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.

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.

Genomic and phylogenetic profiling of RNA of tick-borne arboviruses in Hainan Island, China.

Ticks act as vectors and hosts of numerous arboviruses. Examples of medically important arboviruses include the tick-borne encephalitis virus, Crimean Congo hemorrhagic fever, and severe fever with thrombocytopenia syndrome. Recently, some novel arboviruses have been identified in blood specimens of patients with unexplained fever and a history of tick bites in Inner Mongolia. Consequently, tick-borne viruses are a major focus of infectious disease research. However, the spectrum of tick-borne viruses in subtropical areas of China has yet to be sufficiently characterized. In this study, we collected 855 ticks from canine and bovine hosts in four locations in Hainan Province. The ticks were combined into 18 pools according to genus and location. Viral RNA-sequence libraries were subjected to transcriptome sequencing analysis. Molecular clues from metagenomic analyses were used to classify sequence reads into virus species, genera, or families. The diverse viral reads closely associated with mammals were assigned to 12 viral families and important tick-borne viruses, such as Jingmen, Beiji nairovirus, and Colorado tick fever. Our virome and phylogenetic analyses of the arbovirus strains provide basic data for preventing and controlling human infectious diseases caused by tick-borne viruses in the subtropical areas of China.

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.

Characterizing viral species in mosquitoes (Culicidae) in the Colombian Orinoco: insights from a preliminary metagenomic study.

Mosquitoes (Diptera: Culicidae) are primary vectors of arthropod-borne viruses (arboviruses) that pose significant public health threats. Recent advances in sequencing technology emphasize the importance of understanding the arboviruses and insect-specific viruses (ISVs) hosted by mosquitoes, collectively called the "virome". Colombia, a tropical country with favorable conditions for the development and adaptation of multiple species of Culicidae, offers a favorable scenario for the transmission of epidemiologically important arboviruses. However, entomovirological surveillance studies are scarce in rural areas of the country, where humans, mosquitoes, and animals (both domestic and wild) coexist, leading to a higher risk of transmission of zoonotic diseases to humans. Thus, our study aimed to perform a preliminary metagenomic analysis of the mosquitoes of special relevance to public health belonging to the genera Ochlerotatus, Culex, Limatus, Mansonia, Psorophora, and Sabethes, within a rural savanna ecosystem in the Colombian Orinoco. We employed third-generation sequencing technology (Oxford Nanopore Technologies; ONT) to describe the virome of mosquitoes samples. Our results revealed that the virome was primarily shaped by insect-specific viruses (ISVs), with the Iflaviridae family being the most prevalent across all mosquito samples. Furthermore, we identified a group of ISVs that were common in all mosquito species tested, displaying the highest relative abundance concerning other groups of viruses. Notably, Hanko iflavirus-1 was especially prevalent in Culex eknomios (88.4%) and Ochlerotatus serratus (88.0%). Additionally, other ISVs, such as Guadalupe mosquito virus (GMV), Hubei mosquito virus1 (HMV1), Uxmal virus, Tanay virus, Cordoba virus, and Castlerea virus (all belonging to the Negevirus genus), were found as common viral species among the mosquitoes, although in lower proportions. These initial findings contribute to our understanding of ISVs within mosquito vectors of the Culicidae family in the Eastern Plains of Colombia. We recommend that future research explore deeper into ISV species shared among diverse vector species, and their potential interactions with arboviruses. In addition, we also showed the need for a thorough exploration of the influence of local rural habitat conditions on the shape of the virome in mosquito vectors.

Detection of coinfection with Primate Erythroparvovirus 1 and arboviruses (DENV, CHIKV and ZIKV) in individuals with acute febrile illness in the state of Rio Grande do Norte in 2016.

