-100 million cases annually throughout the world. Dengue infection is caused by four different dengue serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) and the clinical spectrum of disease ranges from dengue fever, dengue hemorrhagic fever (DHF), dengue shock syndrome, and death. Epidemiological information has linked the development of DHF with secondary dengue infections and has also suggested that certain DENV strains are more virulent than others. In 2001, the first DHF cases associated with DENV-2 virus were recognized in the coastal region of Peru. In 2010 severe cases from an outbreak in Iquitos were registered; and resulted in the largest DHF epidemic that region had ever experience. However, studies to address the origins, distribution, and genetic diversity of Peruvian DENV-2 strains from the last ten years have not been performed. To address this knowledge gap, RNA extracted from 30 viruses isolated in C6/36 (Aedes albopictus) cells were performed by RT-PCR. The envelope (E) sequences were determined and used in a phylogenetic comparison with a global sample of DENV-2 viruses. Phylogenetic analysis confirmed the circulation of two DENV-2 genotypes in Peru: American (prior to 2001) and American/Asian (2000-2010). Additionally we identified American/Asian genotype variants from two clades (1 and 2) were introduced into Peru from the north (Ecuador) and the east (Brazil or Bolivia). In light of evidence for increased virulence of clade 2, our results support the need for continuous monitoring for emerging strains of new DENV variants or genotypes that may be associated with severe disease.

Preliminary investigation of Culicidae species in South Pantanal, Brazil and their potential importance in arbovirus transmission / Investigação preliminar das espécies de culicídeos do Pantanal Sul brasileiro e sua potencial importância na transmissão de arbovírus

In view of the high circulation of migratory birds and the environmental and climatic conditions which favor the proliferation of arthropods, the Brazilian Pantanal is susceptible to circulation of arboviruses. However, the amount of data concerning arbovirus vectors in this area is scarce; therefore the aim of this study was to conduct a preliminary investigation of Culicidae species in the Nhecolândia Sub-region of South Pantanal, Brazil and their potential importance in the arbovirus transmission. A total of 3684 specimens of mosquitoes were captured, 1689 of which caught in the rainy season of 2007, were divided into 78 pools and submitted to viral isolation, Semi-Nested RT-PCR and Nested RT-PCR, with a view to identifying the most important arboviruses in Brazil. Simultaneously, 70 specimens of ticks found blood-feeding on horses were also submitted to the same virological assays. No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, a total of 22 Culicidae species were identified, ten of which had previously been reported as vectors of important arboviruses. The diversity of species found blood-feeding on human and horse hosts together with the arboviruses circulation previously reported suggest that the Nhecolândia Sub-region of South Pantanal is an important area for arbovirus surveillance in Brazil.

Regiões como o Pantanal brasileiro, que apresentam fatores como riqueza de fauna silvestre incluindo circulação de aves migratórias e condições ambientais e climáticas favoráveis à proliferação de artrópodes estão potencialmente sujeitas à circulação de arbovírus. Entretanto, poucos trabalhos foram realizados acerca da presença de arbovírus em potenciais vetores no Pantanal. Neste sentido o principal objetivo deste trabalho foi conduzir uma investigação preliminar para presença de arbovírus em amostragens de culicídeos capturados na Sub-região da Nhecolândia no Pantanal Sul. Um total de 3684 mosquitos foi capturado, dos quais 78 grupos compondo uma amostragem de 1789 espécimes foram submetidos às técnicas de isolamento viral e RT-PCR para os mais importantes arbovírus no Brasil. Simultaneamente, 70 espécimes de carrapatos capturados durante hematofagia em cavalos também foram submetidos à pesquisa viral. Não houve isolamento viral em nenhuma amostra analisada e os resultados de detecção de ácido nucléico viral foram também negativos. Entretanto, foram identificadas 22 espécies de culicídeos, dez das quais previamente reportadas como vetores de importantes arbovírus. A competência vetorial de espécies capturadas durante hematofagia em humanos e cavalos aliada ao relato prévio de circulação de arbovírus sugerem a Sub-região da Nhecolândia como uma importante área de vigilância para arbovírus no Centro-Oeste do Brasil.

Reliable detection of St. Louis encephalitis virus by RT-nested PCR

Introducción. El virus de la encefalitis de St. Louis (VESL), arbovirus reemergente en Sudamérica, provocó casos humanos en Argentina y Brasil. Esto pone de manifiesto la necesidad de incrementar el conocimiento sobre arbovirus para poder controlar y prevenir la aparición de futuros casos. Por este motivo, surge la necesidad de realizar exhaustivas investigaciones epidemiológicas y de laboratorio para asegurar la rápida identificación del agente y una apropiada acción de los agentes de salud. En este estudio se describe el desarrollo de una técnica de RT-nested PCR específica para la detección del VESL. Material y métodos. Se procedió a la selección de la región genómica del VESL que aportara mayor información sobre la variabilidad genética natural del virus. Así, se diseñaron cebadores degenerados que amplificaron un fragmento de 234 pb del gen de la envoltura de 9 cepas de VESL (Parton, BeH356964, SPAN11916, AN9275, AN9124 y 78V6507 y tres obtenidas de agrupamientos de mosquitos naturalmente infectados). Resultados. El método amplificó el genoma de todas las cepas del VESL analizadas y no se obtuvo amplificación con otros Flavivirus, tales como el virus de la fiebre amarilla, el virus Ilheus, el virus dengue-2, el virus Bussuquara, el virus del Oeste del Nilo, el virus de la encefalitis japonesa y el virus del valle Murray. Este método fue específico y sensible, con un bajo límite de detección: menos de 10 unidades formadoras de placa. Conclusión. La técnica desarrollada resultó ser confiable y de amplio espectro para la detección del VESL, y puede ser útil para la ejecución de estudios ecológicos, clínicos y de vigilancia virológica (AU)

Introduction. St. Louis encephalitis virus (SLEV) is a re-emerging arbovirus in South America, with reported cases in humans in Argentina and Brazil. This fact indicates that there is an urgent need to increase the current knowledge about this virus in order to control and prevent future cases. Exhaustive epidemiological and laboratory investigation is required to ensure fast, accurate identification of the viral agent and allow prompt surveillance action by health authorities. Herein, we report the development of a species-specific RT-nested PCR to detect SLEV. Material and methods. After selecting the SLEV genomic region providing the greatest information on the natural genetic variability of this virus, degenerated oligonucleotide primers were designed to amplify a 234-bp fragment of the envelope gene from nine SLEV strains (Parton, BeH356964, SPAN11916, AN9275, AN9124, 78V6507 and 3 SLEV strains obtained from naturally infected mosquito pools). Results. The method was able to identify the genome of all the SLEV strains tested and did not amplify unrelated RNA viruses, such as yellow fever virus, Ilheus virus, dengue-2 virus, Bussuquara virus, West Nile virus, Japanese encephalitis virus and Murray Valley encephalitis virus. The method was specific and sensitive, with a lower detection limit of < 10 plaque-forming units. Conclusion. This molecular assay is a reliable procedure with a wide spectrum for detecting the natural diversity of SLEV and may be useful for ecological studies, clinical and laboratory settings and virological surveillance (AU)