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At least 37 records · Page 2Linked to original sources
USGS2013

The role of virulence for in vivo superinfection fitness of a vertebrate RNA virus

We have developed a novel, in vivo superinfection fitness assay to examine superinfection dynamics and the role of virulence in superinfection fitness. This assay involves controlled, sequential infections of a natural, vertebrate host, Oncorhynchus mykiss (rainbow trout), with variants of a co-evolved viral pathogen, infectious hematopoietic necrosis virus (IHNV). Intervals between infections ranged from 12 hours to 7 days, and both frequency of superinfection and viral replication levels were examined. Using virus genotype pairs of equal and unequal virulence, we observed that superinfection generally occurred with decreasing frequency as the interval between exposures to each genotype increased. For both the equal virulence and unequal virulence genotype pairs, the frequency of superinfection was the same regardless of which genotype was used in the primary exposure. However, the ability to replicate in the context of superinfection did not differ between the genotypes of equal or unequal virulence tested here. For both genotype pairs, the mean viral load of the secondary virus was significantly reduced in superinfection, while primary virus replication was unaffected. Our results demonstrate, for the two genotype pairs examined, that superinfection restriction does occur for IHNV, and that higher virulence did not correlate with a significant difference in superinfection fitness. To our knowledge, this is the first assay to examine the role of virulence of an RNA virus in determining superinfection fitness dynamics within a natural, vertebrate host.

Journal of Virology↗
USGS1979

Fish viruses: A double-stranded RNA icosahedral virus from a North American cyprinid

A previously unreported virus disease of cultured golden shiners ( Notemigonus crysoleucas ) is described. The condition is called golden shiner virus (GSV) disease. The virus is icosahedral, measures approximately 70 nm, is ether and heat resistant, stable at pH 3, 7, and 10, and appears to have a double stranded RNA core. It was not neutralized by infectious pancreatic necrosis virus specific antiserum. GSV causes syncytial like cytopathic effect in fathead minnow cells but not brown bullhead, rainbow trout gonad (RTG-2) or channel catfish ovary cells. Severe mortalities in golden shiner culture ponds do not appear to be associated with GSV infections.

Journal of the Fisheries Research Board of Canada↗
USGS1998

Identification of Flavobacterinium and Flexibacterin species by species-specific polymerase chain reaction primers to the 16S ribosomal RNA gene

Species-specific polymerase chain reaction (PCR) primers have been developed for the identification of the causative agents of warmwater and marine finrot in fish: Flavobacterium columnare ( Flexibacter columnaris ) and Flexibacter maritimus. Differences in gene sequence in the bacterial small-subunit (16S) ribosomal RNA (rRNA) were used to design the species-specific PCR primers. The previously reported species-specific PCR primers Psy1 and Psy2 for the identification of Flavobacterium psychrophilum ( Flexibacter psychrophila ), the causative agent of coldwater finrot, were also used to develop a speciation scheme for all three bacterial finrots. These three primer sets were successful in discriminating among yellow-pigmented bacteria as well as in speciating the three major pathogenic flexibacteria to fish. The primer sets were designed to produce uniquely sized subproducts of 16S rRNA for each species: Flavobacterium psychrophilum (1,100 base pairs, bp), F. columnare (800 bp), and Flexibacter maritimus (400 bp). These primers were shown to correctly speciate field isolates in double-blind experiments ( P = 0.01).

