Current knowledge of RNA virus biodiversity is both biased and fragmentary, reflecting a focus on culturable or disease-causing agents. Here we profile the transcriptomes of over 220 invertebrate species sampled across nine animal phyla and report the discovery of 1,445 RNA viruses, including some that are sufficiently divergent to comprise new families. The identified viruses fill major gaps in the RNA virus phylogeny and reveal an evolutionary history that is characterized by both host switching and co-divergence. The invertebrate virome also reveals remarkable genomic flexibility that includes frequent recombination, lateral gene transfer among viruses and hosts, gene gain and loss, and complex genomic rearrangements. Together, these data present a view of the RNA virosphere that is more phylogenetically and genomically diverse than that depicted in current classification schemes and provide a more solid foundation for studies in virus ecology and evolution.

Although arthropods are important viral vectors, the biodiversity of arthropod viruses, as well as the role that arthropods have played in viral origins and evolution, is unclear. Through RNA sequencing of 70 arthropod species we discovered 112 novel viruses that appear to be ancestral to much of the documented genetic diversity of negative-sense RNA viruses, a number of which are also present as endogenous genomic copies. With this greatly enriched diversity we revealed that arthropods contain viruses that fall basal to major virus groups, including the vertebrate-specific arenaviruses, filoviruses, hantaviruses, influenza viruses, lyssaviruses, and paramyxoviruses. We similarly documented a remarkable diversity of genome structures in arthropod viruses, including a putative circular form, that sheds new light on the evolution of genome organization. Hence, arthropods are a major reservoir of viral genetic diversity and have likely been central to viral evolution.DOI: http://dx.doi.org/10.7554/eLife.05378.001

Although segmented and unsegmented RNA viruses are commonplace, the evolutionary links between these two very different forms of genome organization are unclear. We report the discovery and characterization of a tick-borne virus-Jingmen tick virus (JMTV)-that reveals an unexpected connection between segmented and unsegmented RNA viruses. The JMTV genome comprises four segments, two of which are related to the nonstructural protein genes of the genus Flavivirus (family Flaviviridae), whereas the remaining segments are unique to this virus, have no known homologs, and contain a number of features indicative of structural protein genes. Remarkably, homology searching revealed that sequences related to JMTV were present in the cDNA library from Toxocara canis (dog roundworm; Nematoda), and that shared strong sequence and structural resemblances. Epidemiological studies showed that JMTV is distributed in tick populations across China, especially Rhipicephalus and Haemaphysalis spp., and experiences frequent host-switching and genomic reassortment. To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented viral ancestors.evolution | segmentation S egmentation is a common form of genome organization in RNA viruses, although why it has evolved more than once and is maintained in such a diverse array of viruses, including those with both positive-and negative-sense genomes, are unclear (1). Segmented and unsegmented viruses usually belong to different viral families, such that the sequence divergence between them is often too great for any meaningful evolutionary inference. The only example of "recent" genome fragmentation in a single RNA molecule occurred in a laboratory strain of foot-and-mouth disease virus (2, 3), although that this occurred following extensive propagation in cell culture means that its relationship to segmentation in nature is uncertain. Hence, the evolutionary links between unsegmented and segmented viruses, as well as the mechanisms that underpin their genesis, are poorly understood.The Flaviviridae are a family of unsegmented positive sense RNA viruses that infect vertebrate and invertebrate species, including the important human pathogens dengue virus, yellow fever virus, and hepatitis C virus. Despite the substantial sequence divergence between the Flavivirus, Pestivirus, and Hepacivirus genera that make up the Flaviviridae, they exhibit a similar genomic structure characterized by a single ORF with distinct clusters of structural and nonstructural genes. The ORF is translated into a single polyprotein, which is subsequently cleaved by cellular and viral proteases into structural and nonstructural proteins. Among the nonstructural protein products, NS3 and NS5 possess the enzymatic domains essential for RNA capping and genome replication (4), whereas the NS3 and NS2b proteins form a two-component serine protease involved in posttranslational cleavage of the viral polyprotein (5).Herein we describe the discovery and characterization of an...

Herein we reported the development of aptamer-based biosensors (aptasensors) based on label-free aptamers and gold nanoparticles (AuNPs) for detection of Escherichia coli (E. coli) O157:H7 and Salmonella typhimurium. Target bacteria binding aptamers are adsorbed on the surface of unmodified AuNPs to capture target bacteria, and the detection was accomplished by target bacteria-induced aggregation of the aptasensor which is associated as red-to-purple color change upon high-salt conditions. By employing anti-E. coli O157:H7 aptamer and anti-S. typhimurium aptamer, we developed a convenient and rapid approach that could selectively detect bacteria without specialized instrumentation and pretreatment steps such as cell lysis. The aptasensor could detect as low as 105colony-forming units (CFU)/ml target bacteria within 20 min or less and its specificity was 100%. This novel method has a great potential application in rapid detection of bacteria in the near future.

Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterise the complete ‘infectome’ (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing (“metatranscriptomics”) we documented the presence of contaminant viral sequences in multiple ‘blank’ negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank control libraries, as well as contaminating sequences from the Totiviridae, Tombusviridae and Lentiviridae families of RNA virus. These data suggest that viral contamination of common laboratory reagents is likely commonplace and can comprise a wide variety of viruses.

