Complex Virome Research Articles

Insights into the RNA Virome of the Corn Leafhopper Dalbulus maidis, a Major Emergent Threat of Maize in Latin America

The maize leafhopper (Dalbulus maidis) is a significant threat to maize crops in tropical and subtropical regions, causing extensive economic losses. While its ecological interactions and control strategies are well studied, its associated viral diversity remains largely unexplored. Here, we employ high-throughput sequencing data mining to comprehensively characterize the D. maidis RNA virome, revealing novel and diverse RNA viruses. We characterized six new viral members belonging to distinct families, with evolutionary cues of beny-like viruses (Benyviridae), bunya-like viruses (Bunyaviridae) iflaviruses (Iflaviridae), orthomyxo-like viruses (Orthomyxoviridae), and rhabdoviruses (Rhabdoviridae). Phylogenetic analysis of the iflaviruses places them within the genus Iflavirus in affinity with other leafhopper-associated iflaviruses. The five-segmented and highly divergent orthomyxo-like virus showed a relationship with other insect associated orthomyxo-like viruses. The rhabdo virus is related to a leafhopper-associated rhabdo-like virus. Furthermore, the beny-like virus belonged to a cluster of insect-associated beny-like viruses, while the bi-segmented bunya-like virus was related with other bi-segmented insect-associated bunya-like viruses. These results highlight the existence of a complex virome linked to D. maidis and paves the way for future studies investigating the ecological roles, evolutionary dynamics, and potential biocontrol applications of these viruses on the D. maidis—maize pathosystem.

Characterization of Two Novel Viruses Within a Complex Virome from Flowering Ginger in Hawaii.

Flowering ginger (Alpinia purpurata) is economically and culturally important in Hawaii. In the past decade, a slow decline syndrome has impacted the production of this crop in the state. RNA sequencing analyses and virus indexing surveys were done on samples collected from four of the Hawaiian Islands. Viral sequences corresponding to six viruses were recovered from transcriptomic data from samples with virus-like symptoms. Canna yellow mottle virus (CaYMV, genus Badnavirus) and two novel viruses, Alpinia vein clearing virus (ApVCV, genus Ampelovirus) and Alpinia vein streaking virus (ApVSV, genus Betanucleorhabdovirus), were found at a moderate incidence in diseased plants. Conversely, three other viruses, including the two potyviruses, banana bract mosaic virus and bean common mosaic virus, and a badnavirus, banana streak GF virus, were also found but at a low incidence. Virus detection in potential insect vectors and transmission assays identified the mealybug Planococcus citri as a vector of CaYMV and ApVCV, whereas the aphid Pentalonia caladii was identified as a vector of the novel ApVSV. Both P. citri and P. caladii are common pests of flowering ginger in Hawaii. Transmission of ApVSV was achieved using P. caladii colonies either established in the laboratory or naturally feeding on infected plants, although no transmission was obtained using viruliferous aphids originally reared on taro (Colocasia esculenta). Our study provides insights into the potential association between viral infections and the observed decline symptoms of flowering ginger in Hawaii. However, more definitive studies are needed to link single or mixed viral infections with decline symptoms.

The Virome of Cocoa Fermentation-Associated Microorganisms.

Theobroma cacao plantations are of significant economic importance worldwide, primarily for chocolate production. During the harvest and processing of cocoa beans, they are subjected to fermentation either by microorganisms present in the environment (spontaneous fermentation) or the addition of starter cultures, with different strains directly contributing distinct flavor and color characteristics to the beans. In addition to fungi and bacteria, viruses are ubiquitous and can affect the quality of the fermentation process by infecting fermenting organisms, destabilizing microbial diversity, and consequently affecting fermentation quality. Therefore, in this study, we explored publicly available metatranscriptomic libraries of cocoa bean fermentation in Limon Province, Costa Rica, looking for viruses associated with fermenting microorganisms. Libraries were derived from the same sample at different time points: 7, 20, and 68 h of fermentation, corresponding to yeast- and lactic acid bacteria-driven phases. Using a comprehensive pipeline, we identified 68 viral sequences that could be assigned to 62 new viral species and 6 known viruses distributed among at least nine families, with particular abundance of elements from the Lenarviricota phylum. Interestingly, 44 of these sequences were specifically associated with ssRNA phages (Fiersviridae) and mostly fungi-infecting viral families (Botourmiaviridae, Narnaviridae, and Mitoviridae). Of note, viruses from those families show a complex evolutionary relationship, transitioning from infecting bacteria to infecting fungi. We also identified 10 and 3 viruses classified within the Totiviridae and Nodaviridae families, respectively. The quantification of the virus-derived RNAs shows a general pattern of decline, similar to the dynamic profile of some microorganism genera during the fermentation process. Unexpectedly, we identified narnavirus-related elements that showed similarity to segmented viral species. By exploring the molecular characteristics of these viral sequences and applying Hidden Markov Models, we were capable of associating these additional segments with a specific taxon. In summary, our study elucidates the complex virome associated with the microbial consortia engaged in cocoa bean fermentation that could contribute to organism/strain selection, altering metabolite production and, consequently, affecting the sensory characteristics of cocoa beans.

