Metagenomic Exploration of Viruses throughout the Indian Ocean

The characterization of global marine microbial taxonomic and functional diversity is a primary goal of the Global Ocean Sampling Expedition. As part of this study, 19 water samples were collected aboard the Sorcerer II sailing vessel from the southern Indian Ocean in an effort to more thoroughly understand the lifestyle strategies of the microbial inhabitants of this ultra-oligotrophic region. No investigations of whole virioplankton assemblages have been conducted on waters collected from the Indian Ocean or across multiple size fractions thus far. Therefore, the goals of this study were to examine the effect of size fractionation on viral consortia structure and function and understand the diversity and functional potential of the Indian Ocean virome. Five samples were selected for comprehensive metagenomic exploration; and sequencing was performed on the microbes captured on 3.0-, 0.8- and 0.1 µm membrane filters as well as the viral fraction (<0.1 µm). Phylogenetic approaches were also used to identify predicted proteins of viral origin in the larger fractions of data from all Indian Ocean samples, which were included in subsequent metagenomic analyses. Taxonomic profiling of viral sequences suggested that size fractionation of marine microbial communities enriches for specific groups of viruses within the different size classes and functional characterization further substantiated this observation. Functional analyses also revealed a relative enrichment for metabolic proteins of viral origin that potentially reflect the physiological condition of host cells in the Indian Ocean including those involved in nitrogen metabolism and oxidative phosphorylation. A novel classification method, MGTAXA, was used to assess virus-host relationships in the Indian Ocean by predicting the taxonomy of putative host genera, with Prochlorococcus, Acanthochlois and members of the SAR86 cluster comprising the most abundant predictions. This is the first study to holistically explore virioplankton dynamics across multiple size classes and provides unprecedented insight into virus diversity, metabolic potential and virus-host interactions.     

Distribution and Fractionation of Arsenic in Selected Fresh Water Lake Sediments

The distribution and nature of the As-bearing components of the sediments of selected fresh water lakes in Saskatchewan, Canada, were studied. The total As contents ranged from 2.7 to 13.2 ppm As and were found to be present in both colloidal and non-colloidal fractions of the lake sediments. The proportion of the As bound to carbonates, organic matter and sesquioxides accounted for 12 to 74% of the total As of the sediments. The remaining As was apparently associated with apatite and silicates. The data indicate that the bulk of As in the lake sediments resides in sesquioxidic components and apatite. The release of As from the lake sediments would thus be affected by the stability of these As-bearing components in a series of particle size fractions. Furthermore, based on information on the geographical distribution of As in the lake sediments, land erosion and agricultural runoff appear to be contributing significantly to the As concentration in lake sediments. It is thus suggested that close attention should be paid to land management and soil conservation to prevent further enrichment of As in lake sediments and to curtail the subsequent As contamination of the food chain.     

Metagenomic Detection of Viral Pathogens in Spanish Honeybees: Co-Infection by Aphid Lethal Paralysis,Israel Acute Paralysis and Lake Sinai Viruses

The situation in Europe concerning honeybees has in recent years become increasingly aggravated with steady decline in populations and/or catastrophic winter losses. This has largely been attributed to the occurrence of a variety of known and “unknown”, emerging novel diseases. Previous studies have demonstrated that colonies often can harbour more than one pathogen, making identification of etiological agents with classical methods difficult. By employing an unbiased metagenomic approach, which allows the detection of both unexpected and previously unknown infectious agents, the detection of three viruses, Aphid Lethal Paralysis Virus (ALPV), Israel Acute Paralysis Virus (IAPV), and Lake Sinai Virus (LSV), in honeybees from Spain is reported in this article. The existence of a subgroup of ALPV with the ability to infect bees was only recently reported and this is the first identification of such a strain in Europe. Similarly, LSV appear to be a still unclassified group of viruses with unclear impact on colony health and these viruses have not previously been identified outside of the United States. Furthermore, our study also reveals that these bees carried a plant virus, Turnip Ringspot Virus (TuRSV), potentially serving as important vector organisms. Taken together, these results demonstrate the new possibilities opened up by high-throughput sequencing and metagenomic analysis to study emerging new diseases in domestic and wild animal populations, including honeybees.     

