Mosquitoes are the most important vectors of emerging infectious diseases. During the past decade, our understanding of the diversity of viruses they carry has greatly expanded. Most of these viruses are considered mosquito-specific but there is increasing evidence that these viruses may affect the vector competence of mosquitoes. Metagenomics approaches have focused on specific mosquito species for the identification of what is called the core virome. Despite the fact that, in most ecosystems, multiple species may participate in virus emergence and circulation, there is a lack of understanding of the virus-carrier/host network for both vector-borne and mosquito-specific viruses. Here, we studied the core virome of mosquitoes in a diverse local ecosystem that had 24 different mosquito species. Analysis of the viromes of these 24 mosquito species resulted in the identification of 34 viruses, which included 15 novel viruses, as determined according to the species demarcation criteria of the respective virus families. Most of the mosquito species had never been analysed previously and a comparison of the individual viromes of the 24 mosquito species revealed novel relationships among mosquito species and virus families. Groups of related viruses and mosquito species from multiple genera formed a complex web in the local ecosystem. Furthermore, analyses of the virome of mixed-species pools of mosquitoes from representative traps of the local ecosystem showed almost complete overlap with the individual-species viromes identified in the study. Quantitative analysis of viruses’ relative abundance revealed a linear relationship to the abundance of the respective carrier/host mosquito species, supporting the theory of a stable core virome in the most abundant species of the local ecosystem. Finally, our study highlights the importance of using a holistic approach to investigating mosquito viromes relationships in rich and diverse ecosystems.

BackgroundMosquitoes are the most important vectors of emerging infectious diseases. During the past decade, our understanding of the diversity of viruses they carry has greatly expanded. Most of these viruses are considered mosquito-specific, while there is increasing evidence that these viruses may affect mosquito vector potential. Metagenomics approaches have focused on specific mosquito species for the identification of what is called core virome. However, in most ecosystems, multiple species may participate in virus emergence and circulation, while there is lack of understanding on the viruses-carrier/host network for both vector-borne and mosquito-specific viruses. Results Here, we studied the core mosquito virome in a diverse ecosystem comprised of 24 different mosquito species. Analysis of these 24 diverse viromes resulted in the discovery of 35 viruses with known genetic traits and 9 novel viruses. Comparison of the viromes of the 24 individual species revealed novel relationships between mosquito species and virus families, as most of the mosquito species had never been analysed in the past. Groups of related viruses and mosquito species from multiple genera formed a complex network in the ecosystem. Analyses of whole traps of mosquitoes of variable composition not only showed a stable core virome for each species but also a relationship between mosquito population and virome composition.ConclusionsOur study highlighted the importance of a holistic approach regarding mosquito viromes in rich and diverse ecosystems. Our data further supported the idea of a stable core virome, characteristic of each mosquito species. The remarkable stability of the core virome seemed to determine the composition of the total mosquito core virome of a habitat in the ecosystem.

Riboswitches are structured non-coding RNAs found in the 5′ UTR of important genes for bacterial metabolism, virulence and survival. Upon the binding of specific ligands that can vary from simple ions to complex molecules such as nucleotides and tRNAs, riboswitches change their local and global mRNA conformations to affect downstream transcription or translation. Due to their dynamic nature and central regulatory role in bacterial metabolism, riboswitches have been exploited as novel RNA-based targets for the development of new generation antibacterials that can overcome drug-resistance problems. During recent years, several important riboswitch structures from many bacterial representatives, including several prominent human pathogens, have shown that riboswitches are ideal RNA targets for new compounds that can interfere with their structure and function, exhibiting much reduced resistance over time. Most interestingly, mainstream antibiotics that target the ribosome have been shown to effectively modulate the regulatory behavior and capacity of several riboswitches, both in vivo and in vitro, emphasizing the need for more in-depth studies and biological evaluation of new antibiotics. Herein, we summarize the currently known compounds that target several main riboswitches and discuss the role of mainstream antibiotics as modulators of T-box riboswitches, in the dawn of an era of novel inhibitors that target important bacterial regulatory RNAs.

T-box riboswitches (T-boxes) are essential RNA regulatory elements with a remarkable structural diversity, especially among bacterial pathogens. In staphylococci, all glyS T-boxes synchronize glycine supply during synthesis of nascent polypeptides and cell wall formation and are characterized by a conserved and unique insertion in their antiterminator/terminator domain, termed stem Sa. Interestingly, in Staphylococcus aureus the stem Sa can accommodate binding of specific antibiotics, which in turn induce robust and diverse effects on T-box-mediated transcription. In the present study, domain swap mutagenesis and probing analysis were performed to decipher the role of stem Sa. Deletion of stem Sa significantly reduces both the S. aureus glyS T-box-mediated transcription readthrough levels and the ability to discriminate among tRNAGly isoacceptors, both in vitro and in vivo. Moreover, the deletion inverted the previously reported stimulatory effects of specific antibiotics. Interestingly, stem Sa insertion in the terminator/antiterminator domain of Geobacillus kaustophilus glyS T-box, which lacks this domain, resulted in elevated transcription in the presence of tigecycline and facilitated discrimination among proteinogenic and nonproteinogenic tRNAGly isoacceptors. Overall, stem Sa represents a lineage-specific structural feature required for efficient staphylococcal glyS T-box-mediated transcription and it could serve as a species-selective druggable target through its ability to modulate antibiotic binding.

Viral metagenomics, also known as virome studies, have yielded an unprecedented number of novel sequences, essential in recognizing and characterizing the etiological agent and the origin of emerging infectious diseases. Several tools and pipelines have been developed, to date, for the identification and assembly of viral genomes. Assembly pipelines often result in viral genomes contaminated with host genetic material, some of which are currently deposited into public databases. In the current report, we present a group of deposited sequences that encompass ribosomal RNA (rRNA) contamination. We highlight the detrimental role of chimeric next generation sequencing reads, between host rRNA sequences and viral sequences, in virus genome assembly and we present the hindrances these reads may pose to current methodologies. We have further developed a refining pipeline, the Zero Waste Algorithm (ZWA) that assists in the assembly of low abundance viral genomes. ZWA performs context-depended trimming of chimeric reads, precisely removing their rRNA moiety. These, otherwise discarded, reads were fed to the assembly pipeline and assisted in the construction of larger and cleaner contigs making a substantial impact on current assembly methodologies. ZWA pipeline may significantly enhance virus genome assembly from low abundance samples and virus metagenomics approaches in which a small number of reads determine genome quality and integrity.