Viromes of Antarctic fish resemble the diversity found at lower latitudes.

Antarctica harbours some of the most isolated and extreme environments on Earth, concealing a largely unexplored and unique component of the global animal virosphere. To understand the diversity and evolutionary histories of viruses in these polar species, we determined the viromes of gill metatranscriptomes from 11 Antarctic fish species with 248 samples collected from the Ross Sea region spanning the Perciformes, Gadiformes, and Scorpaeniformes orders. The continent's shift southward and cooling temperatures >20 million years ago led to a reduction in biodiversity and subsequent radiation of some marine fauna, such as the notothenioid fishes. Despite decreased host species richness in polar regions, we revealed a surprisingly complex virome diversity in Ross Sea fish, with the types and numbers of viruses per host species and individuals sampled comparable to that of fish in warmer marine environments with higher host community diversity. We also observed a higher number of closely related viruses likely representing instances of recent and historic host-switching events among the Perciformes (all notothenioids) than in the Gadiformes, suggesting that rapid speciation events within this order generated closely related host species with few genetic barriers to cross-species transmission. Additionally, we identified novel genomic variation in an arenavirus with a split nucleoprotein sequence containing a stable helical structure, indicating potential adaptation of viral proteins to extreme temperatures. These findings enhance our understanding of virus evolution and virus-host interactions in response to environmental shifts, especially in less diverse ecosystems that are more vulnerable to the impacts of anthropogenic and climate changes.

Meta-Transcriptomic Analysis Uncovers the Presence of Four Novel Viruses and Multiple Known Virus Genera in a Single Hibiscus rosa-sinensis Plant in Colombia.

Hibiscus is not native to Colombia but well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of its leaves, was collected for virome analysis using meta-transcriptomic high-throughput sequencing technology. Bioinformatic analysis identified 12.5% of the total reads in the Ribo-Zero cDNA library which mapped to viral genomes. BLAST searches revealed the presence of carlavirus, potexvirus, and of known members of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus in the sample; confirmed by RT-PCR with virus-specific primers followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the mixed infection. Both RNA dependent RNA polymerase and coat protein gene sequences of the potex and carla viruses shared less than 72% nucleotide and 80% amino acid identities with any alphaflexi- and betaflexi-virus sequences available in GenBank, identifying three novel carlavirus and one potexvirus species in the Hibiscus rosa-sinensis plant. The detection of physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus are also new reports from Colombia. Overall, the meta-transcriptome analysis identified the complex virome associated with the black spot symptoms on hibiscus leaves and demonstrated the diversity of virus genera tolerated in the mixed infection of a single H. rosa-sinensis plant.

Carrot populations in France and Spain host a complex virome rich in previously uncharacterized viruses.

High-throughput sequencing (HTS) has proven a powerful tool to uncover the virome of cultivated and wild plants and offers the opportunity to study virus movements across the agroecological interface. The carrot model consisting of cultivated (Daucus carota ssp. sativus) and wild carrot (Daucus carota ssp. carota) populations, is particularly interesting with respect to comparisons of virus communities due to the low genetic barrier to virus flow since both population types belong to the same plant species. Using a highly purified double-stranded RNA-based HTS approach, we analyzed on a large scale the virome of 45 carrot populations including cultivated, wild and off-type carrots (carrots growing within the field and likely representing hybrids between cultivated and wild carrots) in France and six additional carrot populations from central Spain. Globally, we identified a very rich virome comprising 45 viruses of which 25 are novel or tentatively novel. Most of the identified novel viruses showed preferential associations with wild carrots, either occurring exclusively in wild populations or infecting only a small proportion of cultivated populations, indicating the role of wild carrots as reservoir of viral diversity. The carrot virome proved particularly rich in viruses involved in complex mutual interdependencies for aphid transmission such as poleroviruses, umbraviruses and associated satellites, which can be the basis for further investigations of synergistic or antagonistic virus-vector-host relationships.