Papillomaviruses (PVs) have been identified in a wide range of animal species and are associated with a variety of disease syndromes including classical papillomatosis, aural plaques, and genital papillomas. In horses, 13 PVs have been described to date, falling into six genera. Using total RNA sequencing (meta-transcriptomics) we identified a novel equine papillomavirus in semen taken from a thoroughbred stallion suffering a genital lesion, which was confirmed by nested RT-PCR. We designate this novel virus Equus caballus papillomavirus 9 (EcPV9). The complete 7656 bp genome of EcPV9 exhibited similar characteristics to those of other horse papillomaviruses. Phylogenetic analysis based on concatenated E1-E2-L2-L1 amino acid sequences revealed that EcPV9 clustered with EcPV2, EcPV4, and EcPV5, although was distinct enough to represent a new viral species within the genus Dyoiotapapillomavirus (69.35%, 59.25%, and 58.00% nucleotide similarity to EcPV2, EcPV4, and EcPV5, respectively). In sum, we demonstrate the presence of a novel equine papillomavirus for which more detailed studies of disease association are merited.

the diversity of pathogens associated with acute respiratory infection (ARi) makes diagnosis challenging. traditional pathogen screening tests have a limited detection range and provide little additional information. We used total RNA sequencing ("meta-transcriptomics") to reveal the full spectrum of microbes associated with paediatric ARI. Throat swabs were collected from 48 paediatric ARI patients and 7 healthy controls. Samples were subjected to meta-transcriptomics to determine the presence and abundance of viral, bacterial, and eukaryotic pathogens, and to reveal mixed infections, pathogen genotypes/subtypes, evolutionary origins, epidemiological history, and antimicrobial resistance. We identified 11 RNA viruses, 4 DNA viruses, 4 species of bacteria, and 1 fungus. While most are known to cause ARIs, others, such as echovirus 6, are rarely associated with respiratory disease. Co-infection of viruses and bacteria and of multiple viruses were commonplace (9/48), with one patient harboring 5 different pathogens, and genome sequence data revealed large intra-species diversity. Expressed resistance against eight classes of antibiotic was detected, with those for MLS, Bla, Tet, Phe at relatively high abundance. In summary, we used a simple total RNA sequencing approach to reveal the complex polymicrobial infectome in ARi. this provided comprehensive and clinically informative information relevant to understanding respiratory disease.Acute respiratory infections (ARI) are a leading cause of morbidity and mortality in newborns and young children, who experience an average of 3 to 6 ARIs annually 1-3 . Identifying the diversity of pathogens responsible for ARIs remains challenging because they involve a diverse set of viruses, bacteria, and fungal pathogens, with co-infection among them commonplace 4,5 . Traditional testing methods such as PCR, serological typing, bacterial culture and antibody detection, are regarded as the "gold standard" and widely used in ARI diagnosis 6,7 . However, despite an ongoing effort to include multiple pathogens in a single assay 8,9 , it remains difficult to simultaneously identify all potential ARI pathogens and capture new or uncommon respiratory pathogens 10 .Metagenomic next-generation sequencing (mNGS) is an unbiased way of discovering a broad range of infectious agents 11-13 , and has been recently introduced into clinical research to investigate the microbial cause of unusual disease cases 14 , perform broad-scale surveys for pathogens in undiagnosed diseases 15,16 , and understand the role of opportunistic infections 17,18 . For example, a study of severe pneumonia revealed that mNGS is both efficient and reliable 19,20 . Importantly, the utility of mNGS goes beyond pathogen identification. In particular, total RNA sequencing ("meta-transcriptomics") has successfully revealed the entire "infectome" (viruses, bacteria and eukaryotes) present within an organism and provided relevant data on genome sequence, gene expression, open Scientific RepoRtS | (2020) 10:3963 | https://doi.o...

Viral pathogens are being increasingly described in association with mass morbidity and mortality events in reptiles. However, our knowledge of reptile viruses remains limited. Herein, we describe the meta-transcriptomic investigation of a mass morbidity and mortality event in a colony of central bearded dragons (Pogona vitticeps) in 2014. Severe, extensive proliferation of the respiratory epithelium was consistently found in affected dragons. Similar proliferative lung lesions were identified in bearded dragons from the same colony in 2020 in association with increased intermittent mortality. Total RNA sequencing identified two divergent DNA viruses: a reptile-infecting circovirus, denoted bearded dragon circovirus (BDCV), and the first exogeneous reptilian chaphamaparvovirus—bearded dragon chaphamaparvovirus (BDchPV). Phylogenetic analysis revealed that BDCV was most closely related to bat-associated circoviruses, exhibiting 70% amino acid sequence identity in the Replicase (Rep) protein. In contrast, in the nonstructural (NS) protein, the newly discovered BDchPV showed approximately 31%–35% identity to parvoviruses obtained from tilapia fish and crocodiles in China. Subsequent specific PCR assays revealed BDCV and BDchPV in both diseased and apparently normal captive reptiles, although only BDCV was found in those animals with proliferative pulmonary lesions and respiratory disease. This study expands our understanding of viral diversity in captive reptiles.