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At least 19 recordsLinked to original sources
USGS2018

A novel technique for isolating DNA from Tempus™ blood RNA tubes after RNA isolation

Objective We use Tempus blood RNA tubes (Applied Biosystems) during health assessments of American moose ( Alces alces spp.) as a minimally invasive means to obtain RNA. Here we describe a novel protocol to additionally isolate high-quality DNA from the supernatant remaining after the RNA isolation methodology. Metrics used to qualify DNA quality included measuring the concentration, obtaining a DNA integrity number from a genomic DNA ScreenTape assay (Agilent), and running the isolated DNA on an agarose gel. Results Of the 23 samples analyzed, the average DNA concentration was 121 ng/µl (range 4–337 ng/µl) and a genomic DNA ScreenTape assay of seven samples indicated high DNA integrity values for 6 of the 7 samples (range 9.1–9.4 out of 10). Of the DNA sent for genotyping by sequencing, all proved to be of sufficient integrity to yield high-quality next-generation sequence results. We recommend this simple procedure to maximize the yield of both RNA and DNA from blood samples.

BMC Research Notes↗
USGS2017

Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.-approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples ( n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA-positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho ( Oncorhynchus kisutch Walbaum) and Chinook ( O. tshawytscha Walbaum) salmon.

Alaska, Washington↗
USGS2013

Relations between DNA- and RNA-based molecular methods for cyanobacteria and microcystin concentration at Maumee Bay State Park Lakeside Beach, Oregon, Ohio, 2012

Water samples were collected from Maumee Bay State Park Lakeside Beach, Oregon, Ohio, during the 2012 recreational season and analyzed for selected cyanobacteria gene sequences by DNA-based quantitative polymerase chain reaction (qPCR) and RNA-based quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results from the four DNA assays (for quantifying total cyanobacteria, total Microcystis , and Microcystis and Planktothrix strains that possess the microcystin synthetase E ( mcyE ) gene) and two RNA assays (for quantifying Microcystis and Planktothrix genera that are expressing the microcystin synthetase E ( mcyE ) gene) were compared to microcystin concentration results determined by an enzyme-linked immunosorbent assay (ELISA). Concentrations of the target in replicate analyses were log10 transformed. The average value of differences in log10 concentrations for the replicates that had at least one detection were found to range from 0.05 to >0.37 copy per 100 milliliters (copy/100 mL) for DNA-based methods and from >0.04 to >0.17 copy/100 mL for RNA-based methods. RNA has a shorter half-life than DNA; consequently, a 24-hour holding-time study was done to determine the effects of holding time on RNA concentrations. Holding-time comparisons for the RNA-based Microcystis toxin mcyE assay showed reductions in the number of copies per 100 milliliters over 24 hours. The log difference between time 2 hours and time 24 hours was >0.37 copy/100 mL, which was higher than the analytical variability (log difference of >0.17 copy/100 mL). Spearman’s correlation analysis indicated that microcystin toxin concentrations were moderately to highly related to DNA-based assay results for total cyanobacteria (rho=0.69), total Microcystis (rho=0.74), and Microcystis strains that possess the mcyE gene (rho=0.81). Microcystin toxin concentrations were strongly related with RNA-based assay results for Microcystis mcyE gene expression (rho=0.95). Correlation analysis could not be done for Planktothrix mcyE gene expression because of too few detections.