A collection ofTrichoderma isolates from natural environments inSardinia reveals a complex virome that includes negative-sense fungal viruses with unprecedented genome organizations.

Trichoderma genus includes soil-inhabiting fungi that provide important ecosystem services in their interaction with plants and other fungi, as well as biocontrol of fungal plant diseases. A collection of Trichoderma isolates from Sardinia has been previously characterized, but here we selected 113 isolates, representatives of the collection, and characterized their viral components. We carried out high-throughput sequencing of ribosome-depleted total RNA following a bioinformatics pipeline that detects virus-derived RNA-directed RNA polymerases (RdRps) and other conserved viral protein sequences. This pipeline detected seventeen viral RdRps with two of them corresponding to viruses already detected in other regions of the world and the remaining fifteen representing isolates of new putative virus species. Surprisingly, eight of them are from new negative-sense RNA viruses, a first in the genus Trichoderma. Among them is a cogu-like virus, closely related to plant-infecting viruses. Regarding the positive-sense viruses, we report the presence of an 'ormycovirus' belonging to a recently characterized group of bisegmented single-stranded RNA viruses with uncertain phylogenetic assignment. Finally, for the first time, we report a bisegmented member of Mononegavirales which infects fungi. The proteins encoded by the second genomic RNA of this virus were used to re-evaluate several viruses in the Penicillimonavirus and Plasmopamonavirus genera, here shown to be bisegmented and encoding a conserved polypeptide that has structural conservation with the nucleocapsid domain of rhabdoviruses.

Determinants of Chikungunya and O'nyong-Nyong Virus Specificity for Infection of Aedes and Anopheles Mosquito Vectors.

Mosquito-borne diseases caused by viruses and parasites are responsible for more than 700 million infections each year. Anopheles and Aedes are the two major vectors for, respectively, malaria and arboviruses. Anopheles mosquitoes are the primary vector of just one known arbovirus, the alphavirus o'nyong-nyong virus (ONNV), which is closely related to the chikungunya virus (CHIKV), vectored by Aedes mosquitoes. However, Anopheles harbor a complex natural virome of RNA viruses, and a number of pathogenic arboviruses have been isolated from Anopheles mosquitoes in nature. CHIKV and ONNV are in the same antigenic group, the Semliki Forest virus complex, are difficult to distinguish via immunodiagnostic assay, and symptomatically cause essentially the same human disease. The major difference between the arboviruses appears to be their differential use of mosquito vectors. The mechanisms governing this vector specificity are poorly understood. Here, we summarize intrinsic and extrinsic factors that could be associated with vector specificity by these viruses. We highlight the complexity and multifactorial aspect of vectorial specificity of the two alphaviruses, and evaluate the level of risk of vector shift by ONNV or CHIKV.

Uncovering a Complex Virome Associated with the Cacao Pathogens Ceratocystis cacaofunesta and Ceratocystis fimbriata.

Theobroma cacao is one of the main crops of economic importance in the world as the source of raw material for producing chocolate and derivatives. The crop is the main source of income for thousands of small farmers, who produce more than 80% of the world's cocoa supply. However, the emergence, re-emergence and proliferation of pathogens, such as Ceratocystis spp., the causative agent of Ceratocystis wilt disease and canker disease, have been affecting the sustainability of many crops. Fungal control is laborious, often depending on fungicides that are expensive and/or toxic to humans, prompting researchers to look for new solutions to counteract the proliferation of these pathogens, including the use of biological agents such as mycoviruses. In this study, we investigated the diversity of microorganisms associated with the T. cacao pathogens Ceratocystis cacaofunesta and Ceratocystis fimbriata with a focus on the virome using RNA sequencing data available in public databases. We used a comprehensive bioinformatics pipeline containing several steps for viral sequence enrichment and took advantage of an integrated assembly step composed of different assemblers followed by sequence similarity searches using NCBI nonredundant databases. Our strategy was able to identify four putative C. cacaofunesta viruses (hypovirus, sclerotimonavirus, alphapartitivirus and narnavirus) and six C. fimbriata viruses (three alphaendornaviruses, one victorivirus and two mitoviruses). All the viral sequences identified showed similarity to viral genomes in public databases only at the amino acid level, likely representing new viral species. Of note, we present the first report of viruses associated with the cacao pathogens C. cacaofunesta and C. fimbriata and the second report of viral species infecting members of the Ceratocystidaceae family. Our findings highlight the need for further prospective studies to uncover the real diversity of fungus-infecting viruses that can contribute to the development of new management strategies.

