Virome Diversity among Mosquito Populations in a Sub-Urban Region of Marseille,France

Some mosquito species have significant public health importance given their ability to transmit major diseases to humans and animals, making them the deadliest animals in the world. Among these, the Aedes (Ae.) genus is a vector of several viruses such as Dengue, Chikungunya, and Zika viruses that can cause serious pathologies in humans. Since 2004, Ae. albopictus has been encountered in the South of France, and autochthonous cases of Dengue, Chikungunya, and Zika diseases have recently been reported, further highlighting the need for a comprehensive survey of the mosquitoes and their associated viruses in this area. Using high throughput sequencing (HTS) techniques, we report an analysis of the DNA and RNA viral communities of three mosquito species Ae. albopictus, Culex (Cx.) pipiens, and Culiseta (Cs.) longiareolata vectors of human infectious diseases in a small sub-urban city in the South of France. Results revealed the presence of a significant diversity of viruses known to infect bacteria, plants, insects, and mammals. Several novel viruses were detected, including novel members of the Rhabdoviridae, Totiviridae, Iflaviviridae, Circoviridae, and Sobemoviridae families. No sequence related to major zoonotic viruses transmitted by mosquitoes was detected. The use of HTS on arthropod vector populations is a promising strategy for monitoring the emergence and circulation of zoonoses and epizooties. This study is a contribution to the knowledge of the mosquito microbiome.     

The Myotis chiloensis Guano Virome: Viral Nucleic Acid Enrichments for High-Resolution Virome Elucidation and Full Alphacoronavirus Genome Assembly

Bats are widespread mammals of the order Chiroptera. They are key for ecosystem functioning, participating in crucial processes. Their unique ability amongst mammals to fly long distances, their frequently large population sizes, and their longevity favor infectious agent persistence and spread. This includes a large variety of viruses, encompassing many important zoonotic ones that cause severe diseases in humans and domestic animals. Despite this, the understanding of the viral ecological diversity residing in bat populations remains unclear, which complicates the determination of the origins of zoonotic viruses. To gain knowledge on the viral community of a widely distributed insectivorous bat species, we characterized the guano virome of a native Chilean bat species (Myotis chiloensis (Waterhouse, 1840)). By applying a novel enrichment strategy, we were able to secure a consequent percentage of viral reads, providing unprecedented resolution for a bat virome. This in turn enabled us to identify and assemble a new bat alphacoronavirus from Chilean bats closely related to PEDV, an important viral pathogen with high mortality rates in suckling piglets. This study highlights the importance of applying and improving high-resolution virome studies in this vital order to ultimately enhance epidemiological surveillance for potentially zoonotic pathogens.     

人类病毒组及研究进展

人类病毒组的研究对象包括人体内部及表面所有病毒相关核酸序列。近年来随着高通量测序技术的快速发展,大量文献描述了不同人体器官或系统相关人类病毒组的组成成分及其作用。本文拟对人体口腔、皮肤、呼吸道、血液、神经系统、胃肠道、泌尿生殖道和肿瘤组织中目前病毒组相关知识进行概述,以便了解人类病毒组的组成、遗传多样性、动态变化,深入认识病毒组对人类疾病和健康的影响。     

Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.     

Exploring the Diversity of the Human Blood Virome

Metagenomics is greatly improving our ability to discover new viruses, as well as their possible associations with disease. However, metagenomics has also changed our understanding of viruses in general. The vast expansion of currently known viral diversity has revealed a large fraction of non-pathogenic viruses, and offers a new perspective in which viruses function as important components of many ecosystems. In this vein, studies of the human blood virome are often motivated by the search for new viral diseases, especially those associated with blood transfusions. However, these studies have revealed the common presence of apparently non-pathogenic viruses in blood, particularly human anelloviruses and, to a lower extent, human pegiviruses (HPgV). To shed light on the diversity of the human blood virome, we subjected pooled plasma samples from 587 healthy donors in Spain to a viral enrichment protocol, followed by massive parallel sequencing. This showed that anelloviruses were clearly the major component of the blood virome and showed remarkable diversity. In total, we assembled 332 complete or near-complete anellovirus genomes, 50 of which could be considered new species. HPgV was much less frequent, but we, nevertheless, recovered 17 different isolates that we subsequently used for characterizing the diversity of this virus. In-depth investigation of the human blood virome should help to elucidate the ecology of these viruses, and to unveil potentially associated diseases.     