Ohio↗
USGS2011

Effects of hypoxia on consumption, growth, and RNA:DNA ratios of young Yellow Perch

As in various freshwater and coastal marine ecosystems worldwide, seasonal bottom water hypoxia is a recurring phenomenon in Lake Erie’s central basin. While bottom hypoxia can strongly affect sessile benthic animals, its effects on mobile organisms such as fish are less understood. We evaluated the potential for bottom hypoxia to affect the growth rates of yellow perch Perca flavescens, a species of ecological and economic importance in the lake. To this end, we (1) conducted laboratory experiments to quantify the effects of reduced dissolved oxygen on consumption, somatic growth, and RNA:DNA ratios (an index of short-term growth) of young yellow perch and (2) explored the effects of bottom hypoxia on young yellow perch growth in Lake Erie’s central basin by collecting individuals in hypoxic and normoxic regions of the lake and quantifying their RNA:DNA ratios. Yellow perch consumption and growth in our experiments declined under hypoxic conditions (≤2 mg O2/L). While yellow perch RNA:DNA ratios responded strongly to experimental temperature, nucleic acid ratios were not significantly affected by dissolved oxygen or feeding ration. We did, however, observe a positive correlation between yellow perch growth and RNA:DNA ratios at low temperatures (11◦C). The nucleic acid ratios of yellow perch collected in Lake Erie varied spatiotemporally, but their patterns were not consistent with hypoxia. In short, while yellow perch consumption and growth rates respond directly and negatively to low oxygen conditions, these responses are not necessarily reflected in RNA:DNA ratios. Moreover, in central Lake Erie, where yellow perch can behaviorally avoid hypoxic areas, the RNA:DNA ratios of yellow perch do not respond strongly to bottom hypoxia. Thus, this study suggests that there is no strong negative effect of bottom hypoxia on the growth of young yellow perch in Lake Erie.

Transactions of the American Fisheries Society↗
USGS1976

Differential inhibition of host protein synthesis in L cells infected with RNA - temperature-sensitive mutants of vesicular stomatitis virus

The response of mouse L cells to infection with wild-type (wt) and temperature-sensitive (ts) mutants of vesicular stomatitis virus was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to delineate the synthesis of host cell and viral proteins. Experiments utilized transcriptase mutants of complementation group I (ts114 and ts13), a group IV mutant (ts44) that is restricted in total RNA synthesis (RNA-1) but not in primary transcription, and a group II mutant (ts52) variably restricted in RNA synthesis (RNA +/-). L cells infected with ts mutants at permissive temperature exhibited the wt response of progressive inhibition of host cell protein synthesis accompanied by accumulation of all five viral proteins. Mutant ts44 (IV) also switched off cell protein synthesis at restrictive temperature and accumulated all five viral proteins, but with disproportionate ratios of N and G proteins. At restrictive temperature, cells infected with group I ts mutants failed to accumulate any viral protein and did not exhibit significant reduction in host cell protein synthesis. These data suggest that vesicular stomatitis virus inhibits cell protein synthesis at a stage of viral infection after transcription and possibly translation but preceding replication of progeny viral RNA.

Journal of Virology↗
USGS2003

Characterization of the mutant spectra of a fish RNA virus within individual hosts during natural infections

Infectious hematopoietic necrosis virus (IHNV) is an RNA virus that causes significant mortalities of salmonids in the Pacific Northwest of North America. RNA virus populations typically contain genetic variants that form a heterogeneous virus pool, referred to as a quasispecies or mutant spectrum. This study characterized the mutant spectra of IHNV populations within individual fish reared in different environmental settings by RT–PCR of genomic viral RNA and determination of partial glycoprotein gene sequences of molecular clones. The diversity of the mutant spectra from ten in vivo populations was low and the average mutation frequencies of duplicate populations did not significantly exceed the background mutation level expected from the methodology. In contrast, two in vitro populations contained variants with an identical mutational hot spot. These results indicated that the mutant spectra of natural IHNV populations is very homogeneous, and does not explain the different magnitudes of genetic diversity observed between the different IHNV genogroups. Overall the mutant frequency of IHNV within its host is one of the lowest reported for RNA viruses.