A catalogue of 1,167 genomes from the human gut archaeome

The human gut microbiome plays an important role in health, but its archaeal diversity remains largely unexplored. In the present study, we report the analysis of 1,167 nonredundant archaeal genomes (608 high-quality genomes) recovered from human gastrointestinal tract, sampled across 24 countries and rural and urban populations. We identified previously undescribed taxa including 3 genera, 15 species and 52 strains. Based on distinct genomic features, we justify the split of the Methanobrevibacter smithii clade into two separate species, with one represented by the previously undescribed ‘CandidatusMethanobrevibacter intestini’. Patterns derived from 28,581 protein clusters showed significant associations with sociodemographic characteristics such as age groups and lifestyle. We additionally show that archaea are characterized by specific genomic and functional adaptations to the host and carry a complex virome. Our work expands our current understanding of the human archaeome and provides a large genome catalogue for future analyses to decipher its impact on human physiology.

ViromeBrowser: A Shiny App for Browsing Virome Sequencing Analysis Results.

Experiments in which complex virome sequencing data is generated remain difficult to explore and unpack for scientists without a background in data science. The processing of raw sequencing data by high throughput sequencing workflows usually results in contigs in FASTA format coupled to an annotation file linking the contigs to a reference sequence or taxonomic identifier. The next step is to compare the virome of different samples based on the metadata of the experimental setup and extract sequences of interest that can be used in subsequent analyses. The viromeBrowser is an application written in the opensource R shiny framework that was developed in collaboration with end-users and is focused on three common data analysis steps. First, the application allows interactive filtering of annotations by default or custom quality thresholds. Next, multiple samples can be visualized to facilitate comparison of contig annotations based on sample specific metadata values. Last, the application makes it easy for users to extract sequences of interest in FASTA format. With the interactive features in the viromeBrowser we aim to enable scientists without a data science background to compare and extract annotation data and sequences from virome sequencing analysis results.

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were winter hardy and largely dominated by interspecific hybrid Vitis sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish V. vinifera L. cultivars (e.g., Pinot noir). We characterized the virome of leafroll-infected interspecific hybrid cultivar and compared it to the virome of V. vinifera cultivar to support and facilitate the transition of the industry. A dsRNA sequencing method was used to sequence symptomatic and asymptomatic grapevine leaves of different cultivars. The results suggested a complex virome in terms of composition, abundance, richness, and phylogenetic diversity. Three viruses, grapevine Rupestris stem pitting-associated virus, grapevine leafroll-associated virus (GLRaV) 3 and 2 and hop stunt viroid (HSVd) largely dominated the virome. However, their presence and abundance varied among grapevine cultivars. The symptomless grapevine cultivar Vidal was frequently infected by multiple virus and viroid species and different strains of the same virus, including GLRaV-3 and 2. Our data show that viruses and viroids associated with the highest number of grapevines expressing symptoms included HSVd, GLRaV-3 and GLRaV-2, in gradient order. However, co-occurrence analysis revealed that the presence of GLRaV species was randomly associated with the development of virus-like symptoms. These findings and their implications for grapevine leafroll disease management are discussed.

The LUCA and its complex virome.

The last universal cellular ancestor (LUCA) is the most recent population of organisms from which all cellular life on Earth descends. The reconstruction of the genome and phenotype of the LUCA is a major challenge in evolutionary biology. Given that all life forms are associated with viruses and/or other mobile genetic elements, there is no doubt that the LUCA was a host to viruses. Here, by projecting back in time using the extant distribution of viruses across the two primary domains of life, bacteria and archaea, and tracing the evolutionary histories of some key virus genes, we attempt a reconstruction of the LUCA virome. Even a conservative version of this reconstruction suggests a remarkably complex virome that already included the main groups of extant viruses of bacteria and archaea. We further present evidence of extensive virus evolution antedating the LUCA. The presence of a highly complex virome implies the substantial genomic and pan-genomic complexity of the LUCA itself.