Human Virome in Cervix Controlled by the Domination of Human Papillomavirus

Although other co-viral infections could also be considered influencing factors, cervical human papillomavirus (HPV) infection is the main cause of cervical cancer. Metagenomics have been employed in the NGS era to study the microbial community in each habitat. Thus, in this investigation, virome capture sequencing was used to examine the virome composition in the HPV-infected cervix. Based on the amount of HPV present in each sample, the results revealed that the cervical virome of HPV-infected individuals could be split into two categories: HPV-dominated (HD; ≥60%) and non-HPV-dominated (NHD; <60%). Cervical samples contained traces of several human viral species, including the molluscum contagiosum virus (MCV), human herpesvirus 4 (HHV4), torque teno virus (TTV), and influenza A virus. When compared to the HD group, the NHD group had a higher abundance of several viruses. Human viral diversity appears to be influenced by HPV dominance. This is the first proof that the diversity of human viruses in the cervix is impacted by HPV abundance. However, more research is required to determine whether human viral variety and the emergence of cancer are related.     

Phage Diving: An Exploration of the Carcharhinid Shark Epidermal Virome

The epidermal microbiome is a critical element of marine organismal immunity, but the epidermal virome of marine organisms remains largely unexplored. The epidermis of sharks represents a unique viromic ecosystem. Sharks secrete a thin layer of mucus which harbors a diverse microbiome, while their hydrodynamic dermal denticles simultaneously repel environmental microbes. Here, we sampled the virome from the epidermis of three shark species in the family Carcharhinidae: the genetically and morphologically similar Carcharhinus obscurus (n = 6) and Carcharhinus galapagensis (n = 10) and the outgroup Galeocerdo cuvier (n = 15). Virome taxonomy was characterized using shotgun metagenomics and compared with a suite of multivariate analyses. All three sharks retain species-specific but highly similar epidermal viromes dominated by uncharacterized bacteriophages which vary slightly in proportional abundance within and among shark species. Intraspecific variation was lower among C. galapagensis than among C. obscurus and G. cuvier. Using both the annotated and unannotated reads, we were able to determine that the Carcharhinus galapagensis viromes were more similar to that of G. cuvier than they were to that of C. obscurus, suggesting that behavioral niche may be a more prominent driver of virome than host phylogeny.     

A catalog of tens of thousands of viruses from human metagenomes reveals hidden associations with chronic diseases

Despite remarkable strides in microbiome research, the viral component of the microbiome has generally presented a more challenging target than the bacteriome. This gap persists, even though many thousands of shotgun sequencing runs from human metagenomic samples exist in public databases, and all of them encompass large amounts of viral sequence data. The lack of a comprehensive database for human-associated viruses has historically stymied efforts to interrogate the impact of the virome on human health. This study probes thousands of datasets to uncover sequences from over 45,000 unique virus taxa, with historically high per-genome completeness. Large publicly available case-control studies are reanalyzed, and over 2,200 strong virus–disease associations are found.