BACKGROUND: Arthropod-borne viruses, known as arboviruses, pose substantial risks to global public health. Dengue (DENV), Chikungunya (CHIKV) and Zika (ZIKV) viruses stand out as significant concerns in Brazil and worldwide. Their overlapping clinical manifestations make accurate diagnosis a challenge, underscoring the need for reliable laboratory support. This study employs a comprehensive molecular diagnostic approach to track viral infections in individuals with acute febrile illness, a period marked by widespread outbreaks of DENV, CHIKV and ZIKV. METHODS: Between January and August 2016, we received a total of 713 serum samples obtained from individuals with acute febrile illness, previously tested for DENV, CHIKV or ZIKV, with initial negative results, from LACEN-NATAL. Of the total 713 samples, 667 were from females (354 of them pregnant) and 46 from males. Molecular diagnosis was conducted using the Multiplex RT-qPCR technique for simultaneous detection of DENV, CHIKV and ZIKV. Additionally, we performed differential diagnosis by RT-qPCR for other viruses of the Flavivirus, Alphavirus Enterovirus genera and qPCR for Primate Erythroparvovirus 1 (B19V) species, in accordance with Ministry of Health guidelines. RESULTS: Among the 713 cases, 78.2% tested positive for viral infections, including 48% with CHIKV viremia, 0.6% with DENV and 0.1% with ZIKV. Arboviral coinfections totaled 2.4%, including DENV-CHIKV (1.7%) and CHIKV-ZIKV (0.7%). Moreover, 8% exhibited B19V viremia. Simultaneous infections were identified in 17.5%, encompassing B19V-CHIKV (17.1%), B19V-DENV (0.1%), and B19V-ZIKV (0.3%) Triple infections were observed in 1.3% of cases with B19V-DENV-CHIKV (1%) and B19V-CHIKV-ZIKV (0.3%). CONCLUSION: Molecular testing demonstrated high efficacy in diagnosing prevalent arboviruses and detecting multiple coinfections. This approach helps to elucidate etiologies for symptomatic cases, especially during arbovirus outbreaks, and aids comprehensive surveillance. Our findings underscore the importance of monitoring co-circulating pathogens, such as B19V, with implications for clinical management, particularly in pregnant individuals. This study enhances our understanding of arbovirus epidemiology and reinforces the critical role of molecular diagnosis in disease surveillance and control.

Vesicular Stomatitis Virus Elicits Early Transcriptome Response in Culicoides sonorensis Cells.

Viruses that are transmitted by arthropods, or arboviruses, have evolved to successfully navigate both the invertebrate and vertebrate hosts, including their immune systems. Biting midges transmit several arboviruses including vesicular stomatitis virus (VSV). To study the interaction between VSV and midges, we characterized the transcriptomic responses of VSV-infected and mock-infected Culicoides sonorensis cells at 1, 8, 24, and 96 h post inoculation (HPI). The transcriptomic response of VSV-infected cells at 1 HPI was significant, but by 8 HPI there were no detectable differences between the transcriptome profiles of VSV-infected and mock-infected cells. Several genes involved in immunity were upregulated (ATG2B and TRAF4) or downregulated (SMAD6 and TOLL7) in VSV-treated cells at 1 HPI. These results indicate that VSV infection in midge cells produces an early immune response that quickly wanes, giving insight into in vivo C. sonorensis VSV tolerance that may underlie their permissiveness as vectors for this virus.

Expanding diversity of bunyaviruses identified in mosquitoes.

Mosquitoes interact with various organisms in the environment, and female mosquitoes in particular serve as vectors that directly transmit a number of microorganisms to humans and animals by blood-sucking. Comprehensive analysis of mosquito-borne viruses has led to the understanding of the existence of diverse viral species and to the identification of zoonotic arboviruses responsible for significant outbreaks and epidemics. In the present study on mosquito-borne bunyaviruses we employed a broad-spectrum RT-PCR approach and identified eighteen different additional species in the Phenuiviridae family and also a number of related but unclassified bunyaviruses in mosquitoes collected in Zambia. The entire RNA genome segments of the newly identified viruses were further analyzed by RNA sequencing with a ribonuclease R (RNase R) treatment to reduce host-derived RNAs and enrich viral RNAs, taking advantage of the dsRNA panhandle structure of the bunyavirus genome. All three or four genome segments were identified in eight bunyavirus species. Furthermore, L segments of three different novel viruses related to the Leishbunyaviridae were found in mosquitoes together with genes from the suspected host, the Crithidia parasite. In summary, our virus detection approach using a combination of broad-spectrum RT-PCR and RNA sequencing analysis with a simple virus enrichment method allowed the discovery of novel bunyaviruses. The diversity of bunyaviruses is still expanding and studies on this will allow a better understanding of the ecology of hematophagous mosquitoes.