A complex virome including two distinct emaraviruses associated with virus-like symptoms in Camellia japonica

Camellia japonica plants manifesting a complex and variable spectrum of viral symptoms like chlorotic ringspots, necrotic rings, yellowing with necrotic rings, yellow mottle, leaves and petals deformations, and flower color-breaking have been studied since 1940, mainly by electron microscopic analyses; however, a strong correlation between the symptoms and one or more well-characterized viruses was never verified. In this work, samples collected from symptomatic plants were analyzed using the next-generation sequencing technique, and a complex virome composed of members of the Betaflexiviridae and Fimoviridae families was identified. In particular, the genomic fragments typical of the emaravirus group were organized in the genomes of two new emaraviruses species, tentatively named Camellia japonica–associated emaravirus 1 and 2. They are the first emaraviruses described in camellia plants and found in symptomatic plants. At the same time, in both symptomatic and asymptomatic plants, five betaflexivirus isolates were detected that, based on amino acid sequence comparisons, can be considered two new isolates of the recently characterized camellia ringspot–associated virus 1 and 2 (CRSaV-1/2). These recently identified betaflexiviruses associated with C. japonica disease show an unusual hyper-conservation of the coat protein at the amino acid level.The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are MN385581, MN532567, MN532565, MN385582, MN532566, MN385573, MN385577, MN385574, MN385578, MN385575, MN385579, MN385576, MN385580, MN557024, MN557025, MN557026, MN557027, and MN557028.

A Comprehensive Analysis of the Global Human Gut Archaeome from a Thousand Genome Catalogue

The human gut microbiome plays an important role in health and disease, but the archaeal diversity therein remains largely unexplored. Here we report the pioneering analysis of 1167 non-redundant archaeal genomes recovered from diverse human gastrointestinal tract microbiomes. We identified 15 novel species including 52 previously unknown archaeal strains. Based on distinct genomic features, we warrant the split of the Methanobrevibacter smithii clade into two separate species. Patterns derived from 1.8 million proteins coded in these genomes showed substantial correlation with socio-demographic characteristics such as age and lifestyle. We infer that archaea are actively replicating in the human gut and are characterized by specific genomic and functional adaptations to the host. The archaeome carries a complex virome, with some species showing unexpected host flexibility. Our work furthers our current understanding of the human archaeome, and provides a large genome catalogue for future analyses to decipher its impact on human physiology.

Virome heterogeneity and connectivity in waterfowl and shorebird communities

Models of host-microbe dynamics typically assume a single-host population infected by a single pathogen. In reality, many hosts form multi-species aggregations and may be infected with an assemblage of pathogens. We used a meta-transcriptomic approach to characterize the viromes of nine avian species in the Anseriformes (ducks) and Charadriiformes (shorebirds). This revealed the presence of 27 viral species, of which 24 were novel, including double-stranded RNA viruses (Picobirnaviridae and Reoviridae), single-stranded RNA viruses (Astroviridae, Caliciviridae, Picornaviridae), a retro-transcribing DNA virus (Hepadnaviridae), and a single-stranded DNA virus (Parvoviridae). These viruses comprise multi-host generalist viruses and those that are host-specific, indicative of both virome connectivity (host sharing) and heterogeneity (host specificity). Virome connectivity was apparent in two well described multi-host virus species -avian coronavirus and influenza A virus- and a novel Rotavirus species that were shared among some Anseriform species, while virome heterogeneity was reflected in the absence of viruses shared between Anseriformes and Charadriiformes, as well as differences in viral abundance and alpha diversity among species. Overall, we demonstrate complex virome structures across host species that co-exist in multi-species aggregations.

Metagenomic Analysis of Riesling Grapevine Reveals a Complex Virome Including Two New and Divergent Variants of Grapevine leafroll-associated virus 3.