Journal of Aquatic Animal Health↗
USGS1998

Determination of phylogenetic relationships of Flavobacterinium psychrophilum (Flexibacterin psychrophilus), Flavobacterinium columnare (Flexibacterin columnaris), and Flexibacterin maritimus by sequence analysis of 16S ribosomal RNA genes amplified by polymerase chain reaction

The nearly complete small subunit 16S ribosomal RNA (rRNA) gene sequence amplified by polymerase chain reaction was determined for Flavobacterium psychrophilum (formerly Flexibacter psychrophilus ) by using automated nucleotide sequencing. The sequence was found to be 1,465 base pairs (bp) in length, a size consistent with previously determined sequences for 14 other bacterial species from various taxa, including the yellow-pigmented bacteria. Sequence signatures confirmed that this organism was a member of the bacterial division Bacteroides–Flavobacterium. Parsimonious and additive phylogenetic trees were constructed with homology, and pairwise evolutionary distances were used to estimate phylogenetic relationships. Data show that F. psychrophilum, F. columnare , and Flexibacter maritimus are closely related, have a common descent, and represent a distinct group within the division Bacteroides–Flavobacterium. This group also included other organisms from the genera Flavobacterium and Cytophaga. Further, Flavobacterium aquatile , the type species for the genus Flavobacterium , was also determined to be a member of this “ Flavobacterium–Cytophaga–Flexibacter subcomplex.” This supports previous assertions that the type strain of Flavobacterium should be changed to a more representative species such as Flavobacterium breve .

Journal of Aquatic Animal Health↗
USGS2000

Correlation of transforming growth factor-β messenger RNA (TGF-β mRNA) expression with cellular immunoassays in Triamcinolone-treated captive hybrid striped bass

Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative–competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis × male white bass M. chrysops ) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

Journal of Aquatic Animal Health↗
USGS2017

Avian influenza virus RNA in groundwater wells supplying poultry farms affected by the 2015 influenza outbreak

During the 2015 outbreak of highly pathogenic avian influenza virus (HPAI) on poultry farms in the midwestern United States, concern was raised about the potential for HPAI to contaminate groundwater. Our study objective was to evaluate the occurrence of HPAI in the groundwater supply wells on 13 outbreak-affected poultry farms in Iowa and Wisconsin. We sampled 20 wells, six waste-storage lagoons, and one pond. Three wells and one lagoon were positive for the matrix gene indicative of influenza A virus. Using a semi-nested qPCR assay specific to the H5 HPAI outbreak strain, one well was H5-positive, matching the outbreak virus hemagglutinin gene. Matrix gene-positive samples analyzed for avian influenza virus (AIV) by cell culture and embryonating egg culture were negative. Seven wells were positive by PCR for a poultry-specific parvovirus, thus providing corroborating evidence of virus transport pathways between poultry fecal wastes and groundwater. Our data suggest it is possible for AIV to be transported to groundwater, and during an outbreak, the potential for poultry farm wells to become contaminated with AIV should be considered.

Iowa, Wisconsin↗
USGS2011

Alphacoronaviruses in new World bats: Prevalence, persistence, phylogeny, and potential for interaction with humans

Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.

PLoS ONE↗
USGS1990

Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus

The polymerase chain reaction [PCR) was used to amplify a portion of the nucleoprotein [NI gene of infectious hematopoietic necrosis virus (IHNV). Using a published sequence for the Round Butte isolate of IHNV, a pair of PCR pnmers was synthesized that spanned a 252 nucleotide region of the N gene from residue 319 to residue 570 of the open reading frame. This region included a 30 nucleotide target sequence for a synthetic oligonucleotide probe developed for detection of IHNV N gene messenger RNA. After 25 cycles of amplification of either messenger or genomic RNA, the PCR product (DNA) of the expected size was easily visible on agarose gels stained with ethidium bromide. The specificity of the amplified DNA was confirmed by Southern and dot-blot analysis using the biotinylated oligonucleotide probe. The PCR was able to amplify the N gene sequence of purified genomic RNA from isolates of IHNV representing 5 different electropherotypes. Using the IHNV primer set, no PCR product was obtained from viral hemorrhagic septicemia virus RNA, but 2 higher molecular weight products were synthesized from hirame rhabdovirus RNA that did not hybridize with the biotinylated probe. The PCR could be efficiently performed with all IHNV genomic RNA template concentrations tested (1 ng to 1 pg). The lowest level of sensitivity was not determined. The PCR was used to amplify RNA extracted from infected cell cultures and selected tissues of Infected rainbow trout. The combination of PCR and nucleic acid probe promises to provide a detection method for IHNV that is rapid, h~ghly specific, and sensitive.