An 11-point time course midgut transcriptome across 72 h after bloodfeeding provides detailed temporal resolution of transcript expression in the arbovirus vector, Aedes aegypti.

As the major vector for dengue, Zika, yellow fever, and chikungunya viruses, the mosquito Aedes aegypti is one of the most important insects in public health. These viruses are transmitted by bloodfeeding, which is also necessary for the reproduction of the mosquito. Thus, the midgut plays an essential role in mosquito physiology as the center for bloodmeal digestion and as an organ that serves as the first line of defense against viruses. Despite its importance, transcriptomic dynamics with fine temporal resolution across the entire digestion cycle have not yet been reported. To fill this gap, we conducted a transcriptomic analysis of A. aegypti female midguts across a 72-h bloodmeal digestion cycle for 11 time points, with a particular focus on the first 24 h. PCA analysis confirmed that 72 h is indeed a complete digestion cycle. Cluster and GO enrichment analysis showed the orchestrated modulation of thousands of genes to accomplish the midgut's role as the center for digestion, as well as nutrient transport with a clear progression with sequential emphasis on transcription, translation, energy production, nutrient metabolism, transport, and finally, autophagy by 24-36 h. We further determined that many serine proteases are robustly expressed as if to prepare for unexpected physiological challenges. This study provides a powerful resource for the analysis of genomic features that coordinate the rapid and complex transcriptional program induced by mosquito bloodfeeding.

Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells.

BACKGROUND: Mosquito-specific viruses (MSVs) comprise a variety of different virus families, some of which are known to interfere with infections of medically important arboviruses. Viruses belonging to the family Mesoniviridae or taxon Negevirus harbor several insect-specific viruses, including MSVs, which are known for their wide geographical distribution and extensive host ranges. Although these viruses are regularly identified in mosquitoes all over the world, their presence in mosquitoes in Germany had not yet been reported. METHODS: A mix of three MSVs (Yichang virus [Mesoniviridae] and two negeviruses [Daeseongdong virus and Dezidougou virus]) in a sample that contained a pool of Coquillettidia richiardii mosquitoes collected in Germany was used to investigate the interaction of these viruses with different arboviruses in Culex-derived cells. In addition, small RNA sequencing and analysis of different mosquito-derived cells infected with this MSV mix were performed. RESULTS: A strain of Yichang virus (Mesoniviridae) and two negeviruses (Daeseongdong virus and Dezidougou virus) were identified in the Cq. richiardii mosquitoes sampled in Germany, expanding current knowledge of their circulation in central Europe. Infection of mosquito-derived cells with these three viruses revealed that they are targeted by the small interfering RNA (siRNA) pathway. In Culex-derived cells, co-infection by these three viruses had varying effects on the representative arboviruses from different virus families (Togaviridae: Semliki forest virus [SFV]; Bunyavirales: Bunyamwera orthobunyavirus [BUNV]; or Flaviviridae: Usutu virus [USUV]). Specifically, persistent MSV co-infection inhibited BUNV infection, as well as USUV infection (but the latter only at specific time points). However, the impact on SFV infection was only noticeable at low multiplicity of infection (MOI 0.1) and at specific time points in combination with the infection status. CONCLUSIONS: Taken together, these results are important findings that will lead to a better understanding of the complex interactions of MSVs, mosquitoes and arboviruses.

Convergent trends and spatiotemporal patterns of Aedes-borne arboviruses in Mexico and Central America.