Nyamanini and Midway Viruses Define a Novel Taxon of RNA Viruses in the Order Mononegavirales

Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.Nyamanini virus (NYMV) was first isolated in 1957 from a cattle egret (Bubulcus ibis) in South Africa (24). It has subsequently been isolated in Nigeria, Egypt, India, and Thailand from cattle egrets and Argas walkerae ticks (14, 16, 24). Although there has been no recognized human infection or disease associated with NYMV, suckling mice succumbed to NYMV infection 7 or 8 days after intracerebral inoculation (14). NYMV has not been definitively characterized or classified to date (10).Midway virus (MIDWV) was first isolated in 1966 from seabird ticks of two species [Ornithodoros (Alectorobius) spp.] collected on the Midway, Kure, and Manana islands in the Central Pacific and from northern Honshu, Japan. In addition, on Aomatsushima Island, nestling seabirds of two seabird species, Larus crassirostris and Nycticorax nycticorax, were found to have antibody to MIDWV. This virus is pathogenic for newborn Swiss mice but not 4-week-old Swiss mice injected intracranially. Also, the virus is cytopathic in BHK-21 cells and produces plaques in Vero cells. Efforts to classify MIDWV have revealed only that MIDWV is antigenically related to NYMV in cross-box complement fixation (CF) assays (22).To date, conventional approaches, such as serological analysis and electron microscopy (EM), have not yielded definitive characterization of MIDWV or NYMV. Heretofore, it was not known whether MIDWV and NYMV are DNA viruses or RNA viruses or to which virus family they belong. In this paper, we describe the application of unbiased high-throughput sequencing to define the genome sequences of NYMV and MIDWV. By analysis of their genomes, NYMV and MIDWV were determined to be negative-stranded RNA viruses highly divergent from all known viruses but most closely related to viruses in the order Mononegavirales. On the basis of the analysis presented herein, we propose that NYMV and MIDWV define a novel taxon within the order Mononegavirales.     

Virome Profiling of Bats from Myanmar by Metagenomic Analysis of Tissue Samples Reveals More Novel Mammalian Viruses

Bats are reservoir animals harboring many important pathogenic viruses and with the capability of transmitting these to humans and other animals. To establish an effective surveillance to monitor transboundary spread of bat viruses between Myanmar and China, complete organs from the thorax and abdomen from 853 bats of six species from two Myanmar counties close to Yunnan province, China, were collected and tested for their virome through metagenomics by Solexa sequencing and bioinformatic analysis. In total, 3,742,314 reads of 114 bases were generated, and over 86% were assembled into 1,649,512 contigs with an average length of 114 bp, of which 26,698 (2%) contigs were recognizable viral sequences belonging to 24 viral families. Of the viral contigs 45% (12,086/26,698) were related to vertebrate viruses, 28% (7,443/26,698) to insect viruses, 27% (7,074/26,698) to phages and 95 contigs to plant viruses. The metagenomic results were confirmed by PCR of selected viruses in all bat samples followed by phylogenetic analysis, which has led to the discovery of some novel bat viruses of the genera Mamastrovirus, Bocavirus, Circovirus, Iflavirus and Orthohepadnavirus and to their prevalence rates in two bat species. In conclusion, the present study aims to present the bat virome in Myanmar, and the results obtained further expand the spectrum of viruses harbored by bats.     

Phylogenetic Properties of RNA Viruses

A new word, phylodynamics, was coined to emphasize the interconnection between phylogenetic properties, as observed for instance in a phylogenetic tree, and the epidemic dynamics of viruses, where selection, mediated by the host immune response, and transmission play a crucial role. The challenges faced when investigating the evolution of RNA viruses call for a virtuous loop of data collection, data analysis and modeling. This already resulted both in the collection of massive sequences databases and in the formulation of hypotheses on the main mechanisms driving qualitative differences observed in the (reconstructed) evolutionary patterns of different RNA viruses. Qualitatively, it has been observed that selection driven by the host immune response induces an uneven survival ability among co-existing strains. As a consequence, the imbalance level of the phylogenetic tree is manifestly more pronounced if compared to the case when the interaction with the host immune system does not play a central role in the evolutive dynamics. While many imbalance metrics have been introduced, reliable methods to discriminate in a quantitative way different level of imbalance are still lacking. In our work, we reconstruct and analyze the phylogenetic trees of six RNA viruses, with a special emphasis on the human Influenza A virus, due to its relevance for vaccine preparation as well as for the theoretical challenges it poses due to its peculiar evolutionary dynamics. We focus in particular on topological properties. We point out the limitation featured by standard imbalance metrics, and we introduce a new methodology with which we assign the correct imbalance level of the phylogenetic trees, in agreement with the phylodynamics of the viruses. Our thorough quantitative analysis allows for a deeper understanding of the evolutionary dynamics of the considered RNA viruses, which is crucial in order to provide a valuable framework for a quantitative assessment of theoretical predictions.     