Diseases of Aquatic Organisms↗
USGS2023

Book review: Quasispecies as a unifying concept in population dynamics

The quasispecies concept had two independent origins. One source was the theoretical ideas of Manfred Eigen and Peter Schuster in the 1970s. Studying the self-organization and evolution of primitive RNA molecules, they defined quasispecies as a distribution of mutant viral genomes generated by the mutation-selection process. In particular, the quasispecies nucleotide distribution consists of a singular fittest genotype, called the master sequence, surrounded by similar mutant spectra. The other source of the quasispecies concept can be traced to the growing empirical knowledge on RNA viruses formulated around the same time. Genomic sequencing of viral RNA indicated a variety of nucleotide sequences, or a distribution of sequences. Mutations in both Eigen’s theoretical system in the RNA viruses are not rare, as RNA is structurally less stable than RNA, but common, preventing the fittest genotype from becoming dominant. Virologists have adopted the quasispecies concept, with some papers on Covid-19 using the construct to explain that virus’s behavior.

Mathematical Biosciences↗
USGS2021

Interlaboratory comparison of SARS-CoV2 molecular detection assays in use by U.S. veterinary diagnostic laboratories

The continued search for intermediate hosts and potential reservoirs for SARS-CoV2 makes it clear that animal surveillance is critical in outbreak response and prevention. Real-time RT-PCR assays for SARS-CoV2 detection can easily be adapted to different host species. U.S. veterinary diagnostic laboratories have used the CDC assays or other national reference laboratory methods to test animal samples. However, these methods have only been evaluated using internal validation protocols. To help the laboratories evaluate their SARS-CoV2 test methods, an interlaboratory comparison (ILC) was performed in collaboration with multiple organizations. Forty-four sets of 19 blind-coded RNA samples in Tris-EDTA (TE) buffer or PrimeStore transport medium were shipped to 42 laboratories. Results were analyzed according to the principles of the International Organization for Standardization (ISO) 16140-2:2016 standard. Qualitative assessment of PrimeStore samples revealed that, in approximately two-thirds of the laboratories, the limit of detection with a probability of 0.95 (LOD95) for detecting the RNA was ≤20 copies per PCR reaction, close to the theoretical LOD of 3 copies per reaction. This level of sensitivity is not expected in clinical samples because of additional factors, such as sample collection, transport, and extraction of RNA from the clinical matrix. Quantitative assessment of Ct values indicated that reproducibility standard deviations for testing the RNA with assays reported as N1 were slightly lower than those for N2, and they were higher for the RNA in PrimeStore medium than those in TE buffer. Analyst experience and the use of either a singleplex or multiplex PCR also affected the quantitative ILC test results.

Journal of Veterinary Diagnostic Investigation↗
USGS2013

Expression analysis and identification of antimicrobial peptide transcripts from six North American frog species

Frogs secrete antimicrobial peptides onto their skin. We describe an assay to preserve and analyze antimicrobial peptide transcripts from field-collected skin secretions that will complement existing methods for peptide analysis. We collected skin secretions from 4 North American species in the field in California and 2 species in the laboratory. Most frogs appeared healthy after release; however, Rana boylii in the Sierra Nevada foothills, but not the Coast Range, showed signs of morbidity and 2 died after handling. The amount of total RNA extracted from skin secretions was higher in R. boylii and R. sierrae compared to R. draytonii, and much higher compared to Pseudacris regilla. Interspecies variation in amount of RNA extracted was not explained by size, but for P. regilla it depended upon collection site and date. RNA extracted from skin secretions from frogs handled with bare hands had poor quality compared to frogs handled with gloves or plastic bags. Thirty-four putative antimicrobial peptide precursor transcripts were identified. This study demonstrates that RNA extracted from skin secretions collected in the field is of high quality suitable for use in sequencing or quantitative PCR (qPCR). However, some species do not secrete profusely, resulting in very little extracted RNA. The ability to measure transcript abundance of antimicrobial peptides in field-collected skin secretions complements proteomic analyses and may provide insight into transcriptional mechanisms that could affect peptide abundance.