BACKGROUND: Aedes-borne arboviruses cause both seasonal epidemics and emerging outbreaks with a significant impact on global health. These viruses share mosquito vector species, often infecting the same host population within overlapping geographic regions. Thus, comparative analyses of the virus evolutionary and epidemiological dynamics across spatial and temporal scales could reveal convergent trends. METHODOLOGY/PRINCIPAL FINDINGS: Focusing on Mexico as a case study, we generated novel chikungunya and dengue (CHIKV, DENV-1 and DENV-2) virus genomes from an epidemiological surveillance-derived historical sample collection, and analysed them together with longitudinally-collected genome and epidemiological data from the Americas. Aedes-borne arboviruses endemically circulating within the country were found to be introduced multiple times from lineages predominantly sampled from the Caribbean and Central America. For CHIKV, at least thirteen introductions were inferred over a year, with six of these leading to persistent transmission chains. For both DENV-1 and DENV-2, at least seven introductions were inferred over a decade. CONCLUSIONS/SIGNIFICANCE: Our results suggest that CHIKV, DENV-1 and DENV-2 in Mexico share evolutionary and epidemiological trajectories. The southwest region of the country was determined to be the most likely location for viral introductions from abroad, with a subsequent spread into the Pacific coast towards the north of Mexico. Virus diffusion patterns observed across the country are likely driven by multiple factors, including mobility linked to human migration from Central towards North America. Considering Mexico's geographic positioning displaying a high human mobility across borders, our results prompt the need to better understand the role of anthropogenic factors in the transmission dynamics of Aedes-borne arboviruses, particularly linked to land-based human migration.

Jingmen tick virus: an emerging arbovirus with a global threat.

Jingmen tick virus (JMTV), belonging to the Flaviviridae family, is a novel segmented RNA virus identified in 2014 in the Jingmen region of Hubei Province, China. Up to now, JMTV has been detected in a variety of countries or regions in Asia, Europe, Africa, and the Americas, involving a wide range of arthropods and mammals, and even humans. The JMTV genome is composed of four linear RNA segments, two of which are derived from flaviviruses, while the other two segments are unique to JMTV and has no matching virus. Currently, JMTV has been shown to have a pathogenic effect on humans. Humans who had been infected would develop viremia and variable degrees of clinical symptoms. However, the pathogenic mechanism of JMTV has not been elucidated yet. Therefore, it is crucial to strengthen the epidemiological surveillance and laboratory studies of JMTV.

Multifaceted contributions of Dicer2 to arbovirus transmission by Aedes aegypti.

Arthropod-borne viruses (arboviruses) transmitted by Aedes aegypti mosquitoes are an increasing threat to global health. The small interfering RNA (siRNA) pathway is considered the main antiviral immune pathway of insects, but its effective impact on arbovirus transmission is surprisingly poorly understood. Here, we use CRISPR-Cas9-mediated gene editing in vivo to mutate Dicer2, a gene encoding the RNA sensor and key component of the siRNA pathway. The loss of Dicer2 enhances early viral replication and systemic viral dissemination of four medically significant arboviruses (chikungunya, Mayaro, dengue, and Zika viruses) representing two viral families. However, Dicer2 mutants and wild-type mosquitoes display overall similar levels of vector competence. In addition, Dicer2 mutants undergo significant virus-induced mortality during infection with chikungunya virus. Together, our results define a multifaceted role for Dicer2 in the transmission of arboviruses by Ae. aegypti mosquitoes and pave the way for further mechanistic investigations.

Molecular surveillance of arboviruses in Nigeria.

