Project description:Mosquitoes, most often recognized for the microbial agents of disease they may carry, harbor diverse microbial communities that include viruses, bacteria, and fungi, collectively called the microbiota. The composition of the microbiota can directly and indirectly affect disease transmission through microbial interactions that could be revealed by its characterization in natural populations of mosquitoes. Furthermore, the use of shotgun metagenomic sequencing (SMS) approaches could allow the discovery of unknown members of the microbiota. In this study, we use RNA SMS to characterize the microbiota of seven individual mosquitoes (species include Culex pipiens, Culiseta incidens, and Ochlerotatus sierrensis) collected from a variety of habitats in California, USA. Sequencing was performed on the Illumina HiSeq platform and the resulting sequences were quality-checked and assembled into contigs using the A5 pipeline. Sequences related to single stranded RNA viruses of the Bunyaviridae and Rhabdoviridae were uncovered, along with an unclassified genus of double-stranded RNA viruses. Phylogenetic analysis finds that in all three cases, the closest relatives of the identified viral sequences are other mosquito-associated viruses, suggesting widespread host-group specificity among disparate viral taxa. Interestingly, we identified a Narnavirus of fungi, also reported elsewhere in mosquitoes, that potentially demonstrates a nested host-parasite association between virus, fungi, and mosquito. Sequences related to 8 bacterial families and 13 fungal families were found across the seven samples. Bacillus and Escherichia/Shigella were identified in all samples and Wolbachia was identified in all Cx. pipiens samples, while no single fungal genus was found in more than two samples. This study exemplifies the utility of RNA SMS in the characterization of the natural microbiota of mosquitoes and, in particular, the value of identifying all microbes associated with a specific host.
Project description:Mosquitoes carry a wide variety of viruses that can cause vector-borne infectious diseases and affect both human and veterinary public health. Although Mozambique can be considered a hot spot for emerging infectious diseases due to factors such as a rich vector population and a close vector/human/wildlife interface, the viral flora in mosquitoes have not previously been investigated. In this study, viral metagenomics was employed to analyze the viral communities in Culex and Mansonia mosquitoes in the Zambezia province of Mozambique. Among the 1.7 and 2.6 million sequences produced from the Culex and Mansonia samples, respectively, 3269 and 983 reads were classified as viral sequences. Viruses belonging to the Flaviviridae, Rhabdoviridae and Iflaviridae families were detected, and different unclassified single- and double-stranded RNA viruses were also identified. A near complete genome of a flavivirus, tentatively named Cuacua virus, was obtained from the Mansonia mosquitoes. Phylogenetic analysis of this flavivirus, using the NS5 amino acid sequence, showed that it grouped with 'insect-specific' viruses and was most closely related to Nakiwogo virus previously identified in Uganda. Both mosquito genera had viral sequences related to Rhabdoviruses, and these were most closely related to Culex tritaeniorhynchus rhabdovirus (CTRV). The results from this study suggest that several viruses specific for insects belonging to, for example, the Flaviviridae and Rhabdoviridae families, as well as a number of unclassified RNA viruses, are present in mosquitoes in Mozambique.
Project description:Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated.
Project description:BackgroundMosquito-borne infectious diseases pose a severe threat to public health in many areas of the world. Current methods for pathogen detection and surveillance are usually dependent on prior knowledge of the etiologic agents involved. Hence, efficient approaches are required for screening wild mosquito populations to detect known and unknown pathogens.Methodology/principal findingsIn this study, we explored the use of Next Generation Sequencing to identify viral agents in wild-caught mosquitoes. We extracted total RNA from different mosquito species from South China. Small 18-30 bp length RNA molecules were purified, reverse-transcribed into cDNA and sequenced using Illumina GAIIx instrumentation. Bioinformatic analyses to identify putative viral agents were conducted and the results confirmed by PCR. We identified a non-enveloped single-stranded DNA densovirus in the wild-caught Culex pipiens molestus mosquitoes. The majority of the viral transcripts (.>80% of the region) were covered by the small viral RNAs, with a few peaks of very high coverage obtained. The +/- strand sequence ratio of the small RNAs was approximately 7∶1, indicating that the molecules were mainly derived from the viral RNA transcripts. The small viral RNAs overlapped, enabling contig assembly of the viral genome sequence. We identified some small RNAs in the reverse repeat regions of the viral 5'- and 3' -untranslated regions where no transcripts were expected.Conclusions/significanceOur results demonstrate for the first time that high throughput sequencing of small RNA is feasible for identifying viral agents in wild-caught mosquitoes. Our results show that it is possible to detect DNA viruses by sequencing the small RNAs obtained from insects, although the underlying mechanism of small viral RNA biogenesis is unclear. Our data and those of other researchers show that high throughput small RNA sequencing can be used for pathogen surveillance in wild mosquito vectors.