The virome of a major white wine grape of cultivar Riesling showing decline and leafroll disease symptoms was analyzed through high-throughput sequencing (HTS) using total RNAs as templates and the Illumina HiSeq 2500 platform. Analysis of HTS data revealed the presence of five viruses and three viroids in the infected vine. These viruses are Grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-3 (genus Ampelovirus, family Closteroviridae) and three viruses of the family Betaflexiviridae (namely, Grapevine virus A [GVA], Grapevine virus B, and Grapevine rupestris stem pitting-associated virus [GRSPaV]). We also show that multiple distinct strains of three viruses (GLRaV-3, GVA, and GRSPaV) were present in this diseased grapevine. The complete genomes of two novel and highly divergent isolates of GLRaV-3 were determined using the draft genomes derived from HTS data and two independent rapid amplification of cDNA ends (RACE) strategies to obtain sequences at both the 5' and the 3' termini of the viral genomes. Questionable genome regions of both isolates were also verified through cloning of reverse transcription polymerase chain reaction products and Sanger sequencing. These two isolates are vastly divergent from all other isolates of GLRaV-3 whose genome sequences are available in GenBank. Isolate ON8415A has up to 76% nucleotide sequence identities to other isolates representing existing variant groups. We also revealed high degrees of variation in both length and sequence in the terminal untranslated regions (UTRs) of GLRaV-3 variants. The 5'-UTR of most GLRaV-3 isolates whose complete genomes have been sequenced contain tandem repeats of 65 nucleotides, a highly unusual feature rarely observed in (+)single-stranded RNA viruses. Mechanisms for the biogenesis of these tandem repeats and their function in virus replication and pathogenesis require investigation. Findings of this research add to the genetic diversity, evolutionary biology, and diagnostics of GLRaV-3 that afflicts the global grape wine industry.

Complex virome in feces\xa0from Amerindian children\xa0in isolated Amazonian villages

The number of viruses circulating in small isolated human populations may be reduced by viral extinctions and rare introductions. Here we used viral metagenomics to characterize the eukaryotic virome in feces from healthy children from a large urban center and from three Amerindian villages with minimal outside contact. Numerous human enteric viruses, mainly from the Picornaviridae and Caliciviridae families, were sequenced from each of the sites. Multiple children from the same villages shed closely related viruses reflecting frequent transmission clusters. Feces of isolated villagers also contained multiple viral genomes of unknown cellular origin from the Picornavirales order and CRESS-DNA group and higher levels of nematode and protozoan DNA. Despite cultural and geographic isolation, the diversity of enteric human viruses was therefore not reduced in these Amazonian villages. Frequent viral introductions and/or increased susceptibility to enteric infections may account for the complex fecal virome of Amerindian children in isolated villages.

A complex virome unveiled by deep sequencing analysis of RNAs from a French Pinot Noir grapevine exhibiting strong leafroll symptoms.

We have characterized the virome of a grapevine Pinot Noir accession (P70) that displayed, over the year, very stable and strong leafroll symptoms. For this, we have used two extraction methods (dsRNA and total RNA) coupled with the high throughput sequencing (HTS) Illumina technique. While a great disparity in viral sequences were observed, both approaches gave similar results, revealing a very complex infection status. Five virus and viroid isolates [Grapevine leafroll-associated viruse-1 (GLRaV-1), Grapevine virus A (GVA), Grapevine rupestris stem pitting-associated virus (GRSPaV), Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1)] were detected in P70 with a grand total of eleven variants being identified and de novo assembled. A comparison between both extraction methods regarding their power to detect viruses and the ease of genome assembly is also provided.

Synonymous Co-Variation across the E1/E2 Gene Junction of Hepatitis C Virus Defines Virion Fitness.

Hepatitis C virus is a positive-sense single-stranded RNA virus. The gene junction partitioning the viral glycoproteins E1 and E2 displays concurrent sequence evolution with the 3′-end of E1 highly conserved and the 5′-end of E2 highly heterogeneous. This gene junction is also believed to contain structured RNA elements, with a growing body of evidence suggesting that such structures can act as an additional level of viral replication and transcriptional control. We have previously used ultradeep pyrosequencing to analyze an amplicon library spanning the E1/E2 gene junction from a treatment naïve patient where samples were collected over 10 years of chronic HCV infection. During this timeframe maintenance of an in-frame insertion, recombination and humoral immune targeting of discrete virus sub-populations was reported. In the current study, we present evidence of epistatic evolution across the E1/E2 gene junction and observe the development of co-varying networks of codons set against a background of a complex virome with periodic shifts in population dominance. Overtime, the number of codons actively mutating decreases for all virus groupings. We identify strong synonymous co-variation between codon sites in a group of sequences harbouring a 3 bp in-frame insertion and propose that synonymous mutation acts to stabilize the RNA structural backbone.