Arboviral infections are fast becoming a global public health concern as a result of its high fatality rate and sporadic spread. From the outbreak of Zika virus in the Americas, the endemicity of Yellow fever in West Africa and South America, outbreaks of West Nile virus in South Africa to the year-round and national risk of Dengue fever in Mainland China and India. The war against emerging and re-emerging viral infection could probably lead to the next pandemic. To be above the pending possible arboviral pandemic, consistent surveillance of these pathogens is necessary in every society. This study was aimed at conducting a surveillance for Yellow fever virus, Zika virus, Chikungunya virus, Dengue virus and Rift Valley fever virus in four states in Nigeria using molecular techniques. A cross-sectional study involving 1600 blood samples collected from febrile patients in Lagos, Kwara, Ondo and Delta States between 2018 and 2021 was conducted using Real time polymerase chain reaction for detection of the pathogens. Extraction and purification of viral RNA were done using Qiagen Viral RNA Mini Kit. Samples were analyzed using One Step PrimeScript III RT-PCR mix (Takara Bio) alongside optimized primers and probes designed in-house. Positive samples were sequenced on MinION platform (Nanopore technologies). Bioinformatic and phylogenetic analysis were performed with DNASTAR Lasergene 17.3. All the RNA extracted from samples collected from the four states were negative for ZIKV RNA, RVFV RNA, CHIKV RNA and DENV RNA. However, twelve of the samples (2%) tested positive for YFV RNA. Three full genomes of sizes 10,751 bp, 10,500 bp and 10,715 bp were generated and deposited in GenBank with accession numbers: ON323052, ON323053 and ON323054 respectively. Phylogenetic analysis shows clustering within lineage 3 of West African genotype. This result shows an active spread of Yellow fever in Delta State, Nigeria. However, there is no emergence of a new genotype There is a need for an intense surveillance of Yellow fever virus in Nigeria to avert a major outbreak.

The Continuous Adaptive Challenge Played by Arboviruses: An In Silico Approach to Identify a Possible Interplay between Conserved Viral RNA Sequences and Host RNA Binding Proteins (RBPs).

Climate change and globalization have raised the risk of vector-borne disease (VBD) introduction and spread in various European nations in recent years. In Italy, viruses carried by tropical vectors have been shown to cause viral encephalitis, one of the symptoms of arboviruses, a spectrum of viral disorders spread by arthropods such as mosquitoes and ticks. Arboviruses are currently causing alarm and attention, and the World Health Organization (WHO) has released recommendations to adopt essential measures, particularly during the hot season, to restrict the spreading of the infectious agents among breeding stocks. In this scenario, rapid analysis systems are required, because they can quickly provide information on potential virus-host interactions, the evolution of the infection, and the onset of disabling clinical symptoms, or serious illnesses. Such systems include bioinformatics approaches integrated with molecular evaluation. Viruses have co-evolved different strategies to transcribe their own genetic material, by changing the host's transcriptional machinery, even in short periods of time. The introduction of genetic alterations, particularly in RNA viruses, results in a continuous adaptive fight against the host's immune system. We propose an in silico pipeline method for performing a comprehensive motif analysis (including motif discovery) on entire genome sequences to uncover viral sequences that may interact with host RNA binding proteins (RBPs) by interrogating the database of known RNA binding proteins, which play important roles in RNA metabolism and biological processes. Indeed, viral RNA sequences, able to bind host RBPs, may compete with cellular RNAs, altering important metabolic processes. Our findings suggest that the proposed in silico approach could be a useful and promising tool to investigate the complex and multiform clinical manifestations of viral encephalitis, and possibly identify altered metabolic pathways as targets of pharmacological treatments and innovative therapeutic protocols.

Presence, Location and Conservation of Putative G-Quadruplex Forming Sequences in Arboviruses Infecting Humans.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17-26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections.

Molecular Detection of Dengue Virus, Zika Virus, and Chikungunya Virus Arboviruses in Neotropical Bats.

Background: The dengue, Zika, and Chikungunya arboviruses have spread in America in the past year, thus becoming global health issues. These viruses are maintained in nature in two transmission cycles: an urban cycle, transmitted from hematophagous mosquitoes to humans, and a wild cycle, recorded only in Africa and Asia, involving mosquitoes and nonhuman primates as natural hosts. The evidence shows that these arboviruses infect other wild mammals in America, such as rodents, marsupials, and bats. This study aimed to determine the potential natural infection of arboviruses in bats captured in contrasting sites (tropical forests, urban areas, and caves) in Oaxaca, Mexico. Materials and Methods: Liver samples were collected from some bats and tested for RNA from dengue, Zika, and Chikungunya with the quantitative real-time PCR assay. We analyzed 162 samples that encompassed 23 bat species. Results: No natural infection with any of the three arboviruses was detected in any sample tested. Conclusion: The existence of a wild cycle of the three arboviruses in the American continent is not ruled out. However, owing to the low or zero prevalence recorded in other studies and the present study, bats are likely involved in the arbovirus transmission cycle as accidental hosts.