Virus Research↗
USGS2023

Comparison of magnetic bead and rapid swab RNA extraction methods for detecting rabbit hemorrhagic disease virus 2 in rabbit liver samples

We compared a bead RNA extraction method with a one-tube method that required only a heat block and ice. RNA was first extracted from liver samples from nine rabbits dying from rabbit hemorrhagic disease virus 2 (RHDV2) using magnetic beads, and RT-PCR was used to detect RHDV2 sequence. Following freezing, RNA was extracted a second time using the SwiftX™ Swabs Viral RNA Extraction Reagent. RHDV2 was detected in all nine samples. Cycle threshold values were higher in the RT-PCR following SwiftX extraction (mean: 3.79), indicating that the second extraction method resulted in approximately a 1 log 10 reduction in sensitivity. A second freeze–thaw for the samples and less tissue extracted using SwiftX may have contributed additionally to the loss in sensitivity.

BioTechniques↗
USGS2025

Exosomal micro RNA isolation in white-tailed deer (Odocoileus virginianus) for diagnostic biomarker discovery

Molecular approaches are becoming more prevalent for the diagnosis of neurodegenerative diseases in human medicine and can be extended to diagnosis of wildlife diseases such as chronic wasting disease and other prion diseases. These diseases have been associated with exosome-bound molecular biomarkers of disease progression, such as proteins and micro RNA molecules (miRNA). We tested and optimized a method for exosomal miRNA isolation from minimally invasive, small-volume serum samples obtained from white-tailed deer ( Odocoileus virginianus ). We confirmed the isolation of exosomes and optimized a commercially available benchtop kit to obtain sufficient and pure RNA for miRNA sequencing. The selected method for RNA extraction combines two 500- m L serum aliquots into one elution column and re-eluting the final product of the column. We identified 137 miRNA present in healthy white-tailed deer that can be used as a baseline to identify putative miRNA biomarkers of disease progression and mechanisms of infection in future comparative disease studies. This approach to biomarker discovery may help to inform biological processes in wildlife populations and provide alternatives to invasive or postmortem samples.

Journal of Wildlife Diseases↗
USGS1977

Virion RNA polymerases of two salmonid rhabdoviruses

RNA-dependent RNA polymerases were found to be associated with two salmonid rhabdoviruses: infectious hematopoietic necrosis (IHN) virus and the virus of hemorrhagic septicemia (VHS). The protein composition of these rhabdoviruses closely resembles that of rabies virus rather than that of vesicular stomatitis virus (McAllister and Wagner, 1975). The optimal temperature for in vitro transcription was found to be approximately 18 degrees C for IHN virus and approximately 15 degrees for VHS, closely approximating optimal temperatures for growth of these viruses in salmonid cells. Unlike vesicular stomatitis virus, manganese ion (1 mM) could be used as a divalent cation substitute for magnesium ion (5 mM). The in vitro transcription products of IHN and VHS viruses hybridized completely to the homologous genome but not at all to the heterologous genome.

Journal of Virology↗
USGS2024

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Nature Methods↗
USGS1968

Herpetic keratoconjunctivitis: Therapy with synthetic double-stranded RNA

A study was undertaken in rabbits to determine how late in the course of keratoconjunctivitis caused by herpes simplex recovery could be effected by an inducer of interferon. Interferon was induced by means of synthetic double-stranded RNA copolymer formed with polynosinic acid: polycytidilic acid RNA. Therapy promotes recovery from severe and fully established keratoconjunctivitis for which treatment was begun as late as 3 days after virus inoculation. No drug toxicity was observed in the therapeutic dose range. These findings further support the proposed role of the interferon mechanism in the natural recovery of already established viral infection. They also suggest the usefulness of interferon inducers in viral infections of man.