Morphological Characterization of Viruses in the Stratified Water Column of Alkaline, Hypersaline Mono Lake

Concentrations of viruses and prokaryotes in the alkaline, moderately hypersaline, seasonally stratified Mono Lake are among the highest reported for a natural aquatic environment. We used electron microscopy to test whether viral morphological characteristics differed among the epilimnion, metalimnion, and the anoxic hypolimnion of the lake and to determine how the properties of viruses in Mono Lake compare to other aquatic environments. Viral capsid size distributions were more similar in the metalimnion and hypolimnion of Mono Lake, while viral tail lengths were more similar in the epilimnion and metalimnion. The percentage of tailed viruses decreased with depth and the relative percentages of tailed phage families changed with depth. The presence of large (>125 nm capsid), untailed viruses in the metalimnion and hypolimnion suggests that eukaryotic viruses are produced in these suboxic and anoxic, hypersaline environments. Capsid diameters of viruses were larger on average in Mono Lake compared to other aquatic environments, and no lemon-shaped or filamentous viruses were found, in contrast to other high-salinity or high-altitude lakes and seas. Our data suggest that the physically and chemically distinct layers of Mono Lake harbor different viral assemblages, and that these assemblages are distinct from other aquatic environments that have been studied. Furthermore, we found that filtration of a sample through a 0.22-μm pore-size filter significantly altered the distribution of viral capsid diameters and tail lengths, resulting in a relative depletion of viruses having larger capsids and longer tails. This observation highlights the potential for bias in molecular surveys of viral diversity, which typically rely on filtration through 0.2- or 0.22-μm pore-size membrane filters to remove bacteria during sample preparation.     

RNA of RNA Tumour Viruses contains Poly A

Molecular hybridization with radioactive polyuridylic acid has been used to detect regions of polyadenylic acid in virus RNA. On the average, RNA from tumour viruses is twenty-five to fifty-fold richer in polyadenylic acid than RNA from non-oncogenic viruses. Polyacrylamide gel electrophoresis permitted a qualitative distinction between RNA preparations from the two virus classes.     

Rates of Molecular Evolution in RNA Viruses: A Quantitative Phylogenetic Analysis

The study of rates of nucleotide substitution in RNA viruses is central to our understanding of their evolution. Herein we report a comprehensive analysis of substitution rates in 50 RNA viruses using a recently developed maximum likelihood phylogenetic method. This analysis revealed a significant relationship between genetic divergence and isolation time for an extensive array of RNA viruses, although more rate variation was usually present among lineages than would be expected under the constraints of a molecular clock. Despite the lack of a molecular clock, the range of statistically significant variation in overall substitution rates was surprisingly narrow for those viruses where a significant relationship between genetic divergence and time was found, as was the case when synonymous sites were considered alone, where the molecular clock was rejected less frequently. An analysis of the ecological and genetic factors that might explain this rate variation revealed some evidence of significantly lower substitution rates in vector-borne viruses, as well as a weak correlation between rate and genome length. Finally, a simulation study revealed that our maximum likelihood estimates of substitution rates are valid, even if the molecular clock is rejected, provided that sufficiently large data sets are analyzed. Received: 23 February 2001 / Accepted: 3 July 2001     

Metagenomic Profiling of Viruses Associated with Rhipicephalus microplus Ticks in Yunnan Province,China