The human virome is the sum total of all viruses that are intimately associated with people. This includes viruses that directly infect human cells (1, 2) but mostly consists of viruses infecting resident bacteria (i.e., phages) (3). While the large majority of microbiome studies have focused on the bacteriome, revealing numerous important functions for bacteria in human physiology (4), information about the human virome has lagged. However, a number of recent studies have begun making inroads into characterizing the virome (5–13).Just as human-tropic viruses can have dramatic effects on people, phages are able to dramatically alter bacterial physiology and regulate host population size. A variety of evolutionary dynamics can be at play in the phage/bacterium arena, including Red Queen (11), arms-race (14), and piggyback-the-winner (15) relationships, to name just a few. In the gut, many phages enter a lysogenic or latent state and are retained as integrated or episomal prophages within the host bacterium (16). In some instances, the prophage can buttress host fitness (at least temporarily) rather than destroy the host cell. To this effect, prophages often encode genes that can dramatically alter the phenotype of the bacteria, such as toxins (17), virulence factors (18), antibiotic resistance genes (19), photosystem components (20), other auxiliary metabolic genes (21), and CRISPR-Cas systems (22), along with countless genes of unknown function. Experimental evidence has shown that bacteria infected with particular phages (i.e., “virocells”) are physiologically distinct from cognate bacteria that lack those particular phages (21).There have been a few documented cases in which phages have been shown to be mechanistically involved in human health and disease, sometimes through direct interactions with human cells. This includes roles in increased bacterial virulence (17), response to cancer immunotherapy (23), clearance of bacterial infection (24), and resistance to antibiotics (25). Furthermore, phage therapy, the targeted killing of specific bacteria using live phage particles, has shown increasing promise for treatment of antibiotic-resistant bacterial infections (26). Considering the progress already made, phages represent attractive targets of and tools for microbiome restructuring in the interest of improving health outcomes.In addition, several studies have conducted massively parallel sequencing on virus-enriched samples of human stool, finding differential abundance of some phages in disease conditions (6, 27–29). A major issue encountered by these studies is that there is not yet a comprehensive database of annotated virus genome sequences, and de novo prediction of virus sequences from metagenomic assemblies remains a daunting challenge (3). Further, though some tools are able to predict virus-derived sequences with high specificity (30, 31), these tools have not been applied to human metagenomes at a large scale [with a possible exception (13)], and, regrettably, most uncovered virus genomes do not end up in central repositories. One study suggests that only 31% of the assembled sequence data in virion-enriched virome surveys could be identified as recognizably viral (32). On the other hand, another study of 12 individuals was able to recruit over 80% of reads from virus-enriched samples to putative virus contigs (11). Still, most of the potential viral contigs from this study were unclassifiable sequences, and a large majority of contigs appeared to represent subgenomic fragments under 10 kb.The current study sought to overcome the traditional challenges of sparse viral databases and poor detection of highly divergent viral sequences by using Cenote-Taker 2, a new virus discovery and annotation tool (33). The pipeline was applied to sequencing data from nearly 6,000 human metagenome samples. Strict criteria identified over 180,000 viral contigs representing 45,033 specific taxa. In most cases, 70 to 99% of reads from virus-enriched stool datasets could be back-aligned to the Cenote-Taker 2–compiled Human Virome Database. Furthermore, the curated database allowed read-alignment–based abundance profiling of the virome in human metagenomic datasets, enabling the reanalysis of a panel of existing case-control studies. The reanalysis revealed previously undetected associations between chronic diseases and the abundance of 2,265 specific virus taxa.     

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.     

Characterisation of the RNA Virome of Nine Ochlerotatus Species in Finland

RNA viromes of nine commonly encountered Ochlerotatus mosquito species collected around Finland in 2015 and 2017 were studied using next-generation sequencing. Mosquito homogenates were sequenced from 91 pools comprising 16–60 morphologically identified adult females of Oc. cantans, Oc. caspius, Oc. communis, Oc. diantaeus, Oc. excrucians, Oc. hexodontus, Oc. intrudens, Oc. pullatus and Oc. punctor/punctodes. In total 514 viral Reverse dependent RNA polymerase (RdRp) sequences of 159 virus species were recovered, belonging to 25 families or equivalent rank, as follows: Aliusviridae, Aspiviridae, Botybirnavirus, Chrysoviridae, Chuviridae, Endornaviridae, Flaviviridae, Iflaviridae, Negevirus, Partitiviridae, Permutotetraviridae, Phasmaviridae, Phenuiviridae, Picornaviridae, Qinviridae, Quenyavirus, Rhabdoviridae, Sedoreoviridae, Solemoviridae, Spinareoviridae, Togaviridae, Totiviridae, Virgaviridae, Xinmoviridae and Yueviridae. Of these, 147 are tentatively novel viruses. One sequence of Sindbis virus, which causes Pogosta disease in humans, was detected from Oc. communis from Pohjois-Karjala. This study greatly increases the number of mosquito-associated viruses known from Finland and presents the northern-most mosquito-associated viruses in Europe to date.     