Science↗
USGS2016

Assessing models of speciation under different biogeographic scenarios; An empirical study using multi-locus and RNA-seq analyses

Evolutionary biology often seeks to decipher the drivers of speciation, and much debate persists over the relative importance of isolation and gene flow in the formation of new species. Genetic studies of closely related species can assess if gene flow was present during speciation, because signatures of past introgression often persist in the genome. We test hypotheses on which mechanisms of speciation drove diversity among three distinct lineages of desert tortoise in the genus Gopherus . These lineages offer a powerful system to study speciation, because different biogeographic patterns (physical vs. ecological segregation) are observed at opposing ends of their distributions. We use 82 samples collected from 38 sites, representing the entire species' distribution and generate sequence data for mtDNA and four nuclear loci. A multilocus phylogenetic analysis in *BEAST estimates the species tree. RNA‐seq data yield 20,126 synonymous variants from 7665 contigs from two individuals of each of the three lineages. Analyses of these data using the demographic inference package ∂a∂i serve to test the null hypothesis of no gene flow during divergence. The best‐fit demographic model for the three taxa is concordant with the *BEAST species tree, and the ∂a∂i analysis does not indicate gene flow among any of the three lineages during their divergence. These analyses suggest that divergence among the lineages occurred in the absence of gene flow and in this scenario the genetic signature of ecological isolation (parapatric model) cannot be differentiated from geographic isolation (allopatric model).

Ecology and Evolution↗
USGS2017

RNA sequencing analysis of transcriptional change in the freshwater mussel Elliptio complanata after environmentally relevant sodium chloride exposure

To identify potential biomarkers of salt stress in a freshwater sentinel species, we examined transcriptional responses of the common mussel Elliptio complanata to controlled sodium chloride (NaCl) exposures. Ribonucleic acid sequencing (RNA-Seq) of mantle tissue identified 481 transcripts differentially expressed in adult mussels exposed to 2 ppt NaCl (1.2 ppt chloride) for 7 d, of which 290 had nonoverlapping intervals. Differentially expressed gene categories included ion and transmembrane transport, oxidoreductase activity, maintenance of protein folding, and amino acid metabolism. The rate-limiting enzyme for synthesis of taurine, an amino acid frequently linked to osmotic stress in aquatic species, was upregulated, as was the transmembrane ion pump sodium/potassium adenosine 5′-triphosphatase. These patterns confirm a primary transcriptional response to the experimental dose, albeit likely overlapping with nonspecific secondary stress responses. Substantial involvement of the heat shock protein 70 chaperone family and the water-transporting aquaporin family was not detected, however, in contrast to some studies in other bivalves. A subset of the most significantly regulated genes was confirmed by quantitative polymerase chain reaction in an independent sample. Cluster analysis showed separation of mussels exposed to 2 ppt NaCl from control mussels in multivariate space, but mussels exposed to 1 ppt NaCl were largely indistinguishable from controls. Transcriptome-scale analysis of salt exposure under laboratory conditions efficiently identified candidate biomarkers for further functional analysis and field validation

Environmental Toxicology and Chemistry↗
USGS2000

Bacterial indicator occurrence and the use of an F+ specific RNA coliphage assay to identify fecal sources in Homosassa Springs, Florida

A microbiological water quality study of Homosassa Springs State Wildlife Park (HSSWP) and surrounding areas was undertaken. Samples were collected in November of 1997 (seven sites) and again in November of 1998 (nine sites). Fecal bacterial concentrations (total and fecal coliforms, Clostridium perfringens, and enterococci) were measured as relative indicators of fecal contamination. F + -specific coliphage genotyping was performed to determine the source of fecal contamination at the study sites. Bacterial levels were considerably higher at most sites in the 1997 sampling compared to the 1998 sampling, probably because of the greater rainfall that year. In November of 1997, 2 of the 7 sites were in violation of all indicator standards and guidance levels. In November of 1998, 1 of 9 sites was in violation of all indicator standard and guidance levels. The highest concentrations of all fecal indicators were found at a station downstream of the animal holding pens in HSSWP. The lowest levels of indicators were found at the Homosassa Main Spring vent. Levels of fecal indicators downstream of HSSWP (near the point of confluence with the river) were equivalent to those found in the Southeastern Fork and areas upstream of the park influences. F + specific RNA coliphage analysis indicated that fecal contamination at all sites that tested positive was from animal sources (mammals and birds). These results suggest that animal (indigenous and those in HSSWP) and not human sources influenced microbial water quality in the area of Homosassa River covered by this study.