Diseases of Aquatic Organisms↗
USGS2017

The Moquah Barrens Research Natural Area: Loss of a pine barrens ecosystem

The Moquah Barrens Research Natural Area (RNA) was established by the Chequamegon National Forest and the Lakes States Forest Experiment Station in 1935 with a research objective well-suited to the needs of the Forest Service and the scientific understanding of ecosystem function prevalent at the time of establishment. The original research plan was never implemented, which led to a joint Forest-Station decision in 1956 to disestablish the RNA. However, that decision was never implemented. A series of management decisions made after 1956 led to the loss of the pine barrens ecosystem originally encompassed by the RNA. This loss is not irretrievable and the work necessary to recover the original ecosystem is possible under existing RNA management guidelines. The experience of the Moquah Barrens RNA can be used by the Forest Service to improve overall management of the entire system of research natural areas. Two main areas of opportunity are identified: 1) implement an improved approach to managing official records associated with RNAs; and 2) adopt a management framework suitable for long-term ecological projects.

Wisconsin↗
USGS2006

Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus

The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.

Journal of Virological Methods↗
USGS2017

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout ( Oncorhynchus mykiss ) and arctic char ( Salvelinus alpinus ). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Journal of Virological Methods↗
USGS2019

Inactivation of viable surrogates for the select agents virulent Newcastle disease virus and highly pathogenic avian influenza virus using either commercial lysis buffer or heat

Introduction: Federal Select Agent Program regulations require laboratories to document a validated procedure for inactivating select agents prior to movement outside registered space. Avian influenza viruses and virulent Newcastle disease virus (vNDV) are cultured in chicken amnio-allantoic fluid (AAF), but the efficacy of commercial lysis buffers to inactivate viruses in protein-rich media has not been documented. Objectives: We assesses the efficacy of MagMAX™ lysis buffer for inactivating highly pathogenic avian influenza virus (HPAIV) and vNDV in chicken AAF and confirm the inactivation of avian influenza in serum using heat. Methods: Low pathogenic avian influenza virus (LPAIV) and avian paramyxovirus subtype-1 (APMV-1) were incubated with lysis buffer and tested for viability. Known viable LPAIV and APMV-1 RNA was extracted from AAF using MagMAX™-96 AI/ND Viral RNA Isolation kit, and the eluate was tested for remaining infectious agent. Finally, inactivation of LPAIV in serum was examined over 3 combinations of temperature and incubation time. Results: MagMAX™ lysis buffer inactivated both LPAIV and APMV-1 in AAF when incubated for 30 minutes at room temperature. The full extraction process eliminated viable virus from the final RNA eluate. LPAIV in serum heated to 70°C for 30 minutes was rendered noninfectious. Conclusion: The ability of a diagnostic laboratory to move samples from one space to another is critical to maintaining biosecurity as well as efficient laboratory workflow. Our study demonstrates a method to ensure the inactivation of viable avian influenza and avian paramyxoviruses in AAF, RNA eluate, and viable avian influenza virus in sera.