Ticks are well known as vectors of many viruses which usually do great harm to human and animal health. Yunnan Province, widely covered by flourishing vegetation and mainly relying on farming husbandry, is abundant with Rhipicephalus microplus ticks. Therefore, it is of great significance to characterize the viral profile present in R. microplus parasitizing on cattle in Yunnan Province. In this study, a total of 7387 R. microplus ticks were collected from cattle and buffalo in the northwest and southeast areas of Yunnan Province from 2015 to 2017. We investigated the virome of R.microplus using next-generation sequencing(NGS) and the prevalence of important identified viruses among tick groups by RT-PCR. It revealed the presence of diverse virus concerning chu-, rhabdo-, phlebo-, flavi-and parvo-viruses in Yunnan. These viruses consist of single-stranded, circular and segmented sense RNAs, showing a greatly diversity in genomic organization. Furthermore, continuous epidemiological survey among ticks reveals broad prevalence of three viruses(Yunnan mivirus 1, Wuhan tick vrius 1 and YN tick-associated phlebovirus 1) and two possible prevalent viruses including a flavivirus-like segmented virus(Jingmen tick virus) and a bovine hokovirus 2 in Yunnan. Serological investigation among cattle indicates that these identified viruses may be infectious to cattle and can elicit corresponding antibody. Our findings on R. microplus-associated viral community will contribute to the prevention of viral disease and tracking the viral evolution. Further analysis is needed to better elucidate the pathogenicity and natural circulation of these viruses.     

Studies on the RNA Isolated from Fresh Water Sponge, Ephydatia meyeni

Protein and DNA-free RNA, isolated from fresh water sponge, Ephydatia meyeni , shows 31 and 52% increase in A₂₆₀ value upon digestion with pancreatic RNAse and alkali, respectively. While total RNA exhibits a catalytic influence upon the aggregation of sponge cells, tRNA alone has a negligible effect. Although rRNA brings about a similar effect to that of total RNA, EDTA-treated rRNA has no effect upon the aggregation of sponge cells. Results are interpreted in the light of a possible role for polyamines.     

Profile Hidden Markov Models for the Detection of Viruses within Metagenomic Sequence Data

Rapid, sensitive, and specific virus detection is an important component of clinical diagnostics. Massively parallel sequencing enables new diagnostic opportunities that complement traditional serological and PCR based techniques. While massively parallel sequencing promises the benefits of being more comprehensive and less biased than traditional approaches, it presents new analytical challenges, especially with respect to detection of pathogen sequences in metagenomic contexts. To a first approximation, the initial detection of viruses can be achieved simply through alignment of sequence reads or assembled contigs to a reference database of pathogen genomes with tools such as BLAST. However, recognition of highly divergent viral sequences is problematic, and may be further complicated by the inherently high mutation rates of some viral types, especially RNA viruses. In these cases, increased sensitivity may be achieved by leveraging position-specific information during the alignment process. Here, we constructed HMMER3-compatible profile hidden Markov models (profile HMMs) from all the virally annotated proteins in RefSeq in an automated fashion using a custom-built bioinformatic pipeline. We then tested the ability of these viral profile HMMs (“vFams”) to accurately classify sequences as viral or non-viral. Cross-validation experiments with full-length gene sequences showed that the vFams were able to recall 91% of left-out viral test sequences without erroneously classifying any non-viral sequences into viral protein clusters. Thorough reanalysis of previously published metagenomic datasets with a set of the best-performing vFams showed that they were more sensitive than BLAST for detecting sequences originating from more distant relatives of known viruses. To facilitate the use of the vFams for rapid detection of remote viral homologs in metagenomic data, we provide two sets of vFams, comprising more than 4,000 vFams each, in the HMMER3 format. We also provide the software necessary to build custom profile HMMs or update the vFams as more viruses are discovered (http://derisilab.ucsf.edu/software/vFam).     

Virus Specific RNA in Cells transformed by RNA Tumour Viruses

Virus specific RNA comprises 5% of the nuclear RNA and 0.5–1.0% of the cytoplasmic RNA of cells transformed by murine sarcoma viruses. Even cryptically transformed cells which possess no detectable virus or viral proteins synthesize detectable amounts of viral RNA.     

Polyprotein Processing as a Strategy for Gene Expression in RNA Viruses

RNA viruses in many families and genera express their genomes in ways which involve the synthesis and subsequent cleavage of precursor polyproteins. This strategem allows the activation of subsets of proteins with different biochemical functions from the same precursor protein. Although the virus-encoded enzymes responsible for processing the polyproteins are structurally diverse, they are all highly specific for their substrates. The resulting processing cascade is a tightly controlled process, which in several cases involves the action of protein cofactors to modulate the activity of the proteinase.     

Metagenomic Assessment of Prokaryotic Diversity within Hypersaline Tuz Lake,Turkey

Microbiology - Tuz Lake is the second biggest lake with an extreme hypersaline environment (over 32% (w/v) salt) in Turkey. It is reported that the lake was of marine origin, and its formation...