Virome of Three Termite Species from Southern Vietnam

Modern metagenomic approaches enable the effective discovery of novel viruses in previously unexplored organisms. Termites are significant ecosystem converters and influencers. As with the majority of tropical forest insects, termites are studied insufficiently, and termite virome remains especially understudied. Here, we studied the virome of lichenophagous and mycophagous termites (Hospitalitermes bicolor, Macrotermes carbonarius and Odontotermes wallonensis) collected in the Cat Tien National Park (Vietnam). We assembled four full genomes of novel viruses related to Solemoviridae, Lispiviridae, Polycipiviridae and Kolmioviridae. We also found several contigs with relation to Chuviridae and Deltaflexiviridae that did not correspond to complete virus genomes. All the novel viruses clustered phylogenetically with previously identified viruses of the termites. Deltaflexi-like contigs were identified in the fungi-cultivating M. carbonarius and showed homology with viruses recently discovered in the edible basidiomycete mushrooms.     

The Human Virome: Viral Metagenomics,Relations with Human Diseases,and Therapeutic Applications

The human body is colonized by a wide range of microorganisms. The field of viromics has expanded since the first reports on the detection of viruses via metagenomic sequencing in 2002. With the continued development of reference materials and databases, viral metagenomic approaches have been used to explore known components of the virome and discover new viruses from various types of samples. The virome has attracted substantial interest since the outbreak of the coronavirus disease 2019 (COVID-19) pandemic. Increasing numbers of studies and review articles have documented the diverse virome in various sites in the human body, as well as interactions between the human host and the virome with regard to health and disease. However, there have been few studies of direct causal relationships. Viral metagenomic analyses often lack standard references and are potentially subject to bias. Moreover, most virome-related review articles have focused on the gut virome and did not investigate the roles of the virome in other sites of the body in human disease. This review presents an overview of viral metagenomics, with updates regarding the relations between alterations in the human virome and the pathogenesis of human diseases, recent findings related to COVID-19, and therapeutic applications related to the human virome.     

Metagenomics Analysis of the Wheat Virome Identifies Novel Plant and Fungal-Associated Viral Sequences

Wheat viruses including wheat streak mosaic virus, Triticum mosaic virus, and barley yellow dwarf virus cost substantial losses in crop yields every year. Although there have been extensive studies conducted on these known wheat viruses, currently, there is limited knowledge about all components of the wheat (Triticum aestivum L.) virome. Here, we determined the composition of the wheat virome through total RNA deep sequencing of field-collected leaf samples. Sequences were de novo assembled after removing the host reads, and BLASTx searches were conducted. In addition to the documented wheat viruses, novel plant and fungal-associated viral sequences were identified. We obtained the full genome sequence of the first umbra-like associated RNA virus tentatively named wheat umbra-like virus in cereals. Moreover, a novel bi-segmented putative virus tentatively named wheat-associated vipovirus sharing low but significant similarity with both plant and fungal-associated viruses was identified. Additionally, a new putative fungal-associated tobamo-like virus and novel putative Mitovirus were discovered in wheat samples. The discovery and characterization of novel viral sequences associated with wheat is important to determine if these putative viruses may pose a threat to the wheat industry or have the potential to be used as new biological control agents for wheat pathogens either as wild-type or recombinant viruses.     