Project description:There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.
Project description:Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O'nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.
Project description:Viruses are ubiquitous and diverse microorganisms arising as a result of interactions within their vertebrate and invertebrate hosts. Here we report the presence of different viruses in the salivary glands of 1657 mosquitoes classified over 28 culicinae species from the North region of the Brazilian Pantanal wetland through metagenomics, viral isolation, and RT-PCR. In total, 12 viruses were found, eight putative novel viruses with relatively low similarity with pre-existing species of viruses within their families, named Pirizal iflavirus, Furrundu phlebovirus, Pixé phlebovirus, Guampa vesiculovirus, Chacororé flavivirus, Rasqueado orbivirus, Uru chuvirus, and Bororo circovirus. We also found the already described Lobeira dielmorhabdovirus, Sabethes flavivirus, Araticum partitivirus, and Murici totivirus. Therefore, these findings underscore the vast diversity of culicinae and novel viruses yet to be explored in Pantanal, the largest wetland on the planet.
Project description:RNA viruses are a leading cause of human infectious diseases and the prediction of where new RNA viruses are likely to be discovered is a significant public health concern. Here, we geocoded the first peer-reviewed reports of 223 human RNA viruses. Using a boosted regression tree model, we matched these virus data with 33 explanatory factors related to natural virus distribution and research effort to predict the probability of virus discovery across the globe in 2010-2019. Stratified analyses by virus transmissibility and transmission mode were also performed. The historical discovery of human RNA viruses has been concentrated in eastern North America, Europe, central Africa, eastern Australia, and north-eastern South America. The virus discovery can be predicted by a combination of socio-economic, land use, climate, and biodiversity variables. Remarkably, vector-borne viruses and strictly zoonotic viruses are more associated with climate and biodiversity whereas non-vector-borne viruses and human transmissible viruses are more associated with GDP and urbanization. The areas with the highest predicted probability for 2010-2019 include three new regions including East and Southeast Asia, India, and Central America, which likely reflect both increasing surveillance and diversity of their virome. Our findings can inform priority regions for investment in surveillance systems for new human RNA viruses.
Project description:We collected 8,700 mosquitoes in three sites in China, which belonged to seven species. Their viromes were tested using metagenomic sequencing and bioinformatic analysis. The abundant viral sequences were detected and annotated belonging to more than 50 viral taxonomic families. The results were verified by PCR, followed by phylogenetic analysis. In the present study, we identified partial viral genes of dengue virus (DENV), a novel circovirus (CCV), densovirus (DNV), Japanese encephalitis virus (JEV), and Wuhan mosquito virus (WMV) in mosquitoes. Metagenomic analysis and PCR amplification revealed three DENV sequences, which were as homologous to the NS3 gene of DENV from Singapore isolated in 2005, with at least 91% nucleotide (nt) identity. Seven fragments of JEV encoding structural proteins were identified belonging to genotype I. They all shared high homology with structural protein genes of JEV isolated from Laos in 2009. The production of infectious virus particles of the newly isolated virus YunnanJEV2017-4 increased after passage from the BHK-21 cell line to the Vero cell line. Novel circovirus-related genes were identified and as being related to an unnamed gene of a mosquito circovirus (MCCV) sequence from the USA isolated in 2011, with at least 41% nt identity: this distant relationship suggests that the parent virus might belong to a novel circovirus genus. Additionally, numerous known viruses and some unknown viruses were also detected in mosquitoes from Yunnan province, China, which will be tested for propagation.