Florida↗
USGS2017

Relative abundance of deformed wing virus, Varroa destructor virus 1, and their recombinants in honey bees (Apis mellifera) assessed by kmer analysis of public RNA-Seq data

Deformed wing virus (DWV) is a major pathogen of concern to apiculture, and recent reports have indicated the local predominance and potential virulence of recombinants between DWV and a related virus, Varroa destructor virus 1 (VDV). However, little is known about the frequency and titer of VDV and recombinants relative to DWV generally. In this study, I assessed the relative occurrence and titer of DWV and VDV in public RNA-seq accessions of honey bee using a rapid, kmer-based approach. Three recombinant types were detectable graphically and corroborated by de novo assembly. Recombination breakpoints did not disrupt the capsid-encoding region, consistent with previous reports, and both VDV- and DWV-derived capsids were observed in recombinant backgrounds. High abundance of VDV kmers was largely restricted to recombinant forms. Non-metric multidimensional scaling identified genotypic clusters among DWV isolates, which was corroborated by read mapping and consensus generation. The recently described DWV-C lineage was not detected in the searched accessions. The data further highlight the utility of high-throughput sequencing to monitor viral polymorphisms and statistically test biological predictors of titer, and point to the need for consistent methodologies and sampling schemes.

Journal of Invertebrate Pathology↗
USGS2017

The family Rhabdoviridae: Mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins

The family Rhabdoviridae consists of mostly enveloped, bullet-shaped or bacilliform viruses with a negative-sense, single-stranded RNA genome that infect vertebrates, invertebrates or plants. This ecological diversity is reflected by the diversity and complexity of their genomes. Five canonical structural protein genes are conserved in all rhabdoviruses, but may be overprinted, overlapped or interspersed with several novel and diverse accessory genes. This review gives an overview of the characteristics and diversity of rhabdoviruses, their taxonomic classification, replication mechanism, properties of classical rhabdoviruses such as rabies virus and rhabdoviruses with complex genomes, rhabdoviruses infecting aquatic species, and plant rhabdoviruses with both mono- and bipartite genomes.

Virus Research↗
USGS2002

Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

Fish Physiology and Biochemistry↗
USGS2022

RNA-seq reveals potential gene biomarkers in fathead minnows (Pimephales promelas) for exposure to treated wastewater effluent

Discharged wastewater treatment plant (WWTP) effluent greatly contributes to the generation of complex mixtures of contaminants of emerging concern (CECs) in aquatic environments which often contain neuropharmaceuticals and other emerging contaminants that may impact neurological function. However, there is a paucity of knowledge on the neurological impacts of these exposures to aquatic organisms. In this study, caged fathead minnows ( Pimephales promelas ) were exposed in situ in a temperate-region effluent-dominated stream ( i.e. , Muddy Creek) in Coralville, Iowa, USA upstream and downstream of a WWTP effluent outfall. The pharmaceutical composition of Muddy Creek was recently characterized by our team and revealed many compounds there were at a low microgram to high nanogram per liter concentration. Total RNA sequencing analysis on brain tissues revealed 280 gene isoforms that were significantly differentially expressed in male fish and 293 gene isoforms in female fish between the upstream and downstream site. Only 66 (13%) of such gene isoforms overlapped amongst male and female fish, demonstrating sex-dependent impacts on neuronal gene expression. By using a systems biology approach paired with functional enrichment analyses, we identified several potential novel gene biomarkers for treated effluent exposure that could be used to expand monitoring of environmental effects with respect to complex CEC mixtures. Lastly, when comparing the results of this study to those that relied on a single-compound approach, there was relatively little overlap in terms of gene-specific effects. This discovery brings into question the application of single-compound exposures in accurately characterizing environmental risks of complex mixtures and for gene biomarker identification.

Iowa↗