Possible Arbovirus Found in Virome of Melophagus ovinus

Members of the Lipopteninae subfamily are blood-sucking ectoparasites of mammals. The sheep ked (Melophagus ovinus) is a widely distributed ectoparasite of sheep. It can be found in most sheep-rearing areas and can cause skin irritation, restlessness, anemia, weight loss and skin injuries. Various bacteria and some viruses have been detected in M. ovinus; however, the virome of this ked has never been studied using modern approaches. Here, we study the virome of M. ovinus collected in the Republic of Tuva, Russia. In our research, we were able to assemble full genomes for five novel viruses, related to the Rhabdoviridae (Sigmavirus), Iflaviridae, Reoviridae and Solemoviridae families. Four viruses were found in all five of the studied pools, while one virus was found in two pools. Phylogenetically, all of the novel viruses clustered together with various recently described arthropod viruses. All the discovered viruses were tested on their ability to replicate in the mammalian porcine embryo kidney (PEK) cell line. Aksy-Durug Melophagus sigmavirus RNA was detected in the PEK cell line cultural supernate after the first, second and third passages. Such data imply that this virus might be able to replicate in mammalian cells, and thus, can be considered as a possible arbovirus.     

Metagenomic Analysis of the Enteric RNA Virome of Infants from the Oukasie Clinic,North West Province,South Africa,Reveals Diverse Eukaryotic Viruses

Establishing a diverse gut microbiota after birth is essential for preventing illnesses later in life. However, little knowledge exists about the total viral population (virome) present in the gut of infants during the early developmental stage, with RNA viruses being generally overlooked. Therefore, this small pilot longitudinal study investigated the diversity and changes in the enteric RNA virome in healthy infants from South Africa. Faecal samples (n = 12) were collected from four infants at three time points (on average at 8, 13, and 25 weeks), and then sequenced on an Illumina MiSeq platform. The genomic analysis revealed a diverse population of human enteric viruses from the infants’ stools, and changes in the enteric virome composition were observed over time. The Reoviridae family, more specifically the Rotavirus genus, was the most common and could be linked to viral shedding due to the administration of live-attenuated oral vaccines in South Africa, followed by the Picornaviridae family including parechoviruses, echoviruses, coxsackieviruses, enteroviruses, and polioviruses. Polioviruses were also linked to vaccine-related shedding. Astroviridae (astroviruses) and Caliciviridae (noroviruses) were present at low abundance. It is evident that an infant’s gut is colonized by distinct viral populations irrespective of their health state. Further characterization of the human virome (with a larger participant pool) is imperative to provide more conclusive insights into the viral community structure and diversity that has been shown in the current study, despite the smaller sample size.     

Virome Analysis of Signal Crayfish (Pacifastacus leniusculus) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal.     

Targeted Virome Sequencing Enhances Unbiased Detection and Genome Assembly of Known and Emerging Viruses—The Example of SARS-CoV-2

Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known—up to 2015—vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits’ design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits’ panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses.     

Dynamics of the Enteric Virome in a Swine Herd Affected by Non-PCV2/PRRSV Postweaning Wasting Syndrome

A commercial pig farm with no history of porcine circovirus 2 (PCV2) or porcine reproductive and respiratory syndrome virus (PRRSV) repeatedly reported a significant reduction in body weight gain and wasting symptoms in approximately 20–30% of the pigs in the period between three and six weeks after weaning. As standard clinical interventions failed to tackle symptomatology, viral metagenomics were used to describe and monitor the enteric virome at birth, 3 weeks, 4 weeks, 6 weeks, and 9 weeks of age. The latter four sampling points were 7 days, 3 weeks, and 6 weeks post weaning, respectively. Fourteen distinct enteric viruses were identified within the herd, which all have previously been linked to enteric diseases. Here we show that wasting is associated with alterations in the enteric virome of the pigs, characterized by: (1) the presence of enterovirus G at 3 weeks of age, followed by a higher prevalence of the virus in wasting pigs at 6 weeks after weaning; (2) rotaviruses at 3 weeks of age; and (3) porcine sapovirus one week after weaning. However, the data do not provide a causal link between specific viral infections and the postweaning clinical problems on the farm. Together, our results offer evidence that disturbances in the enteric virome at the preweaning stage and early after weaning have a determining role in the development of intestinal barrier dysfunctions and nutrient uptake in the postweaning growth phase. Moreover, we show that the enteric viral load sharply increases in the week after weaning in both healthy and wasting pigs. This study is also the first to report the dynamics and co-infection of porcine rotavirus species and porcine astrovirus genetic lineages during the first 9 weeks of the life of domestic pigs.