Applied Biosafety↗
USGS2022

Revised taxonomy of rhabdoviruses infecting fish and marine mammals

Rhabdoviridae is a large family of negative-sense (-) RNA viruses that includes important pathogens of ray-finned fish and marine mammals. As for all viruses, the taxonomic assignment of rhabdoviruses occurs through a process implemented by the International Committee on Taxonomy of Viruses (ICTV). A recent revision of taxonomy conducted in conjunction with the ICTV Rhabdoviridae Study Group has resulted in the establishment of three new subfamilies ( Alpharhabdovirinae , Betarhabdovirinae , and Gammarhabdovirinae ) within the Rhabdoviridae , as well as three new genera ( Cetarhavirus , Siniperhavirus , and Scophrhavirus ) and seven new species for viruses infecting fish or marine mammals. All rhabdovirus species have also now been named or renamed to comply with the binomial format adopted by the ICTV in 2021, comprising the genus name followed by a species epithet. Phylogenetic analyses of L protein (RNA-dependent RNA polymerase) sequences of (-) RNA viruses indicate that members of the genus Novirhabdovirus (subfamily Gammarhabdovirinae ) do not cluster within the Rhabdoviridae , suggesting the need for a review of their current classification.

Animals↗
USGS1995

Mutational analyses of molecularly cloned satellite tobacco mosaic virus during serial passage in plants: Evidence for hotspots of genetic change

The high level of genetic diversity and rapid evolution of viral RNA genomes are well documented, but few studies have characterized the rate and nature of ongoing genetic change over time under controlled experimental conditions, especially in plant hosts. The RNA genome of satellite tobacco mosaic virus (STMV) was used as an effective model for such studies because of advantageous features of its genome structure and because the extant genetic heterogeneity of STMV has been characterized previously. In the present study, the process of genetic change over time was studied by monitoring multiple serial passage lines of STMV populations for changes in their consensus sequences. A total of 42 passage lines were initiated by inoculation of tobacco plants with a helper tobamovirus and one of four STMV RNA inocula that were transcribed from full-length infectious STMV clones or extracted from purified STMV type strain virions. Ten serial passages were carried out for each line and the consensus genotypes of progeny STMV populations were assessed for genetic change by RNase protection analyses of the entire 1,059-nt STMV genome. Three different types of genetic change were observed, including the fixation of novel mutations in 9 of 42 lines, mutation at the major heterogeneity site near nt 751 in 5 of the 19 lines inoculated with a single genotype, and selection of a single major genotype in 6 of the 23 lines inoculated with mixed genotypes. Sequence analyses showed that the majority of mutations were single base substitutions. The distribution of mutation sites included three clusters in which mutations occurred at or very near the same site, suggesting hot spots of genetic change in the STMV genome. The diversity of genetic changes in sibling lines is clear evidence for the important role of chance and random sampling events in the process of genetic diversification of STMV virus populations.

RNA↗
USGS1987

Toxicity of paraquat in nestling birds: effects on plasma and tissue biochemistry in American kestrels

Beginning the day after hatching, American kestrel ( Falco sparverius ) nestlings were orally dosed daily for 10 days with 5 μL/g of distilled water (controls), 10 mg/kg, 25 mg/kg, or 60 mg/kg of paraquat dichloride (1,1′-dimethyl-4,4′-bipyridinium dichloride) in distilled water. Forty-four percent of the nestlings receiving 60 mg/kg died after 4 days. Plasma LDH activity and total protein concentration were elevated, and plasma alkaline phosphatase activity was lower in survivors of the 60 mg/kg group at 10 days. Lung total sulfhydryl (TSH) and protein-bound sulfhydryl (PBSH) concentrations were significantly higher in the 10 mg/kg, 25 mg/kg, or 60 mg/kg groups. Lung DNA, RNA, protein, and hydroxyproline (collagen) concentrations were not significantly affected by treatment. Liver NPSH was lower in the 60 mg/kg group while liver glycogen concentration was not affected by treatment. Kidney DNA, RNA, and RNA to protein concentration ratio were higher in the 25 mg/kg or 60 mg/kg groups. These findings in combination with recently reported effects on growth and histopathology suggest that altricial nestling kestrels are more sensitive to paraquat exposure than young or adult birds of precocial species. From a comparative viewpoint, lungs of nestling kestrels are less sensitive to paraquat than mammalian lungs.

Maryland↗